Becker & Hickl GmbH

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October 2017

 

 

This brochure is subject to copyright. However, reproduction of small portions of the material in scientific papers or other non-commercial publications is considered fair use under the copyright law. It is requested that a complete citation be included in the publication. If you require confirmation please feel free to contact Becker & Hickl GmbH.

 

 

 

 

 

FLIM Systems for Zeiss LSM 710 / 780 / 880 Family

 Laser Scanning Microscopes

An Overview

 

Abstract: The FLIM systems for the Zeiss LSM 710 /780 / 880 family laser scanning microscopes are based on bh’s Multi-Dimensional TCSPC technique and 64 bit Megapixel technology [21, 30]. The systems feature single-photon sensitivity, excellent spatial and temporal resolution, multi-exponential decay analysis, and short acquisition time. The systems are compatible with multiphoton excitation, diode laser excitation, and tuneable excitation by the Zeiss Intune laser. The system fully exploits the fast beam scanning capability of the Zeiss LSM. The functions include advanced techniques like multi-wavelength detection, Z stack FLIM, time-series FLIM, spatial and temporal mosaic FLIM, fluorescence lifetime-transient scanning (FLITS), and phosphorescence lifetime imaging (PLIM). We describe the use of the system for the measurement of molecular environment parameters, FRET measurements, autofluorescence FLIM of tissue, and plant physiology. This brochure is a condensed version of the handbook of the bh FLIM systems for the Zeiss LSM series microscopes [6].

 

Introduction

Becker & Hickl introduced their multi-dimensional TCSPC technique in 1993. Fluorescence lifetime imaging started in 1996 with applications in ophthalmology [159]. The first FLIM module for laser scanning microscopes was introduced in 1998, bh FLIM systems for the Zeiss LSM laser scanning microscopes are available since 2000 [16, 17]. Since then, several new generations of LSM family laser scanning microscopes, and several generations of bh FLIM modules have been introduced. As a result, a wide variety of bh FLIM systems and of FLIM system configurations are in use [21, 30]. The excitation light source can be the Ti:Sapphire laser of a multiphoton microscope, a picosecond diode laser attached to or integrated in the microscope, or a visible-range tuneable solid state laser. The fluorescence light may be detected via a confocal port of the scan head or via a non-descanned port of a multiphoton microscope. Signals may be detected by one detector, simultaneously by two, three, or four detectors, or by the 16 channels of a bh multi-wavelength detector.

 

Fig. 1: LSM 710 Multiphoton NDD FLIM systems

The bh FLIM systems are using highly efficient GaAsP hybrid detectors. By combining extremely high efficiency with large active area, high counting speed, high time-resolution, and low background, these detectors have triggered a breakthrough in FLIM recording. Another step was made by the introduction of 64-bit data acquisition software. FLIM data are now recorded at unprecedented pixel numbers, high dynamic range, short acquisition time, and minimum exposure of the sample. New hardware and software functions have resulted in advanced FLIM functions, like time-series FLIM, Z stack FLIM, spatial and temporal Mosaic FLIM, combined fluorescence and phosphorescence lifetime imaging (FLIM/PLIM), and fluorescence lifetime-transient scanning (FLITS). [30, 180].

This brochure gives an overview on the bh TCSPC FLIM systems for the Zeiss laser scanning microscopes. It applies to all versions of the LSM 710, LSM 710 NLO, LSM 7 MP, LSM 780, LSM 780 NLO, LSM 880, and LSM 880 NLO microscopes. For a detailed description of the systems please see [6]. We also recommend [21, 30] and [32] as basic literature about the bh FLIM technique and its application.

 

Architecture of the LSM 710 / 780 / 880  FLIM Systems

Optical Configuration

All TCSPC FLIM systems have in common that the sample is excited by a pulsed laser of high repetition rate, scanned at high pixel rate by the optical scanner of the microscope, and that the fluorescence light is detected by one or several fast photon counting detectors connected to the microscope. Typical FLIM configurations for the LSM 710/780/880 family microscopes are shown in Fig. 2, Fig. 3 and Fig. 4.

Fig. 2 shows FLIM configurations for inverted microscopes. The configuration on the left uses multiphoton excitation by a femtosecond titanium-sapphire laser for excitation. The fluorescence light is detected via a non-descanned detection (NDD) beam path. Typically, the light is split in two spectral components by a Zeiss 'T Adapter', and detected by two parallel detectors and TCSPC channels. The configuration shown on the right uses picosecond diode lasers or the tuneable ‘Intune’ laser. The sample is excited by one-photon excitation. The fluorescence light is detected back through the confocal beam path of the scanner and sent out of the scan head via an optical port. It is split into two spectral channels by a bh beamsplitter module and fed into two FLIM detectors. The signals from the detectors are recorded by two separate TCSPC channels of the FLIM system.

Fig. 2: LSM 710/780 family FLIM systems, inverted microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon FLIM with confocal detection.

Fig. 3 shows FLIM at an LSM 710/780 in the upright version. The configuration on the left uses multiphoton excitation and non-descanned detection, the configuration on the right uses one-photon excitation and confocal detection.

Fig. 3: LSM 710/780/880 family FLIM systems, upright microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon excitation  FLIM with confocal detection.

Multiphoton and the one-photon FLIM configurations for the LSM 880 are shown in Fig. 4, left and right. The detectors of the one-photon systems are connected to a beam switch between the scan head and the Zeiss Airy-Scan detector, see Fig. 4, right.

Fig. 4: LSM 880 FLIM systems, inverted microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon excitation  FLIM with confocal detection.

Standard bh FLIM systems for the LSM 710, 780 and 880 use the bh HPM‑100-40 hybrid detectors [30, 24]. However, the bh systems work also with the Zeiss BIG 2 detector [12, 30], with the NIR versions of the HPM‑100 detectors [10, 11, 31], and with the bh MW-FLIM GaAsP multi-spectral FLIM detectors [30]. The optical configuration for multiphoton multi-wavelength FLIM is shown in Fig. 5. The light is collected from an NDD port by a fibre bundle. The light is dispersed spectrally, and detected by a bh PML-16 GaAsP (16-channel) PMT module. Similarly, the multi-wavelength FLIM detector assembly can be attached to the confocal port of the scan head.

Fig. 5: Multi-wavelength FLIM

FLIM Technique

Multi-Dimensional TCSPC

he bh FLIM systems use a combination of bh’s multidimensional time-correlated single-photon counting process with confocal or multiphoton laser scanning [21, 30, 33, 34]. The principle is shown in Fig. 6. The laser scanning microscope scans the sample with a focused beam of a high-repetition-rate pulsed laser. Depending on the laser used, the fluorescence in the sample can either be excited by one-photon or by multiphoton excitation. The FLIM detector is attached either to a confocal or non-descanned port of the laser scanning microscope [6, 16, 17, 18, 19, 20, 30]. For every detected photon the detector sends an electrical pulse into the TCSPC module. Moreover, the TCSPC module receives scan clock signals (pixel, line, and frame clock) from the scanning unit of the microscope.

Fig. 6: Multidimensional TCSPC architecture for FLIM

For each photon pulse from the detector, the TCSPC module determines the time within the laser pulse sequence (i.e. in the fluorescence decay) and the location within the scanning area, x and y. The photon times, t, and the spatial coordinates, x and y, are used to address a memory in which the detection events are accumulated. Thus, in the memory the distribution of the photon density over x, y, and t builds up. The result is a data array representing the pixel array of the scan, with every pixel containing a large number of time channels with photon numbers  for consecutive times after the excitation pulse. In other words, the result is an image that contains a fluorescence decay curve in each pixel [20, 21]. Because the full decay profile is recorded and no photons are suppressed the recording process works at near-ideal photon efficiency [21, 32, 81, 109].

The TCSPC module can also process the photon data to obtain fluorescence-correlation data (FCS) data [30, 22], photon counting histograms (PCH) or photon-counting-lifetime histograms. Moreover the ‘parameter-tagged’ single photon data can be stored in a file for off-line processing by single-molecule spectroscopy techniques [150, 199].

It should be explicitly noted that FLIM by multi-dimensional TCSPC does not require that the scanner stays in one pixel until enough photons for a full fluorescence decay curve have been acquired. It is only necessary that the total pixel time, over a large number of subsequent frames, is large enough to record a reasonable number of photons per pixel. Thus, TCSPC FLIM works even at the highest scan rates available in laser scanning microscopes. At the pixel rates used in practice, the recording process is more or less random: A photon is just stored in a memory location according to its time in the fluorescence decay, its detector channel number, and the location of the laser spot in the sample in the moment of detection.

Standard bh FLIM systems for the LSM 710/780/880 family microscopes have two parallel channels of the architecture shown in Fig. 6. The systems thus deliver high throughput rates. Another advantage is that the channels are independent. If one channel overloads the other one still delivers correct data. Please see [6] and [30] for details.

Multi-Wavelength FLIM

The principle shown in Fig. 6, page 6, can be extended to simultaneously detect in 16 wavelength channels. The optical spectrum of the fluorescence light is spread over an array of 16 detector channels. The TCSPC system and determines the detection times, the channel numbers in the detector array, and the position, x, and y, of the laser spot for the individual photons. These pieces of information are used to build up a photon distribution over the time of the photons in the fluorescence decay, the wavelength, and the coordinates of the image [18, 21, 30, 23]. The principle of multi-wavelength FLIM is shown in Fig. 7.

Fig. 7: Principle of Multi-Wavelength TCSPC FLIM

As for single-wavelength FLIM, the result of the recording process is an array of pixels. However, the pixels now contain several decay curves for different wavelength. Each decay curve contains a large number of time channels; the time channels contain photon numbers for consecutive times after the excitation pulse.

Mosaic FLIM

Mosaic FLIM is part of bh’s new Megapixel FLIM technology. It records a sequence of lifetime images into a single, large photon distribution. The principle is shown in Fig. 8. Mosaic FLIM is used to record image of large sample areas via the Tile Imaging function of the Zeiss LSMs, to record Z stacks, or to record fast time-series of FLIM images. Time series can even be recorded by triggered accumulation to resolve transient lifetime changes in the a sample at a resolution down to less than 50 ms, see Fig. 36.

Fig. 8: Mosaic FLIM. The TCSPC system records a series of lifetime images in to a single, large photon distribution.

FLIM Functions in Brief

Data Acquisition Software

The FLIM systems for the Zeiss LSM 710/780 family use the SPCM data acquisition software. Since 2013 the SPCM software is available in a 64-bit version. SPCM 64 bit exploits the full capability of Windows 64 bit, resulting in faster data processing, capability of recording images of extremely large pixel numbers, and availability of additional multi-dimensional FLIM modes [9, 30, 180]. The bh SPCM software and the Zeiss ZEN software are operated via the same keyboard and the same mouse.

The main panel of the SPCM data acquisition software is configurable by the user [30]. Four configurations for FLIM systems are shown in Fig. 9. During the acquisition the SPCM software displays intermediate results in predefined intervals, usually every few seconds. The acquisition can be stopped after a defined acquisition time or by a user commend when the desired signal-to-noise ratio has been reached [6, 30]. Frequently used operation modes and user interface configurations are selected from a panel of predefined setups.

  

  

Fig. 9: SPCM software panel. Top left to bottom right: FLIM with two detector channels, multi-spectral FLIM, combined fluorescence / phosphorescence lifetime imaging (FLIM/PLIM), fluorescence correlation (FCS).

 

 

FLIM Data Acquisition

From the user point of view, FLIM in a laser scanning microscope is performed by the same general procedures as steady-state imaging. First, the sample is brought in focus by looking through the eyepieces and manually turning the microscope focus buttons. Then a fast repetitive scan is run in which the FLIM system displays fast preview images. While observing these images, the exact location and size of the scan area are selected, and the focal plane is fine-adjusted. Then an appropriate FLIM mode is selected, and the final FLIM acquisition is started.

Fast preview function

When FLIM is applied to live samples the time and the sample exposure needed for positioning, focusing, laser power adjustment, and selection of the scan region has to minimised. Therefore, the FLIM systems have a fast preview function. The preview function displays images in intervals on the order of 1 second and less, see Fig. 10.



Fig. 10: SPCM software in fast preview mode. 1 second per image. Two parallel FLIM channels recording in separate wavelength intervals.

Whatever you change in the microscope: The position of the samples, the scan area, the zoom factor, the focal plane, pinhole size or the laser power - the result becomes immediately visible in the preview images, see Fig. 11.

Fig. 11: When the scan area definition in the Zeiss ZEN software is changed the result is shown in the images immediately.

Easy Switching Between Acquisition Modes and User Interface Configurations

Frequently used operation modes and user interface configuration can be selected from a panel of predefined setups. Switching between Preview modes, FLIM acquisition, different pixel and time-channel numbers, time-series recording, Z-stack recording, FCS, or any other conceivable recording procedure is a matter of a single mouse click, see Fig. 12.

 

Fig. 12: Switching the instrument configuration via the ‘Predefined Setup’ panel

 

High-Efficiency GaAsP Detectors: bh Hybrid Detectors, Zeiss BiG Detector, and bh MW-FLIM Multi-Wavelength Detector

The bh HPM‑100‑40 GaAsP hybrid detectors combine superior time resolution and SPAD-like sensitivity with the large active area of a PMT [30, 24]. The large area avoids alignment problems, and allows light to be efficiently collected from non-descanned ports of multiphoton microscopes. In contrast to SPADs, there is no ‘diffusion tail’ in the temporal response. Moreover, the hybrid detectors are free of afterpulsing. The absence of afterpulsing results in improved contrast, higher dynamic range of the decay curves recorded, and in the capability to obtain FCS data from a single detector.

         

Fig. 13: Fluorescence lifetime images recorded with an HPM‑100-40 hybrid detector (left) and with an id‑100‑50 SPAD (right). Images and decay functions at selected cursor position.

The bh FLIM systems also work with the Zeiss GaAsP BIG-2 detectors. The advantage of this detector is that it can be used for FLIM and conventional imaging as well. Additionally, there is the bh MW FLIM detector for recording multi-spectral FLIM data. Also this detector is now available with a highly efficient GaAsP cathode [30].

 

 

 

Fast Beam Scanning - Fast Acquisition

The bh FLIM systems are perfectly compatible with the fast beam scanning used in the Zeiss LSM 710/780/880 family microscopes. Frame times can be from about 50 ms to a few seconds, with pixel dwell times down to less than one microsecond. The multi-dimensional TCSPC process used in the bh FLIM systems delivers identical results for different scan rates, provided the total acquisition time is the same. FLIM can be acquired at short acquisition time. Fig. 14 shows lifetime images of a BPAE cell recorded within 5 seconds acquisition time.



Fig. 14: FLIM acquired within 5 seconds of acquisition time. Left 485 to 560 nm, right 560 to 650 nm. BPAE cell stained with Alexa 488 and Mito Tracker Red.

Acquisition at high scan rate is also the basis of the fast preview mode (see above) and of fast FLIM time-series recording. Time-series can be recorded as fast as two images per second [30, 106], with temporal mosaic FLIM and triggered accumulation even faster. Fig. 15 shows time-series FLIM of an amoeba. The images are 0.4-second snapshots taken very one second.

Fig. 15: Moving amoeba. Autofluorescence, acquisition time 0.4 s, image rate 1 image per second.

 

Online FLIM Display

Starting from Version 9.72 SPCM software the FLIM systems for the Zeiss LSM 710 / 780 / 880 family are able to display lifetime images online, both during the accumulation of FLIM data and for the individual steps of a fast image sequence. An example is shown in Fig. 16. The calculation of the lifetime images is based on the first moment of the decay data in the pixels of the images [30]. The first-moment technique combines short calculation times with near-ideal photon efficiency. It does not require to reduce the time resolution (time channels per pixel) to obtain high image rates. Even if the fast online lifetime function is used during the FLIM acquisition the data can later be processed by precision SPCImage multi-exponential data analysis.

Fig. 16: Intensity image (left) and online lifetime image (right) calculated online by SPCM software

 

Bi-Directional Scanning

With Version 9.73 SPCM Software, the bh TCSPC / FLIM systems support the bidirectional scanning function of the Zeiss LSMs. As usual, data recording is synchronised with the scanning by frame clock, line clock, and pixel clock pulses from the scanner. Each first line clock pulse indicates the beginning of a forward scan, each second one the beginning of a backward scan. The recording procedure automatically reverses the data from the backward scan and compensates for the line shift caused by the dynamic behaviour of the scanner. The FLIM data structure is the same as for unidirectional scanning. Thus, standard online intensity and lifetime display functions of the SPCM software are available, and data can be analysed by SPCImage as usual [30].

     

Fig. 17: FLIM of Convallaria sample (left, 512x512 pixels) and BPAE Cell sample (right, 1024x1024 pixels), recorded with bidirectional scanning.  Images created by online-lifetime function of SPCM software.

Megapixel FLIM Images

With bh’s megapixel technology, pixel numbers can be increased up to 2048 x 2048 while maintaining a temporal resolution of 256 time channels. Thus, the useful pixel resolution is rather limited by the optical resolution and the maximum field of view of the microscope lens than by the capabilities of the bh FLIM system. By using the ‘Mosaic FLIM’ option, the useful pixel number can be increase even beyond the capabilities of the microscope lens, see page 22. Alternatively, the number of time channels can be increased up to 1024 for images of 1024x1024 pixels, and up to 4096 for images of 512x512 pixels or less.

Fig. 18 shows a FLIM image of a BPAE sample recorded at a resolution of 1024 x 1024 pixels. The image on the left shows the entire area of the scan. The image on the right is a zoom into the data shown left.

 

Fig. 18: Left: Image recorded with 1024 x 1024 pixels. Right: Digital zoom into the data of Fig. 18, showing the two cells on the upper left. Effective resolution 256x256 pixels. LSM 710 Intune system, excitation 535 nm, emission from 550 nm up. BPAE cell stained with Alexa 488 and Mito Tracker Red.

Large pixel numbers are important especially for tissue imaging. They are also useful in cases when a large number of cells have to be investigated and the FLIM results to be compared. Megapixel FLIM records images of many cells simultaneously, and under identical environment conditions. Moreover, the data are analysed in a single analysis run, with identical IRFs and fit parameters. The results are therefore exactly comparable for all cells in the image area.

 

 

 

 

 

Two fully parallel TCSPC FLIM Channels

Standard bh FLIM systems record in two wavelength intervals simultaneously. The signals are detected by separate detectors and processed by separate TCSPC modules [30]. There is no intensity or lifetime crosstalk. Even if one channel overloads the other channel is still able to produce correct data.

         

Fig. 19: Dual-channel detection. BPAE cells stained with Alexa 488 phalloidin and Mito Tracker Red. Left: 460 nm to 550 nm. Right: 550 nm to 650 nm.

 

Laser Wavelength Multiplexing

The bh FLIM system can synchronise two ps diode lasers or a ps diode laser and the Zeiss Intune laser for wavelength-multiplexed FLIM by pulse-interleaved excitation (PIE). An example is shown in Fig. 20.



Fig. 20: FLIM by PIE, Zeiss LSM 710 Intune laser (‘green laser’)  and 405 nm ps diode laser (‘blue laser’). Images recorded by the two parallel SPC‑150 TCSPC modules of the FLIM system.

 

Multiphoton NDD FLIM: Clean Images from Deep Tissue Layers

The bh Multiphoton FLIM systems use the non-descanned detection (NDD) path of the LSM 710/780/880 NLO family microscopes. With non-descanned detection, fluorescence photons scattered on the way out of the sample are detected far more efficiently than in a confocal system. The result is that clear images are obtained from deep tissue layers. An example is shown in Fig. 21. The images show a pig skin sample exited by two-photon excitation at 800 nm. The left image shows the wavelength channel below 480 nm. This channel contains both fluorescence and SHG signals. The SHG fraction of the signal has been extracted from the FLIM data and displayed by colour. The right image is from the channel >480 nm. It contains only fluorescence, the colour corresponds to the amplitude-weighted mean lifetime of the multi-exponential decay functions.

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Fig. 21: Two-photon FLIM of pig skin. LSM 710 NLO, HPM‑100‑40, NDD. Left: Wavelength channel <480nm, colour shows  percentage of SHG. Right: Wavelength channel >480nm, colour shows amplitude-weighted mean lifetime.

 

FLIM with Ultra-Fast Detectors

In 2017 bh introduced ultra-fast hybrid detectors with a timing jitter of less than 9 ps rms. The instrument-response function of a multiphoton FLIM system with these detectors has a full-width at half maximum (FWHM) of less than 20 ps [15, 30]. The fast response greatly improves the accuracy at which fast decay components can be extracted from a multi-exponential decay. Applications are mainly in the field of metabolic FLIM, which requires separation of the decay components bound and unbound NADH, and in the range of FRET, which requires the separation of interacting and non-interacting donor components. An NADH FLIM image recorded with an ultra-fast FLIM system on a Zeiss LSM 880 is shown in Fig. 21. Images of the images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH) are shown in Fig. 23.

   

Fig. 22: Left: NADH Lifetime image, amplitude-weighted lifetime of double-exponential fit. Right: Decay curve in selected spot, 9x9 pixel area. FLIM data format 512x512 pixels, 1024 time channels. The IRF width is 19 ps, the time-channel width 10ps.

    

Fig. 23: Left to right: Images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH). FLIM data format 512x512 pixels, 1024 time channels. Time-channel width 10ps.

FLIM with Diode-Laser Excitation

The LSM 710/780/880 confocal microscopes are available with fully integrated FLIM lasers based on bh / Lasos BDL‑SMC picosecond diode lasers. Together with the superior efficiency of the bh hybrid detectors and the Zeiss LSM 710 / 780 / 880 scan heads FLIM is performed at excellent sensitivity, see Fig. 24.

          

Fig. 24: Confocal FLIM with diode-laser excitation. Left: Plant tissue, autofluorescence. Right: HEK cell, interacting proteins, FRET from GFP into RFP.

Intune FLIM: FLIM with tuneable excitation

Tuneable-excitation FLIM uses the ‘Intune’ laser of the Zeiss LSM 710 systems. With the Intune laser FLIM images of the same sample can be obtained for different excitation wavelength, see Fig. 25.

    

      

Fig. 25: Confocal FLIM with tuneable ‘Intune’ laser. BPAE cells stained with Alexa 488 phalloidin and Mito Tracker Red. Amplitude weighted lifetime of double-exponential model. Excitation at 490, 520, 535, and 556 nm, all images 1024 x 1024 pixels.

By tuning the excitation wavelength to exactly the absorption maximum of the fluorophores maximum excitation efficiency is obtained at minimum sample exposure. High laser power, excellent spectral purity of the laser, high efficiency of the confocal detection path, and high efficiency of the GaAsP hybrid detectors result in FLIM data of excellent special and temporal resolution.

Near-Infrared FLIM: One-Photon Excitation by Ti:Sapphire Laser

Near-infrared (NIR) dyes are used as fluorescence markers in small-animal imaging and in diffuse optical tomography of the human brain. In these applications it is important to know whether the dyes bind to proteins or other tissue constituents, and whether their fluorescence lifetimes depend on the targets they bind to. NIR FLIM is possible by using HPM‑100-50 detectors and Ti:Sapphire laser excitation. Different than for multiphoton FLIM, the Ti:Sapphire laser is used as a one-photon excitation source [30, 28, 31].

         

Fig. 26: Pig skin samples stained with 3,3’-diethylthiatricarbocyanine. Excitation at 780nm, detection 800nm to 900nm

Near-Infrared FLIM: Multiphoton Excitation with OPO

With the Zeiss LSM NLO OPO systems near-infrared FLIM can be performed by two-photon excitation. The fluorescence signals are detected by HPM-100-50 NIR detectors and non-descanned detection. With excitation wavelengths in the range of 1000 to 1330 nm, the typical NIR dyes are excited at high efficiency [30, 28, 31]. Fluorescence is detected up to 900 nm. An example is shown in Fig. 27.

Fig. 27: Pig skin stained with Indocyanin Green. LSM 7 OPO system, two-photon excitation at 1200 nm, non-descanned detection, 780 to 850 nm. Depth from top of tissue 10 µm (left) and 40 µm (right). 512x512 pixels, 256 time channels.

Confocal Multispectral FLIM

The bh multispectral FLIM system detects simultaneously in 16 wavelength intervals [21]. By using bh’s multi-dimensional TCSPC process it avoids to reject any photons by time gating or wavelength scanning. The systems thus reach near-ideal recording efficiency. Dynamic effects in the sample or photobleaching do not cause distortions of the spectra or decay functions. An example for confocal detection with one-photon (diode-laser) excitation is shown in Fig. 28.

Fig. 28: Confocal multispectral FLIM. Part of a water flee, excitation by 405 nm ps diode laser, LSM 710 confocal port, bh MW FLIM detector

Multiphoton Multispectral NDD FLIM

bh’s MW FLIM was the world’s first simultaneously detecting multiphoton multispectral NDD FLIM system. It uses an optical interface that connects to the NDD ports of the LSM 710/780/880 NLO microscopes [23], see Fig. 5. A typical result is shown in Fig. 29.



Fig. 29: Multiphoton Multispectral NDD FLIM. Lifetime images and decay curves in selected pixels and wavelength channels. LSM 710 NLO, bh MW FLIM detector

Multispectral FLIM got a new push by the introduction of the new PML‑16 GaAsP 16-channel detector. This detector has five time the efficiency of the older PML‑16 detectors with conventional cathodes. Another improvement came from bh’s Megapixel FLIM technology. Multi-spectral FLIM can now be obtained at an image size of 512 x 512 pixels in each wavelength channel while maintaining the usual 256-channel time resolution [30].

Z-Stack FLIM

For many years, Z-stack FLIM had been prevented by photobleaching and photodamage caused by the high excitation dose. With the LSM 710/780/880 microscopes and the bh GaAsP hybrid detectors photobleaching is no longer a problem. Z‑stack FLIM is particularly interesting in combination with the deep-tissue imaging capability of multiphoton NDD FLIM. An example is shown in Fig. 30.



Fig. 30: FLIM Z-stack, planes recorded into a sequence of files. Pig skin, autofluorescence, 5 µm to 60µm below the surface. LSM 710 Multiphoton NDD FLIM, HPM-100-40 GaAsP hybrid detector.

 

Infinite-Focus Depth FLIM

Sometimes it is desirable to project the tree-dimensional structure of a tissue sample into a single image. A frequent example is neuronal tissue where neurons are not necessarily located within a particular image plane. In these cases the SPCM software is able to record a Z stack of the 3-dimensional structure of the sample, and then project a selected number images of different Z planes into a single FLIM data set. An example is shown in Fig. 31.

Fig. 31: Infinite-focus depth FLIM. A Z stack of a 3-dimensional sample is recorded, and the image of several planes are projected into a singe FLIM image. Brain tissue, LSM‑710 NLO, Simple-Tau 150 FLIM system.

 

Time-Series FLIM

Time-series  FLIM is available for all system versions, and all detectors. As for Z stack FLIM, a time series can be acquired both by recording into a series of data files and by Mosaic FLIM. The maximum speed for recording a series of files is about 2 images per second [106]. The speed of temporal mosaic FLIM can be even faster, especially if the lifetime change in the sample is repeatable and can be triggered by an external stimulation. Two time series taken at a moss leafs are shown in Fig. 32 and Fig. 35, page 23. Fig. 32 was acquired by recording into a series of files, Fig. 35, page 23, by recording into the elements of a FLIM mosaic. In both examples, the fluorescence lifetime of the chloroplasts changes due to the Kautski effect induced by the illumination.



Fig. 32: Time-series FLIM, recorded into sequence of files. Acquisition time 2 seconds per image. Chloroplasts in moss leaf, the lifetime changes due to Kautski effect

Spatial Mosaic FLIM

Mosaic FLIM is based on bh’s ‘Megapixel FLIM’ technology introduced in 2014. Mosaic FLIM records a large number of images into a single FLIM data array. The individual images within this array can be for different displacement of the sample (spatial mosaic), different depth within the sample (z-stack mosaic), of for different times after a stimulation of the sample (temporal mosaic). Spatial mosaic FLIM combines favourably with the Tile Imaging capability of the Zeiss LSM 710/780 systems. An example is shown in Fig. 33. The complete data array has 2048 x 2048 pixels, and 256 time channels per pixel. Compared to a similar image taken through a low-magnification lens the advantage of mosaic FLIM is that a lens of high numerical aperture can be used, resulting in high detection efficiency and high spatial resolution.

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Fig. 33: Mosaic FLIM of a Convallaria sample. The mosaic has 4x4 elements, each element has 512x512 pixels with 256 time channels. The complete mosaic has 2048 x 2048 pixels, each pixel holding 256 time channels. Zeiss LSM 710 Intune system with bh Simple-Tau 150 FLIM system. Total sample size covered by the mosaic 2.5 x 2.5 mm.

Z Stack Mosaic FLIM

The Mosaic FLIM function can be used to record Z Stacks of FLIM images. As the microscope scans consecutive images planes in the sample the FLIM system records the data into consecutive elements of a FLIM mosaic. The advantage over the traditional record-and-save procedure is that no time has to be reserved for save operations, and that the entire array can be analysed in a single analysis run.

Fig. 34: FLIM Z-stack, recorded by Mosaic FLIM. Pig skin stained with DTTC. 16 planes, 0 to 60 um from top of tissue. Each element of the FLIM mosaic has 512x512 pixels and 256 time channels per pixel. Plane 8 is shown magnified on the right. LSM 7 OPO system, HPM-100-50 GaAs hybrid detector.

Temporal Mosaic FLIM

Mosaic FLIM can be used to record FLIM time series. An example is shown in Fig. 35.

Fig. 35: Time series acquired by mosaic FLIM. Recorded at a speed of 1 mosaic element per second. 64 elements, each element 128 x 128 pixels, 256 time channels, double-exponential fit of decay data. Sequence starts at upper left. Moss leaf, lifetime changes by non-photochemical chlorophyll transient.

Also here, the advantage is that no time has to be reserved for save operations during the recording sequence. A Mosaic-FLIM time series can therefore be made very fast. The most important advantage is, however, that temporal Mosaic FLIM data can be accumulated. A lifetime change in the sample is stimulated periodically, and a mosaic recording sequence started for each stimulation. Because the entire photon distribution is kept in the memory the photons from the subsequent runs are automatically accumulated. The result is that the signal-to-noise ratio no longer depends on the speed of the series. The only speed limitation is the minimum frame time of the scanner. For the Zeiss LSMs frame times of less than 40 milliseconds can be achieved. This brings the transient-time resolution down to the range where Ca2+ transients in neurons occur. Experiments have shown that these can indeed by recorded. An example is shown in Fig. 36.

Fig. 36: Temporal mosaic FLIM of the Ca2+ transient in cultured neurons after stimulation with an electrical signal. The time per mosaic element is 38 milliseconds, the entire mosaic covers 2.43 seconds. Experiment time runs from upper left to lower right. Photons were accumulated over 100 stimulation periods. Zeiss LSM 7 MP multiphoton microscope and bh SPC‑150 TCSPC module. Data courtesy of Inna Slutsky and Samuel Frere, Tel Aviv University, Sackler Faculty of Medicine.

Mosaic FLIM with Routing

The elements of a FLIM mosaic can be assigned to information delivered to the TCSC modules via their routing inputs. The traditional application is multi-wavelength FLIM. Examples for multi-wavelength FLIM by the Mosaic FLIM function are shown in [9] and [30]. A potential application is the combination of FLIM with electro-physiology. In that case, one ore more electrophysiology signals would be measured by micro-electrodes, digitized, and used to control the mosaic element into which the photons are recorded. The result would be a mosaic of FLIM images for different physiological states of the cells.

 

FLITS: Fluorescence Lifetime-Transient Scanning

FLITS records transient effects in the fluorescence lifetime of a sample along a one-dimensional scan. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With repetitive stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond [30, 26]. Typical applications are recording of chlorophyll transients and Ca2+ transients in neurons or neuronal tissue [29]. Examples are shown in Fig. 37 and Fig. 38.

  

Fig. 37: FLITS of chloroplasts in a grass blade, change of fluorescence lifetime after start of illumination. Experiment time runs bottom up. Left: Non-photochemical transient, transient resolution 60 ms. Right: Photochemical transient. Triggered accumulation, transient-time resolution 1 ms.



Fig. 38: FLITS of Ca2+ concentration in cultured neurons. Ca2+ sensor Oregon Green, LSM 7 MP, electrical stimulation, 100 stimulation periods accumulated. Transient-time resolution 2 ms.

 

Simultaneous FLIM / PLIM

The bh FLIM systems are able to simultaneously record fluorescence (FLIM) and phosphorescence lifetime images (PLIM). The technique is based on modulating a high-frequency pulsed excitation laser synchronously with the pixel clock of the scanner. Photon times are determined both with reference to the laser pulses and the laser modulation period [13, 25, 30]. Fluorescence is recorded during the ‘on’ time, phosphorescence during the ‘off’ time of the laser. The technique does not require a reduction of the  laser repetition rate, works with two-photon excitation and non-descanned detection, and delivers an extremely high PLIM sensitivity. Unlike other PLIM techniques, it does not cause moiré in the images and can thus be used at scan rates no lower than the reciprocal phosphorescence decay time. For procedures and parameter setup in combination with the Zeiss LSMs please see [6]. A typical result is shown in Fig. 39.

                         

                   

Fig. 39: Yeast cells stained with a Ruthenium dye. Left: FLIM image and fluorescence decay curve in selected spot.  Right: PLIM image and phosphorescence decay curve in selected spot.

 

FCS

The bh GaAsP hybrid detectors deliver highly efficient FCS [6, 24, 30]. Because the detectors are free of afterpulsing diffusion times are obtained from a single detector, without the loss in correlation events that occurs when the signals from two detectors are cross-correlated. FCS is be obtained with the diode-laser systems, the Intune system, and even with the multiphoton NDD systems. The bh SPCM data acquisition software calculates FCS online and fits the data with a user-configurable model function [30].

       

Fig. 40: FCS with GaAsP hybrid detectors. Left to right: Confocal FCS with ps diode laser, Confocal FCS with Intune laser, Two-photon NDD FCS

SPCImage FLIM Data Analysis

The bh FLIM systems for the Zeiss LSMs use bh SPCImage data analysis software [6, 7]. SPCImage runs a de-convolution on the decay data in the pixels of FLIM data. It uses single, double, or triple-exponential decay analysis to produce pseudo-colour images of lifetimes, amplitudes, or intensities of decay components, or of ratios of these parameters. An ‘incomplete decay’ model is available to determine long fluorescence lifetimes within the short pulse period of the Ti:Sa laser of a multiphoton system. Moreover, SPCImage avoids troublesome recording of an instrument response function (IRF) by extracting the IRF from the FLIM data themselves. Please see [6] or [30] for details.

The main panel of the SPCImage data analysis is shown in Fig. 41. It shows a lifetime image (left) a parameter histogram over the pixels of a region of interest (upper right), and the fluorescence decay curve in a selected spot of the image (lower right). The basic model parameters (one, two or three exponential components) are selected in the upper right.



Fig. 41: Main panel of the SPCImage data analysis (Version 5.4 and later)

Moreover, SPCImage can extract phosphorescence intensities from normal FLIM data, or distinguish regions with single-exponential decay from regions where the decay is multi-exponential. SPCImage is also able to display time-gated images, e.g. to extract SHG images from FLIM data, or to reject un-depleted fluorescence in STED-FLIM images. A batch-processing function and a batch export function are available for analysing a large number of FLIM data sets automatically and to convert them into bmp of tif images. Please see [6] or [30] for details.

Histogram functions

SPCImage has a histogram function for the decay parameter selected for coulour-coding the lifetime image. The histogram shows how often pixels of a given parameter value (lifetime, lifetime of a decay component, amplitude of a component, or combinations of these values) occur in the lifetime image. Histograms can be displayed for several regions of interest. An example is shown in Fig. 42.

          

Fig. 42: Decay-parameter histogram, for two different regions of interest

SPCImage has a function that displays two-dimensional histograms of decay parameter. 2D histograms display a density plot of the pixels over two selectable decay parameters. The decay parameters can be lifetimes, t1, t2, t3, amplitudes, a1, a2, a3, of decay components, amplitude or intensity-weighted lifetimes, tm or ti, or arithmetic conjunctions of these parameters. A histogram of the amplitude ratio, a1/a2, over the amplitude-weighted lifetime, tm, is shown in Fig. 43.

     

Fig. 43: Main panel of SPCImage showing a FRET cell (left) and 2D Histogram of lifetime versus amplitude ration of components

Areas of parameter combinations of a multi-exponential decay function can be selected in the 2D histograms and back-annotated in the images, see Fig. 44.

    

Fig. 44: Images and 2D histograms. Left: amplitude ratio versus amplitude-weighted lifetime. Right: Phasor plot.

Phasor Plot

Version 6.0 SPCImage FLIM analysis software combines time-domain multi-exponential decay analysis with the phasor plot. In the phasor plot, the decay data in the individual pixels are expressed as phase and amplitude values in a polar diagram [62, 63]. Independently of their location in the image, pixels with similar decay signature form clusters in the phasor plot. Different phasor clusters can be selected, and the corresponding pixels back-annotated in the time-domain FLIM images, see Fig. 45. Moreover, the decay functions of the pixels within the selected phasor range can be combined into a single decay curve of high photon number. This curve can be analysed at high accuracy, revealing decay components that are not visible by normal pixel-by pixel analysis, see Fig. 46. Please see [14] and [30] for details.

     

Fig. 45: Left: Lifetime image and lifetime histogram. Right: Phasor plot. The clusters in the phasor plot represent pixels of different lifetime in the lifetime image. Recorded by bh Simple-Tau 152 FLIM system with Zeiss LSM 880.

   

Fig. 46: Left: Selecting a cluster of phasors in the phasor plot. Middle: Combination of the decay data of the corresponding pixels in a single decay curve. Right: Display of the pixels corresponding to the selected cluster in the phasor plot.

 

 

Single-Molecule Burst Analysis

The bh ‘Burst Analyser’ software is used for data analysis of single-molecule fluorescence. It uses parameter-tag data files recorded in the FIFO mode of the SPC‑830, SPC‑130EM, SPC‑150, SPC‑150N, or SPC‑160 TCSPC modules. Photon bursts from single molecules travelling through a femtoliter detection volume are identified in the parameter-tag data. Within the bursts, intensities, intensity variations, fluorescence lifetimes, and ratios of these parameters between several detection channels or different time windows of a PIE recording are determined, and time-traces and histograms of the parameters are calculated. The results are used to obtain histograms and time traces of FRET efficiencies, and to calculate FCS and FCCS data. The Burst Analyser is described in a separate handbook, see [8].

 



Fig. 47: bh Burst Analyzer software for single-molecule data analysis

 

FLIM Applications

If fluorescence in a sample is excited (Fig. 48, left) the emission intensity depends both on the concentration of the fluorophore and on possible interaction of the fluorophore with its molecular environment. If only the intensity of the fluorescence is recorded changes in the concentration cannot be distinguished from changes in the molecular environment. Intensity imaging (second left) is thus an excellent way to resolve the spatial structure of a sample but it does not tell much about effects on the molecular scale. Spectral measurements (second right) are able to distinguish between different fluorophores. However, changes in the local environment usually do not cause changes in the shape of the spectrum. Information about molecular effects is thus difficult to obtain. The fluorescence lifetime of a fluorophore (Fig. 48, right), within reasonable limits, does not depends on the concentration but systematically changes on interaction with the molecular environment. By using the fluorescence lifetime, or, more precisely, the shape of the fluorescence decay function, molecular effects can therefore be investigated independently of the unknown and usually variable fluorophore concentration [27, 30, 35, 44, 124, 153, 182].

Fig. 48: Fluorescence. Left to right: Excitation light is absorbed by a fluorophore, and fluorescence is emitted at a longer wavelength. The fluorescence intensity varies with concentration. The fluorescence spectrum is characteristic of the type of the fluorophore. The fluorescence decay function is an indicator of interaction of the fluorophore with its molecular environment.

Common FLIM applications are measurements of the molecular environment of the fluorophores, pH and ion concentration measurements, probing of protein interaction via FRET, and investigation of metabolic activity and cell viability via the fluorescence lifetimes of NADH and FAD. FLIM may also find application in plant physiology because the fluorescence lifetime of chlorophyll changes with the photosynthesis activity.

 

Measurement of Molecular Environment Parameters

pH Imaging

An example of recording variation in local environment parameters is shown in Fig. 49. A skin sample was stained with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). BCECF has a protonated and a deprotonated form. Both forms have different fluorescence lifetimes. The average lifetime is a direct indicator of the pH in the immediate molecular environment of the fluorophore [91, 124   Lakowicz book]. Similar approaches can be used to measure concentrations of various ions. An application for determining absolute calcium concentrations in mice brains has been described in [118]. FLIM is also used for measuring the concentration of intracellular Cl- in neurons [80, 104]. Changes of the Cl- concentration during maturation of chloride homeostasis in neurons were investigated in [87], changes on inflammation in [77].

             

Fig. 49: Lifetime image of skin tissue stained with BCECF. The lifetime is an indicator of the pH. Right: Fluorescence decay curves in an area of low pH (top) and high pH (bottom). LSM 510 NLO, Data Courtesy of Theodora Mauro, University of San Francisco.

Calcium Imaging

Ca2+ ions are involved in a large number of cell functions, such as intracellular transport, membrane potential, muscle contraction, gene expression, and cell differentiation. There is a wide variety of Ca2+ sensors [124], most of which change their fluorescence lifetime with the Ca2+ concentration. The mechanism of the Ca2+-dependent lifetime change is that the fluorophore has a Ca-bound and an Ca-unbound form of different fluorescence quantum efficiency and thus different fluorescence lifetimes. Consequently, the net fluorescence lifetime depends on the Ca2+ concentration. An example of mapping of the Ca2+ concentration by FLIM is shown in Fig. 50.

Fig. 50: FLIM image of cultured neurons stained with Oregon green OGB-1 AM. Colour range from tm = 1200 ps (blue) to 2400 ps (red). Decay curves of regions with low Ca (top) and high Ca (bottom) shown on the right. LSM 7 MP, Data courtesy of Inna Slutsky and Samuel Frere, Tel Aviv University, Sackler School of Medicine.

The Ca2+ concentration in cells can change within remarkably short periods of time. The Ca2+ response of live neurons to stimulation occurs within milliseconds. Nevertheless, the effects can be measured by FLITS [29, 76] and Temporal Mosaic FLIM [30, 32]. An example is shown in Fig. 51.

Fig. 51: Ca2+ transient in cultured neurons. Oregon Green Bapta, data recorded by temporal mosaic FLIM, 38 ms per mosaic element. Zeiss LSM 7 MP, bh Simple-Tau 152 TCSPC FLIM system.

Also other fluorophores, especially those with medium or low fluorescence quantum efficiency show variations of their fluorescence decay time depending on the local environment and the binding target [28, 31]. It is therefore likely that lifetime-based probes and markers can be found for a wide variety of cell and tissue parameters.

Lifetime imaging can also be used to obtain images of refractive index changes or viscosity changes in the vicinity of a fluorophore [95, 119, 126, 186]. Moreover, there are fluorophores that change their fluorescence behaviour depending on the redox potential of the local environment. An improved redox sensor based on a modified GFP (roGFPiE) was developed by Avezov et al. Test results obtained on various cells by TCSPC FLIM are described in [3].

Ion concentrations in cells can be influenced by physiological processes. Changes can be very fast, with transition times down to the millisecond range. The bh FLIM systems can record such changes by Mosaic FLIM and FLITS. Please see examples in Fig. 36, Fig. 37, and Fig. 38 in Section ‘FLIM Functions’.

Förster Resonance Energy Transfer (FRET)

FRET is an interaction of two fluorophore molecules with the emission band of one dye overlapping the absorption band of the other. In this case the energy from the first dye, the donor, can be  transferred to the second one, the acceptor. The energy transfer itself is a dipole-dipole interaction. It does not involve any light emission and absorption [72, 73, 124]. Fluorescence resonance energy transfer (FRET), or resonance energy transfer (RET), are synonyms of the same effect. The energy transfer rate from the donor to the acceptor decreases with the sixth power of the distance. Therefore it is noticeable only at distances shorter than 10 nm [124 Lakowicz Principles 1999]. FRET results in an extremely efficient quenching of the donor fluorescence and, consequently, decrease of the donor lifetime, see Fig. 52, right.

 

Fig. 52: Fluorescence resonance energy transfer

Because of its dependence on the distance FRET has become an important tool of cell biology [143, 144]. Different proteins are labelled with the donor and the acceptor; FRET is then used to verify whether the proteins are physically linked and to determine distances on the nm scale.

The problem of steady-state FRET techniques is that the concentration of the donor and the acceptor changes throughout the sample. The techniques therefore depend on ratios of the donor and acceptor intensities. However, the FRET-excited acceptor intensity is not directly available. There is ‘donor bleedthrough’ due to the overlap of the donor fluorescence into the acceptor emission band. For the CFP / YFP FRET system this signal component can be more than 30%. Moreover, some of the acceptor molecules are excited directly, not via energy transfer for the donor. Directly-excited acceptor fluorescence is a problem especially when the acceptor concentration is made high to guarantee that all acceptor targets are labelled. Steady-state FRET techniques therefore require careful calibration, including measurements of samples containing only the donor and only the acceptor.

The calibration problems can partially be solved by the acceptor photobleaching technique. An image of the donor is taken, then the acceptor is destroyed by photobleaching, and another donor image is taken. The increase of the donor intensity is an indicator of FRET. The drawback is that this technique is destructive, and that it is difficult to use in live cells. In fixed cells, however, the protein structure is changed already by the fixation so that the results are not necessarily correct.

All steady-state techniques have the problem that there is usually a mixture of interacting and non-interacting proteins. Both the fraction of interacting proteins and the distance between the proteins influence the FRET efficiency. It therefore cannot be told whether a variation in FRET efficiency is due to a variation in the distance or a variation in the fraction of interacting proteins.

 

 

Single-Exponential FLIM FRET

The use of FLIM for FRET has the obvious benefit that the FRET intensity is obtained from a single lifetime image of the donor. Donor bleedthrough and directly excited acceptor fluorescence therefore have no influence of FLIM-FRET measurements. The only reference value needed is the donor lifetime in absence of the acceptor [41, 51, 70, 147]. It will be shown later that even this reference lifetime can be obtained from the FLIM-FRET data themselves.

Fig. 53 shows a single-exponential lifetime image of a cultured HEK (human embryonic kidney) cell expressing two interacting proteins labelled with CFP and YFP.

Fig. 53: FRET in an HEK cell. Blue to red corresponds to a lifetime range of 1.5 to 2.2 ns. (Christoph Biskup, University Jena, Germany)

The classic FRET efficiency can be directly calculated from the decrease in the fluorescence lifetime. Single-exponential lifetime images as the one shown in Fig. 53 are very useful to locate the areas in a cell where the labelled proteins interact. It has been shown that for a given efficiency of the optical system and detector and a given excitation power FLIM-based FRET measurements give better accuracy than steady-state techniques [141]. Single-exponential decay measurements do, however, not solve the general problem of the FRET techniques that the total decrease of the donor fluorescence intensity or fluorescence lifetime depends both on the distance of donor and acceptor and the fraction of interacting donor molecules. In the simplest case, a fraction of the donor molecules may not be linked to their targets, or not all of the acceptor targets may be labelled with an acceptor. This can happen especially in specimens with conventional antibody labelling [122 Lakowicz Gryczinski;  Lakowicz]. But even if the labelling is complete not all of the labelled proteins in a cell are interacting, and the fraction of interacting protein pairs varies throughout the cell.

 

Double-exponential FLIM FRET

TCSPC FLIM solves the problem of interacting and non-interacting donor by double-exponential lifetime analysis. The resulting donor decay functions can be approximated by a double exponential model, with a slow lifetime component from the non-interacting (unquenched) and a fast component from the interacting (quenched) donor molecules. If the labelling is complete, as it can be expected if the cell is expressing fusion proteins of the GFP variants, the decay components directly represent the fractions of interacting and non-interacting proteins. The composition of the donor decay function is illustrated in Fig. 54.

Fig. 54: Fluorescence decay components in FRET systems

Double exponential decay analysis delivers the lifetimes, t0 and tfret, and the intensity factors (amplitudes), a and b, of the two decay components. From these parameters can be derived the true FRET efficiency, Efret , the ratio of the distance and the Förster radius, r/r0 , and the ratio of the number of interacting and non-interacting donor molecules, Nfret / N0 :

                                                                              

                        or                

                                                                                

Fig. 55 shows the fluorescence decay curves in a selected spot (array of 4 adjacent pixels, selected by the blue crosshair) of Fig. 53. The fluorescence decay is indeed double-exponential, with a fast lifetime component, tfret, of 590 ps, and a slow lifetime component, t0, of 2.41 ns.

 

Fig. 55: Fluorescence decay curve in a selected spot of  Fig. 53. The decay profile is clearly double-exponential.

Fig. 56 shows the result of a double-exponential analysis of the data. The left image shows the ratio of the lifetimes of the non-interacting and interacting donor fractions, t0/tfret. The distribution of t0/tfret in different regions is shown far left. The locations of the maxima differ by only 10%, corresponding to a distance variation of about 2%. However, the variation in the intensity coefficients, a/b, is about 10:1.

Fig. 56: FRET results obtained by double exponential lifetime analysis. Left: t0/tfret, Right: Nfret/N0

The results show that the variation in the single-exponential lifetime (Fig. 53) is almost entirely caused by a variation in the fraction of interacting proteins, not by a change in distance. In other words, interpreting variations in the single-exponential lifetime (or classic FRET efficiencies from steady-state experiments!) as distance variations leads to wrong results.

Similar double exponential decay behaviour is commonly found in FRET experiments based on multi-dimensional TCSPC [4, 16, 17, 20, 41, 147]. Double-exponential decay profiles have also been confirmed by streak-camera measurements [40  Biskup Böhmer Pusch J. Cell Sci; Biskup, Nature]. There is no doubt that the double-exponential behaviour is due to the presence of an interacting and a non-interacting donor fraction. It should be noted, however, that real FRET experiments can be more complicated. There is the problem that a protein can be expressing (or be labelled with) several donor or acceptor molecules. Energy may then migrate between the donors, and an the energy transfer rate may increase due to interaction with several acceptor molecules.

An enormous amount of FRET papers based on the bh FLIM technique has been published in the last few years. General characterisations of TCSPC-FLIM FRET for monitoring protein interactions can be found in [41, 51, 147  Biskup Kelbauskas JBO 2004; Chen Periasamy Micr. Res. Tech. 2004;  Chen Periasamy SPIE; Peter Ameer-Beg Biol. Cell. 2004, Snippe Borst J. Vir. Meth 2005]. An overview on monitoring HIV-1 Protein Oligomerization by FLIM FRET was given in [152]. FRET measurements by multi-wavelength FLIM are described in [42, 43].

Almost all of the FRET applications are more or less related to protein interactions or protein organisation, please see [1, 41, 48, 78, 92, 96, 121, 136, 142, 145, 171, 172, 179] for a (somewhat arbitrary) selection.

There is a number of FRET applications which are directly related clinical research [132]. Because FRET yields information about protein interaction it can be used to investigate the formation - and possible dissolution - of amyloid plaques of Alzheimer’s disease [4, 4, 5, 36, 36, 37, 38, 94, 88, 94, 101, 129, 128, 129, 189, 206  Bacskai JBO 2004; Bacskai SPIE; Berezovska J. Neurosci. 2003; Berezovska, SAGE KE 2003, Berezovska, Lleo 2005]. Mechanisms of Huntington disease (another progressive neuro-degenerative disorder) were investigated in [75] and [135].

Another potential application is the investigation of infection mechanisms. The technique was demonstrated by Ghukasyan et al. for the infection of HeLa cells with enterovirus 71 [84].

FLIM FRET applications in synaptic structures have been published in [46, 68, 74, 134, 187, 188]. These experiments are difficult because FRET occurs in sub-resolution volumes of the sample. Nevertheless, Bosch et al. were not only able to detect protein interaction, they could even observe the re-organisation of post-synaptic substructures during long-term potentiation by time-series FLIM [46]. Fogel used a combination of intensity FRET measurement, FLIM FRET measurement, and single-molecule imaging to show that the amyloid precursor protein homo-dimer constitutes a resynaptic receptor that transduces a signal from amyloid-beta peptides to glutamate release [74].

There are currently a number of attempts to detect FRET between endogenous fluorophores, and use the results to gain structural information about proteins and protein complexes. The problem is that these fluorophores are hard to reach by one-photon and even two-photon excitation. It has been shown recently that three-photon excitation of tryptophane - and the detection of its fluorescence - is possible with the Zeiss LSM 710/780/880 NLO microscopes. With UV-optimised optics in the NDD beam path, hybrid detectors, and carefully adjusted laser power three-photon excited TCSPC FLIM images of tryptophane were obtained. FRET was expected to occur between the tryptophane and the bound component of the NADH. Indeed, FLIM experiments have shown a decrease in the tryptophane lifetime correlated with an increase in the bound-unbound ratio of the NADH [2, 102, 144].

For a detailed description of FLIM FRET and a review of the related literature please see ‘The bh TCSPC Handbook’, 6th edition, 2015 [30] and ‘FLIM Systems for Zeiss LSM 710/780/880 microscopes’ [6].

 

Autofluorescence Microscopy of Cells and Tissue

Biological tissue contains a wide variety of endogenous fluorophores [59, 86, 111, 151, 160König Riemann JBO 2003; Marcu in Mycek; Richards-Kortum in Mycek;   Schweitzer Kolb Hammer, Int Ophthalmol. 2001; Schweitzer Hammer Scweitzer Anders JBO 2004;   Rovati JBO 2004;    Urayama & Mycek in Mycek;   Wagnieres in Mycek;   Zheng in Mycek;]. For many years, autofluorescence measurements of cells and tissue have mainly been performed by spectrally resolved imaging techniques. The problem of purely spectrally resolved techniques is that the emission and excitation spectra of the endogenous fluorophores are broad and poorly defined. Many of the fluorophores are mixtures of closely related compounds. The shape of the spectra can therefore vary. Moreover, absorbers present in the tissue may change the apparent fluorescence spectra. It is therefore difficult to disentangle the fluorescence components by their emission spectra alone. The most serious drawback of purely spectral techniques is, however, that different binding states of fluorophores cannot be distinguished.

Fluorescence lifetime imaging is likely to improve the contrast of separation of the different fluorophores. This has been demonstrated by Krasievy et al. [117] who used TCSPC FLIM and phasor plot analysis to separate the fluorescence components of different melanin types, and the melanin fluorescence form the NADH fluorescence. The most important argument for using lifetime imaging is, however, that the lifetimes of many endogenous fluorophores depend on local environment parameters, such as pH, oxygen saturation, and on the binding to proteins. Autofluorescence lifetime detection therefore not only adds an additional separation parameter but also yields direct information about the metabolic state, the microenvironment, and the binding state of the endogenous fluorophores. There is a steadily increasing number of papers about this subject [39, 45, 52, 53, 89, 123, 124, 133, 140, 146, 155, 161, 175, 176, 183, 184, 190, 192, 193, 194, 200  Lakowicz 1992, Lakowicz 1999; Paul & Schneckenburger, Naturwissenschaften 1996].

Autofluorescence imaging has also benefits in cases when the reaction of tissue to optical radiation is to be investigated, such as skin ageing [157] or photodamage and tumor induction by UV [110] and IR irradiation [113]. Such experiments forbid the use of exogenous fluorophores because energy or electron transfer between the fluorophores and the proteins can induce photoreactions. Moreover, TCSPC FLIM is more and more introduced into clinical applications [60, 111, 112, 114, 153, 157, 158, 160, 161, 161]. In these applications ‘label-free’ imaging is needed because staining the tissue with  exogenous fluorophores is either not possible or not permitted.

NADH and FAD Lifetime Imaging

The use of fluorescence-lifetime variations is especially promising for NADH and FAD. These coenzymes are involved in the electron transfer mechanism of the cell metabolism. Both NADH and FAD form redox pairs. NADH is fluorescent in its reduced form but loses fluorescence when oxidised. FAD is fluorescent when oxidised, and loses fluorescence when reduced. The fluorescence intensity ratio of NADH and FAD therefore changes with the redox state of the tissue. Chance et al. defined a ‘Redox Ratio’ that shows whether the metabolism in a cell is more oxidative (phosphorylation) or more reductive (glycolysis) [49, 50]. Measurements of the redox state are related to the ‘Warburg Effect’: Normal cells have an oxidative, cancer cells a reductive metabolism [197, 198]. For practical applications please see [177, 178, 191].

Redox Ratio

The influence of the redox state can be shown by recording FLIM from yeast cells. Fig. 57, left, shows yeast cells in a medium saturated with CO2 and virtually free of oxygen. Except for a few (probably apoptotic) cells the fluorescence is extremely week. When the sample has taken up oxygen from the air the fluorescence intensity increases, accompanied by a change in the lifetimes.

                    

Fig. 57: Yeast cells in a cell dish saturated with CO2 (left) and saturated with O2 (right). Confocal FLIM, excitation 405 nm, detection by bh MW FLIM, 540 nm

NADH Lifetimes

It is known that the fluorescence lifetimes of NADH and FAD depend on the binding to proteins [123, 124, 140]. Unbound NADH has a fluorescence lifetime of about 0.4 ns. When NADH binds to proteins the lifetime typically increases to about 1.2 ns [124]. However, the bound-NADH lifetime depends on the proteins, and can reach 4 to 5 ns [124]. For FAD the effect of binding is opposite: Bound FAD has a lifetime of a few 100 ps, unbound FAD of a few ns.

The fluorescence decay parameters, especially those of NADH, can thus be used to obtain information about the binding state of NADH. The ratio of bound and unbound NADH depends on the metabolic activity. The NADH lifetime or, better, the amplitude ratio of the two decay components is therefore an indicator of the metabolic state. This information is not available from fluorescence spectra or fluorescence intensity images. Additional information about NADH binding can be obtained by recording also fluorescence anisotropy decay data  of intracellular NADH [93, 190, 202]

An overview on NADH / FAD fluorescence dynamics has been given by Chorvat and Chorvatova [55] and by Heikal at al. [93]. Ghukasyan and Kao [85] have shown that the NADH decay parameters, in particular amplitudes of the fast and slow lifetime components, are indicators of physiological and pathological states of the cells. Differentiation between apoptosis and necrosis by the NADH lifetime components was descried by Wang et al. [196].

Skala et al. [175, 176] used two-photon excited autofluorescence FLIM to record lifetime images of NADH in normal and pre-cancerous epithelia. They found a significantly decreased lifetime of the slow decay component of (bound) NADH, in precancerous tissue. A significant decrease of the lifetime of the protein-bound FAD was detected in high-grade pre-cancers [176]. Bird et al. used a similar approach for metabolic mapping of human breast cells [39].

Szaszak et al. [184] used the NADH fluorescence to track metabolic changes in the host and in the pathogen during intracellular infectious by chlamydia trachomatis.

Kantelhardt et al. [105] and Leppert et al. [125] recorded two-photon excited lifetime images of glioma and the surrounding brain tissue. They found a significantly increased mean (amplitude-weighted) lifetime for the glioma cells compared to the surrounding brain cells.

Metabolic imaging of melanoma progression was performed by Pastore et al. [138]. They confirm a significant increase in the unbound / bound (a1/a2) ratio of NADH with the growth of a melanoma tumor, while the lifetimes of the decay components remained constant.

OMI Index

Skala and Walsh combined the normalised value of the redox ratio RR, the normalised amplitude-weighted fluorescence lifetime, tm NADH, and the normalised tm FAD, of NAD(P)H and FAD into a single ‘Optical Molecular Imaging’, or OMI index. High OMI means a shift toward glycolysis and cancer metabolism.

OMI = RR + tm NADH - tm FAD      (all parameters normalised to reference values)

The OMI index turned out to be a robust parameter to characterise the metabolism of a cell. The authors use it successfully to check the response of human cancer cells from clinical biopsies to various anti-cancer drugs. The cells are cultured, the cell cultures are treated with the drugs, the cells are repeatedly imaged by FLIM, and the OMI index is used as an indicator of the response to the drugs [191, 192, 193, 194, 195]. Within a few days, the most efficient drug can be determined and a treatment strategy for the patient be developed. The technique has the potential to revolutionise cancer therapy, and, notably, can be performed by a normal bh FLIM system in combination with a two-photon laser scanning microscope.

Response to Drugs

NADH FLIM is closely related to effect of cancer drugs in chemotherapy. Skala and Walsh used it to evaluate the effect of different cancer drugs on cells from biopsies from patients, and to develop the best treatment strategy, see above. Stunz et al. [181] studied the response of cultured rat astrocytes and neurons to manganese treatment.

Shirmanova et. al. [174] demonstrated the relationships between the intracellular pH and the metabolic state derived from NAD(P)H / FAD FLIM in response to treatment with cisplatin. They found a metabolic shift from glycolysis towards oxidative metabolism during cisplatin-induced inhibition of cancer cell growth in vitro and in vivo.

Sergeeva et al. [170] performed metabolic imaging of NAD(P)H and FAD, and simultaneously used the genetically encoded pH-indicator SypHer1 and the FRET-based mKate2-DEVD-iRFP sensor for pH imaging and caspase-3 activity imaging. They found a relationship between the metabolic state derived from NAD(P)H FLIM, intracellular pH and caspase-2 activation during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation.

The effect of Ouabain, a pharmaceutical drug, and of 4-hydroxynonenal on live cardio-myocytes was studied by Chorvatova et al., see [57] and [58].

Stem Cells Research

Gou at al. used FLIM of the decay components of NADH to distinguish human mesenchymal stem cells from differentiated progenies [90]. Uchugonowa & König applied two-photon autofluorescence lifetime imaging to human and animal stem cells [114, 115]. They found significant changes in the fluorescence lifetimes on differentiation and maturation of the cells. An example is shown in Fig. 58. The differentiated cells have smaller a1/a2 ratios and longer mean lifetime, tm.

             

Fig. 58: FLIM of human salivary gland stem cells. Differentiated cells have significantly lower a1/a2 ratio and longer mean lifetime. Data courtesy of Aisada Uchugonova and Karsten König, Saarland University, Saarbrücken.

 

NADH / FAD Multi-Wavelength FLIM

A problem of NADH / FAD FLIM is that there is a strong overlap in the excitation and emission spectra [124]. An improvement can be achieved by using the bh multi-wavelength FLIM detector [23, 154]. Fig. 59 shows autofluorescence FLIM data of a fresh mouse kidney section recorded by a bh MW-FLIM system. Two-photon excitation at a wavelength of 750 nm was used. Each of the images covers a wavelength range of about 12.5 nm. The colour of the images represents the lifetime obtained from a single-exponential fit.



Fig. 59: Multi-wavelength fluorescence lifetime images of a mouse kidney section. Two-photon excitation at 750 nm. Images in subsequent wavelength intervals from 350 nm to 550 nm. (Christoph Biskup, University Jena, Germany)

Due to the different fluorophores present in the tissue, the lifetimes in the individual wavelength intervals are different. The third left image contains a strong SHG component. SHG reveals itself as an infinitely short lifetime component, see the blue features in the third to left image.

An interesting application of multi-wavelength lifetime detection of NADH has been published by Chorvat et al. [56]: The authors used the data to detect early stages in the metabolism of isolated live cardiomyocytes during rejection of transplanted hearts. The effect of Ouabain, a pharmaceutical drug, on live cardiomyocytes was studied by Chorvatova et al., see [57].

An overview about autofluorescence multi-wavelength FLIM is given in [55]. Full exploitation of multi-wavelength autofluorescence FLIM data requires a global fit with a suitable model that includes the effective spectra of the expected fluorophores. The task is extremely difficult because some of the endogenous fluorophores are in fact mixtures of slightly different compounds, with different absorption and emission spectra [161]. Moreover, there may be absorbers of unknown absorption spectra, inhomogeneous distribution, and unknown concentration in the tissue. Nevertheless, there is currently a number of approaches that are likely to solve at least a part of the problem [53, 54, 55, 148].

 

Two-Photon FLIM of Skin

A promising application of two-photon excited FLIM measurements of tissue autofluorescence is optical tomography of skin [108, 111, 112]. The technique has reached the state of clinical evaluation, and there is a growing number of publications in this field [60, 114, 139, 153, 157, 158]. Multiphoton FLIM images of skin are surprisingly rich in detail, both in terms of morphological and fluorescence-lifetime information. An example is shown in Fig. 60.

ො

Fig. 60: Two-photon FLIM of pig skin. LSM 710 NLO, excitation 800nm, HPM‑100‑40, NDD. Left: Wavelength channel <480nm, colour shows  percentage of SHG in the recorded signal. Right: Wavelength channel >480nm, colour shows amplitude-weighted mean lifetime.

Measurements as the ones described above can favourably be performed by Z stack FLIM. Fig. 61 shows Z-stack FLIM data of a pig skin sample in the wavelength channel above 480 nm. The data were analysed by the batch-processing function of the SPCImage. The colour of images shows the average (intensity-weighted) lifetime of a double-exponential decay fitted to the decay data. The intensity of the images was normalised to the intensity of the brightest pixel.

Fig. 62 shows the SHG intensity extracted from the FLIM data in the channel below 480 nm. Also here, intensity of the images was normalised to the intensity of the brightest pixel.



Fig. 61: FLIM Z stack recorded at a pig skin sample, excitation at 800 nm, emission above 480 nm. Z step width 5.09 µm, scan area 212 x 212 µm. Images from 5 µm to about 60µm below the surface. Colour represents average (intensity-weighted) lifetime of a double-exponential fit. FLIM data format 256 x256 pixels, 256 time channels. Normalised intensity.

Fig. 62: Z stack recorded at a pig skin sample, excitation at 800 nm, emission below 480 nm, SHG signal, extracted by selecting photons in an early time window. Z step width is 5.09 µm, scan area 212 x 212 µm. Images from 5 µm to about 60µm depth. Normalised intensity.

In all autofluorescence lifetime experiments it is important that the metabolism of the tissue be not changed before or during the measurement. This is critical especially for ex-vivo samples. Sanchez et al. have therefore studied the degradation of skin samples over the storage time and under different storage conditions [156].

 

Autofluorescence of the Ocular Fundus

TCSPC FLIM has recently be introduced in ophthalmic scanners. The instruments have produced FLIM images of amazing quality. Clinical trials have shown that FLIM is able to detect metabolic changes that are early indications of a number of eye diseases [71, 160, 161, 163, 164, 161, 163, 164, 166, 167, 168]. Ophthalmic FLIM has produced absolutely tantalizing results. Ophthalmic scanner do, however, not deliver spatial resolution at the cell level. Clinical research has therefore to be supported by FLIM microscopy of ex-vivo samples. A FLIM study of extra-macular drusen has been published by Schweitzer et al. [165]. Based on the FLIM data, the authors were able to clearly discriminate the RPE from Bruch’s membrane, drusen, and choroidal connective tissue. An example of a FLIM image of the RPE with hard drusen is shown in Fig. 63.

             

Fig. 63: Autofluorescence FLIM image of a human fundus sample. Triple-exponential analysis, left to right: Lifetimes of the fast, medium, and slow decay component. Courtesy of Dietrich Schweitzer, Martin Hammer, Sven Peters, Christoph Biskup, University Jena, Germany. LSM 710, bh MW FLIM detector and SPC‑150 TCSPC module.

The data were acquired with the MW FLIM multi-wavelength detector. The figure shows the lifetime data in all combined wavelength channels. Triple-exponential decay analysis was used; the images show the fast decay component, the medium component, and the slow decay component. Interestingly, in the drusen and in the Bruch’s membrane an extremely slow component with about 8 ns decay time occurs. The lifetimes in the RPE are much shorter.

Spectrally resolved data are shown in Fig. 64. It shows 6 consecutive wavelength channels of the MW FLIM detector from 450 nm to 575 nm. The lifetime shown is the amplitude weighted average of a triple-exponential decay model. The intensities were normalised to the brightest pixel.



Fig. 64: Lifetime images of the amplitude-weighted lifetime, tm, for single wavelength channels from 450 nm to 575 nm. The lifetime scale is blue to red, 0 to 2500 ps. LSM 710, bh MW FLIM detector and SPC‑150 TCSPC module.

 

FLIM of Other Endogenous Fluorophores

There are other endogenous fluorophores which can possibly be used for the identification of the tissue state and of cancerous and pre-cancerous situations. Melanin and its relationship to melanoma has been described above. The use of the tryptophane fluorescence, including FRET from tryptophane into NAD(P)H has been described in [2, 102, 144]. The problem of tryptophane is that it cannot easily be excited by wavelength available in one-photon and two-photon laser microscopes. The authors therefore used three-photon excitation. FLIM with endogenous bilirubins has been described by Shen et al. [173]. Bilirubin is present at high concentration in liver tissue. The authors excited it by two-photon excitation at 1230 nm. In differentiated hepatocellular carcinoma they found a fluorescence lifetime of 500 ps compared to 300 ps in non-tumor tissue. Endogenous fluorescence is also recorded from porphyrines. Porphyrines exhibit a strongly multi-exponential fluorescence decay which indicates that they are a mixture of slightly different compounds, different conformations, or, possibly, aggregates of different size. The composition of these mixtures may depend on the tissue state. The composition of the decay function may therefore contain biological information. Other endogenous fluorophores, such as lipofuscin or advanced glycation end products (AGEs) are important as indicators in ophthalmic FLIM [30, 168].

 

Diffusion of Nanoparticles in Skin

An important issue in dermatology is diffusion of drugs and nano-particles through human skin. On the one hand, diffusion through the skin may be desirable as a way of drug delivery. On the other hand, nanoparticles contained in sunscreens or cosmetic products should not permeate through the stratum corneum of human skin. Moreover, it is important whether the nanoparticles cause changes in their molecular environment. The use of two-photon imaging and FLIM to study these effects has been described in [82, 127, 149, 153, 158].

 

NADH FLIM with Oxygen Sensing by PLIM

Cancer cells and normal cells can be distinguished by recording NADH FLIM images, and determining either the amplitude-weighted lifetime or (more accurately) the amplitude ratio of the fast and slow decay component. The amplitude ratio reflects the metabolic state of the cell. Cancer cells are preferentially running glycolysis, normal cells oxidative phosphorylation. However, the metabolic state of a cell also varies with the oxygen concentration (or the oxygen partial pressure). It is therefore important to track the oxygen concentration simultaneously with the NADH FLIM recording. The oxygen concentration can be obtained from PLIM data of an exogenous phosphorescence dye. Due to its high sensitivity and its ability to record FLIM and PLIM simultaneously the bh FLIM/PLIM technique is excellently suitable for these applications, and the number of publications is steeply increasing.

The first publications concentrated on the characterisation of phosphorescent probes. In [66] and [69] Dmitriev and Papkovski give a summary on available phosphorescent probes, their O2 sensitivity, brightness, phosphorescence lifetimes, cell-staining capability, and toxicity.

The problem of sensitivity of phosphorescent dyes to other cell parameters, such as pH, viscosity, and oxygen diffusion time constant, is addressed by embedding the dyes in nanoparticles and nanostructured cell substrates. Phosphorescent nanoparticles, nanostructured films, scaffolds and fibres as pO2-sensors are explored in [65, 67, 97, 98, 137, 185, 201]. Phosphorescent gold clusters as probes for lipid droplets were described in [116].

Oxygen imaging of neurospheres is described in [64]. Applications to Giant Umbrella Cells and to healthy and inflamed colon tissue are described in [203] and [205]. Changes in intracellular oxygen concentration with the metabolic activity were observed by Zhdanov et al. [204]. Jenkins et al. studied the function of SPCA ATPases in HCT116 cells. They found correlations of the expression of SPCA1 and SPCA2 with the O2 concentration [99].

Kurokawa et al. [120] provided a detailed evaluation of TCSPC PLIM with Platinum(II) 5,10,15,20-tatrakis-[4-carboxyphenyl)porphyrin. They tested the sensor with MKN45 cells, colon26 spheroids under different O2 concentration. They were able to correlate low pO2 regions with mitochondria, to track the change in O2 concentration in Colon26 cells after addition of antimycin A, and to visualise the O2 change in MIN6 cells after glucose stimulation.

Simultaneous measurements of NADH FLIM and PLIM and the correlation of metabolic parameters and O2 concentration were published first by Kalinina et al. [103]. The authors observed changes in the NADH lifetime with the O2 concentration and after addition of antimycin A. Changes of the NADH decay functions with the intracellular O2 concentration were also demonstrated by Shcheslavskiy et al. [169].

Lukina et al. [130] used a fibre-based TCSPC system with implantable fibre tips [30] to record NAD(P)H decay curves simultaneously with phosphorescence decay curves of an oxygen sensor in mouse tumors. The authors found characteristic changes in the NADH decay parameters and the O2 concentration between healthy tissue and tumor tissue.

Plant Physiology

Plant tissue contains a large number of endogenous fluorophores. In practice the fluorescence is dominated by the fluorescence of chlorophyll and the fluorescence of flavines. The flavines emit in a broad range from about 480 nm to 580 nm. Chlorophyll is excited at any wavelength throughout the visible spectrum and emits a strong fluorescence signal around 700 nm. FLIM images recorded with the bh multi-spectral FLIM system at a Zeiss LSM 710 NLO are shown in Fig. 65.

Fig. 65: Multi-spectral FLIM of plant tissue. Moss leaf, LSM 710 NLO, two-photon excitation at 850 nm. Amplitude-weighted mean lifetime of double-exponential fit. Intensity normalised to brightest pixel of each wavelength interval.

The fluorescence of chlorophyll competes with the energy transfer into the photosynthesis channels. Thus, the fluorescence lifetime is a sensitive indicator of the photosynthesis efficiency. Therefore, the fluorescence lifetime of the chlorophyll in live plant tissue not only depends on the state of the tissue but also on the light intensity, and the time of exposure.

The changes in the fluorescence intensity of chlorophyll with the exposure to light have been found 1931 by Kautsky and Hirsch [107]. The effects have been termed fluorescence induction, fluorescence transients, or Kautsky effect [83, 131Govindjee Aust. J. Plant Physiol. 1995; Govindjee& Seufferheld Funct. Plant Biol. 200; Maxwell & Johnson, J. Exp. Bot. 2000]. When a dark-adapted leaf is exposed to light the intensity of the chlorophyll fluorescence starts to increase. After a steep rise the intensity falls again and finally reaches a steady-state level. The rise time is of the order of a few milliseconds to a second, the fall time can be from several seconds to minutes.

The initial rise of the fluorescence intensity is attributed to the progressive closing of reaction centres in the photosynthesis pathway. Therefore the quenching of the fluorescence by the photosynthesis decreases with the time of illumination, with a corresponding increase of the fluorescence intensity. The fluorescence quenching by the photosynthesis pathway is termed ‘photochemical quenching’. The slow decrease of the fluorescence intensity at later times is termed ‘non-photochemical quenching’. It is believed that it is a mechanism the plant uses to protect itself from photodamage. Because the chlorophyll transients are closely related to the metabolism of the plant they are a far better indicator of the health state than the fluorescence lifetime itself [21, 30].

By using a fast scan and a reasonable short acquisition time non-photochemical transients can be measured by time-series FLIM [6, 106]. After getting the sample in the focus the sample is shifted to an area that has not been exposed to light. Then a time-series of FLIM images is recorded. A typical result is shown in Fig. 66. The acquisition time per image was 1 second, the image size 256 x 256 pixels x 64 time channels. The illumination by the laser initiates an increase in non-photochemical quenching. The result is a decrease in the fluorescence lifetime of the chloroplasts which can clearly be seen in Fig. 66.



Fig. 66: Change of the fluorescence lifetime of chlorophyll with time of exposure. Moss leaf, 1-photon excitation at 445 nm, 256x256 pixels.Data mossleaflet-wq00 to wq50 analysed by Axel Bergmann with bh Optispec 7.4.09. 2exp decay, binning 5

The lifetime changes of chlorophyll can also be recorded by temporal mosaic FLIM, see page 23. Non-repeatable processes can be recorded at a speed of about two images per second. Repeatable processes can be recorded at images rates down to the sub-50 ms range. Even faster effects can be recorded by FLITS. The technique records transient effects in the fluorescence lifetime of a sample along a one-dimensional scan. An example is shown in Fig. 67. It shows decay curves for one location within the line for times of 0.4 s, 8 s, and 14 s after the turn-on of the laser.  The mean lifetimes, tm, obtained from a double-exponential fit are 862 ps, 471 ps, and 402 ps, respectively. The changes in the decay profiles are clearly visible.

Fig. 67: Decay curves for a selected pixel within the line for times of 0.4 s, 8 s, and 14 s after the turn-on of the laser

FLITS can even be used to record the photochemical transients. A time resolution of about 1 millisecond is required. To obtain enough photons within a time interval this short the laser must be on-off modulated and the data acquired over a large number of on-off periods. Please see [30, 26] for details.

 

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166.     D. Schweitzer, Autofluorescence diagnostics of ophthalmic diseases. In: V.V. Ghukasyan, A.H. Heikal, eds., Natural biomarkers for cellular metabolism. Biology, techniques, and applications.  CRC Press, Taylor and Francis Group, Boca Raton, London, New York (2015)

167.     D. Schweitzer, M. Hammer, Fluorescence Lifetime Imaging in Ophthalmology. In: W. Becker (ed.) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York (2015)

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174.     M. V. Shirmanova, I. N. Druzhkova, M. M. Lukina, V. V. Dudenkova, N. I. Ignatova, L. B. Snopova, V. I. Shcheslavskiy, V. V. Belousov, E. V. Zagaynova, Chemotherapy with cisplatin: insights into intracellular pH and metabolic landscape of cancer cells in vitro and in vivo. Scientific Reports 7, 8911-1 to -11

175.     M. C. Skala, K. M. Riching, D. K. Bird, A. Dendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, N. Ramanujam, In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J. Biomed. Opt. 12 02401-1 to 10 (2007)

176.     M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, N. Ramanujam, In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia, PNAS 104, 19494-19499 (2007)

177.     M.C. Skala, A. Fontanella, L. Lan, J.A. Izatt, M.W. Dewhirst, Longitudinal optical imaging of tumor metabolism and hemodynamics. J. Biomed. Opt. 15(1) 011112-1 to -8 (2010)

178.     M.C. Skala, N. Ramanujam, Multiphoton Redox Ratio Imaging for Metabolic Monitoring in vivo. Methods Mol. Biol. 594, 155-162 (2010)

179.     J. F. Soderholm, S. L. Bird, P. Kalab, Y.i Sampathkumar, K Hasegawa, M. Uehara-Bingen, Karsten Weis, R. Heald, Importazole, a small molecule inhibitor of the transport receptorimportin-β. ACS Chem Biol. 15, 6, 700–708 (2011)

180.     Studier, H., Becker, W.  Megapixel FLIM. Proc. SPIE 8948, (2014)

181.     E. Stuntz, Y. Gong1, D. Sood, V. Liaudanskaya, D. Pouli, K. P. Quinn, C. Alonzo, Z. Liu1, D. L. Kaplan, I. Georgakoudi, Endogenous Two-Photon Excited Fluorescence Imaging Characterizes Neuron and Astrocyte Metabolic Responses to Manganese Toxicity. Scientific Reports 7, 1041-1 to -15 (2017)

182.     K. Suhling, L. M. Hirvonen, J. A. Levitt, P.-H. Chung, C. Tregido, A. le Marois, D. Rusakov, K. Zheng, Fluorescence Lifetime Imaging (FLIM): Basic Concepts and Recent Applications. In: W. Becker (ed.) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York (2015)

183.     Y. Sun, J. Qu, G. Xu, L. Zhao, H. Niu, Autofluorescence lifetime imaging of Lipofuscin and melanin in retinal pigment epithelium cells. Invest Ophthalmol Vis Sci, IOVS 48, 2965 (2007)

184.     M. Szaszak, P. Steven, K. Shima, R. Orzekowsky-Schröder, Gereon Hüttmann, I.R. König, W. Solbach, J. Rupp, Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell. PLOS Pathogens 7, e1002108-1 to 12 (2011)

185.     C. Toncelli, O. V. Arzhakova, A. Dolgova, A. L. Volynskii, N. F. Bakeev, J. P. Kerry, D. B. Papkovsky, Oxygen-sensitive phosphorescent nanomaterials produced from high density polyethylene films by local solvent-crazing. Anal. Chem. 86(3), 1917-23 (2014)

186.     C. Tregido, J.A. Levitt, K. Suhling, Effect of refractive index on the fluorescence lifetime of green fluorescent protein. J. Biomed. Opt. 13(3), 031218-1 to -8 (2008)

187.     Y. Ueda, S. Kwok, Y. Hayashi, Application of FRET probes in the analysis of neuronal plasticity, Frontiers in neural circuits 7, 163 (2013)

188.     Y. Ueda, Y. Hayashi, PIP3 regulates spinule formation in dentritic spines structural long-term potentiation. J. Neurosci. 33(27) 11040-11047 (2013)

189.     K. Uemura, C. M. Lill, M. Banks, M. Asada, N. Aoyagi, K. Ando, M. Kubota, T. Kihara, T. Nishimoto, H. Sugimoto, R. Talahashi, B. T. Hyman, S. Shimohama, O. Berezovska, A. Kinoshita, N-cadherin-based adhesion anhances Ab releases and decreases Ab42/40 ratio. J. Neurochem. 108, 350-360 (2009)

190.     H.D. Vishwasrao, A. A. Heikal, K.A. Kasischke, W. W. Webb, Conformational Dependence of Intracellular NADH on Metabolic State Revealed by Associated Fluorescence Anisotropy. J. Biol. Chem. 280, 25119-25126 (2005)

191.     A. Walsh, R.C. Cook, B. Rexer, C.L. Arteaga, M.C. Skala, Optical Imaging of metabolism in HER2 overexpressing breast cancer cells. Biomedical Optics Express 3(1), 75-85 (2012)

192.     A. J. Walsh, R. S. Cook, H. C. Manning, D. J. Hicks, A. Lafontant, C. L. Arteaga, M. C. Skala, Optical Metabolic Imaging Identifies Glycolytic Levels, Subtypes, and Early-Treatment Response in Breast Cancer. Cancer Res. 73, 6164-6174 (2013)

193.     A. J. Walsh, R. S. Cook, M. E. Sanders, L. Aurisicchio, G. Ciliberto, C. L. Arteaga, M. C. Skala, Quantitative Optical Imaging of Primary Tumor Organoid Metabolism Predicts Drug Response in Breast Cancer. Cancer Res 74, OF1-OF11 (2014)

194.     A. J. Walsh, A. T. Shah, J. T. Sharick, M. C. Skala, Fluorescence Lifetime measurements of NADH in live cells and tissue. In: W. Becker (ed.) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York (2015)

195.     A. J. Walsh, M. C. Skala, Optical metabolic imaging quantifies heterogeneous cell populations. Biomed. Opt. Expr. 6, 559-573 (2015)

196.     H-W. Wang, V. Ghukassyan, C.T. Chen, Y.H. Wei, H.W. Guo, J.S. Yu, F.J. Kao, Differentiation of appoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells. J. Biomed. Opt. 13(5), 054011-1 to 9 (2008)

197.     O. Warburg, On the origin of cancer cells. Science 123, 309-314 (1956)

198.     O. Warburg, On respiratory impairment in cancer cells. Science 124, 269-270 (1956)

199.     J. Widengren, V. Kudryavtsev, M. Antonik, S. Berger. M. Gerken, C. A. M. Seidel, Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal.Chem. 78, 2039-2050 (2006)

200.     M.A. Yaseen, S. Sakadzic, w. Wu, W. Becker, K.A. Kasischke, D.A. Boas, In vivo imaging of cerebral enery matabolism with two-photon fluorescence lifetime imaging microscopy of NADH. Biomed. Opt. Expr. 4, 307-321 (2013)

201.     G. Yazgan, R. I. Dmitriev, V Tyagi, J.Jenkins, G-M. Rotaru, M.Rottmar, R. . Rossi, C. Toncelli, D. B. Papkovsky, K. Maniura-Weber,  G. Fortunato, Steering surface topographies of electrospun fibers: understanding the mechanisms. Scietific Reports 7, 158-1 to -13 (2017)

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Specifications

                                                                                                                    General Principle

Lifetime measurement                                                                                             time-domain

Excitation                                                                                                    high-frequency pulsed lasers

Buildup of lifetime images                                                  Single-photon detection by multi-dimensional TCSPC [30]

                                                                                            Builds up distribution of photons over photon arrival time

                                                                                                               after laser pulses, scan coordinates,

                                                                                            time from laser modulation, time from start of experiment.

Multi-wavelength FLIM                                       uses wavelength of photons as additional coordinate of photon distribution

Excitation wavelength multiplexing                               uses laser number as additional coordinate of photon distribution

Scan rate                                                                                                          works at any scan rate

Buildup of fluorescence correlation data                                          correlation of absolute photon times [30]

General operation modes                                                               FLIM, two spectral or polarisation channels

                                                                                                                       Multi-wavelength FLIM

                                                                                                Time-series FLIM, microscope-controlled time series

                                                                                                                               Z-Stack FLIM

                                                                                                                  Mosaic FLIM, x,y, z, temporal

                                                                                                          Excitation-wavelength multiplexed FLIM

                                                                                                    FLITS (fluorescence lifetime-transient scanning)

                                                                                    PLIM (phosphorescence lifetime imaging) simultaneous with FLIM

                                                                                                                FCS, cross FCS, gated FCS, PCH

                                                                                                          Single-point fluorescence decay recording

 

                                                                                      Data recording hardware, please see [30] for details

TCSPC System                                                        bh Simple Tau 152 TCSPC system, extension box coupled to laptop PC

Coupling to PC                                                                               bus extension cable or thunderbolt interface

Number of parallel TCSPC / FLIM channels                                                   typ. 2, min. 1, max 4

Number of detector (routing) channels in FLIM modes                               16 for each FLIM channel

Principle                                                                                                   Advanced TAC/ADC principle

Electrical time resolution                                                                               2.3 ps rms / 6.8 ps fwhm

Minimum time channel width                                                                                      813 fs

Timing stability over 30 minutes                                                                      typ. better than 5ps

Dead time                                                                                                                     100 ns

Saturated count rate                                                                                          10 MHz per channel

Dual-time-base operation                                                         via micro times from TAC and via macro time clock

Source of macro time clock                                                                   internal 40MHz clock or from laser

Input from detector                                                                                  constant-fraction discriminator

Reference (SYNC) input                                                                           constant-fraction discriminator

Synchronisation with scanning                                                   via frame clock, line clock and pixel clock pulses

Scan rate                                                                                                                  any scan rate

Synchronisation with laser multiplexing                                                            via routing function

Recording of multi-wavelength data                                           simultaneous in 16 channels, via routing function

Experiment trigger function                                         TTL, used for Z stack FLIM and microscope-controlled time series

Basic acquisition principles                                                              on-board-buildup of photon distributions

                                                                                                buildup of photon distributions in computer memory

                                                                                                   generation of parameter-tagged single-photon data

                                                                                                          online auto or cross correlation and PCH

Operation modes                                                                     f(t), oscilloscope, f(txy), f(t,T), f(t) continuous flow

                                                                                                             FIFO (correlation / FCS / MCS) mode

                                                                                             Scan Sync In imaging, Scan Sync In with continuous flow

                                                                                   FIFO imaging, with MCS imaging, mosaic imaging, time-series imaging

                                                                                                Multi-detector operation, laser multiplexing operation

                                                                                                       cycle and repeat function, autosave function

Max. Image size, pixels (SPCM 64 bit software)                     4096x4096        2048x2048          512x512           256x256

No of time channels, see [30]                                                          64                    256                  1024                 4096

 

                                                                                     Data Acquisition Software, please see [30] for details

Operating system                                                                         Windows XP, Windows 7, Windows 8, 64 bit

Loading of system configuration                                                          single click in predefined setup panel

Start / stop of measurement                                   by operator or by timer, starts with start of scan, stops with end of frame

Online calculation and display, FLIM, PLIM                                in intervals of Display Time, min. 1 second

Online calculation and display, FCS, PCH                                     in intervals of Display Time, min. 1 second

Number of images diplayed simultaneously                                                                 max 8

Number of curves (Decay, FCS, PCH, Multiscaler)                                       8 in one curve window

Cycle, repeat, autosave functions                                                                    user-defined, used for

                                                                                                          for time-series recording, Z stack FLIM,

                                                                                                                microscope-controlled time series

Saving of measurement data                                                                  User command or autosave function

                                                                                              Optional saving of parameter-tagged single-photon data

Link to SPCImage data analysis                                        automatically after end of measurement or by user command

 

                                                              Data Analysis: bh SPCImage, integrated in bh TCSPC package, see [6] or [30]

Data types processed                                                      FLIM, PLIM, MW FLIM, time-series, Z stacks, single curves

Procedure                                                                                    iterative convolution or first-moment calculation

IRF                                                                                                          synthethic IRF or measured IRF

Model functions                                                                                 single, double, triple exponential decay

                                                                                            single, double, triple exponential incomplete decay models

Parameters displayed                                                     amplitude- or intensity-weighted average of component lifetimes

                                                                                                   ratios of lifetimes or amplitudes, FRET efficiency

                                                                                    fractional intensities of components or ratios of fractional intensities

                                                                                                                        parameter distributions

Parameter histograms, one-dimensional                    Pixel frequency over any decay parameter or ratio of decay parameters

Parameter histograms, two-dimensional                               Pixel frequency over two decay parameters, Phasor plot

 

                                                                 Excitation Sources, One-Photon Excitation, please see [6] for details

Picosecond Diode Lasers                                                                        Modified bh BDL-SMC lasers

Number of lasers simultaneously operated                                                                      2

Wavelengths                                                                   405nm, 445nm, 473nm, 488nm, 515nm, 640nm, 685nm, 785nm

Mode of operation                                                                                        picosecond pulses or CW

Pulse width, typical                                                                                                40 to 100 ps

Pulse frequency                                                                                   selectable, 20MHz, 50MHz, 80MHz

Power in picosecond mode                                         0.4mW to 1mW injected into fibre. Depends on wavelength version.

Power in CW mode                                                        20 to 40mW injected into fibre. Depends on wavelength version.

 

Tuneable Lasers in visible range                                                                   Zeiss Intune Laser

Wavelength                                                                                                            480 to 640 nm

Repetition rate                                                                                                            40 MHz

 

Other Vis-Range Lasers

Visible and UV range                                                               any ps pulsed laser of  20 to 80 MHz repetition rate

     Coupling requirements                                                            Point Source-Kineflex compatible fibre adapter

     Wavelength                                                                                    any wavelength from 400nm to 800nm

 

Synchronisation / Modulation of lasers

Laser Multiplexing                                                                Diode lasers, pixel by pixel, line by line, frame by frame

                                                                                                  requires bh DDG-210 card and multiplexing indimo

Interleaved excitation                                                        sync of diode laser to diode laser or diode laser to intune laser

                                                                                                                    requires bh laser sync option

Laser Modulation for PLIM                                              Diode lasers, requires bh DDG-210 card and PLIM indimo

 

                                                                            Excitation Sources, Multi-Photon Excitation, please see [6] for details

Femtosecond NIR Lasers                                                 any femtosecond Ti:Sa laser or Ti:Sa pumped OPO

Wavelength                                                                                                              650 to 1000

Repetition rate                                                                                                        typ. 80 MHz

Laser Modulation for PLIM                                                    requires bh DDG-210 card and Zeiss PLIM indimo

 

                                                                                                                     Detectors

Interface to LSM 710 family microscopes                                                        NDD / BIG adapter

                                                                                               DC port adapter (older LSMs only, not recommended)

Beamsplitter, NDD port                                                                                 Zeiss NDD T Adapter

Beamsplitter, confocal port                                                   bh beamsplitter assembly with Zeiss-type filter cubes

                                                                                         detectors are portable between NDD and (confocal) BIG port

 

Hybrid Detectors (standard)                                                                bh HPM-100-40 hybrid detector

Spectral Range                                                                                                        300 to 710nm

Peak quantum efficiency                                                                                           40 to 50%

IRF width (fwhm)                                                                                                  120 to 130 ps

Detector area                                                                                                                 3mm

Background count rate, thermal                                                                300 to 2000 counts per second

Background from afterpulsing                                                                                not detectable

Afterpulsing peak in FCS                                                                                       not detectable

Power supply and overload shutdown                                           via DCC-100 controller of TCSPC system

 

Hybrid Detectors (ultra-fast, optional)                                        bh HPM-100-06 and -07 hybrid detector

Spectral Range                                                                                    -06: 300 to 600nm   -07: 300 to 750nm

Peak quantum efficiency                                                                                               20 %

IRF width (fwhm, with Ti:Sa laser)                                                                              19 ps

Detector area                                                                                                                 3mm

Background count rate, thermal                                                                 100 to 500 counts per second

Background from afterpulsing                                                                                not detectable

Power supply and overload shutdown                                           via DCC-100 controller of TCSPC system

 

Hybrid Detectors for NIR (optional)                                                   bh HPM-100-50 hybrid detector

Spectral Range                                                                                                        400 to 900nm

Peak quantum efficiency                                                                                           12 to 15%

IRF width (fwhm)                                                                                                  130 to 160 ps

Detector area                                                                                                                 3mm

Background count rate, thermal                                                               1000 to 8000 counts per second

Background from afterpulsing                                                                                not detectable

Power supply and overload shutdown                                           via DCC-100 controller of TCSPC system

 

Multi-Wavelength FLIM Detector (optional)                                       bh MW FLIM assembly

Spectral range                                                                                           380 to 630nm or 380 to 750nm

Number of wavelength channels                                                                                      16

Spectral width of wavelength channels                                                                       12.5 nm

IRF width (fwhm)                                                                                                        250 ps

Power supply and overload shutdown                                           via DCC-100 controller of TCSPC system

 

Detectors (optional)                                       PMZ-100 PMT modules   id100-50 SPAD modules    R3809U MCP PMTs

IRF width (fwhm, with Ti:Sa laser)                                200ps                                      60ps                                    28ps

Other specifications                                                                                               please see [30]

 

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

Becker & Hickl GmbH

Nunsdorfer Ring 7-9

12277 Berlin, Berlin

Tel.   +49 / 30 / 212 800 20, Fax. +49 / 30 / 212 800 213

email: info@becker-hickl.com, www.becker-hickl.com