DCS-120 Confocal and Multiphoton FLIM Systems

with

SPCImage NG FLIM Data Analyis

The DCS-120 is a family of confocal and multiphoton laser scanning microscopes for fluorescence lifetime imaging. Confocal systems use picosecond diode lasers for excitation, multiphoton systems use femtosecond fibre lasers or Titanium Sapphire lasers. The recording process is based on fast scanning by galvanometer mirrors, confocal or non-descanned detection, and bhs multidimensional TCSPC technique. The systems record fluorescence-lifetime images at ultra-high temporal resolution, high spatial resolution, and ultra-high sensitivity [1, 2]. Data analysis is performed by bh's SPCImage NG FLIM data analysis software. Maximum-Likelihood Estimation (MLE) and GPU processing deliver multi-exponential fit results of unprecedented accuracy within calculation times of seconds.

The DCS‑120 system is available with inverted and upright microscopes of Nikon, Zeiss, and Olympus. Due to their fast beam scanning and high sensitivity the DCS-120 systems are perfectly compatible with live-cell imaging. The DCS-120 systems cover the whole range from entry-level lifetime imaging to advanced multi-dimensional FLIM applications. These include simultaneous recording of FLIM or steady-state fluorescence images in two fully parallel wavelength channels, laser wavelength multiplexing, multi-wavelength FLIM, fast time-series recording, Z stack FLIM, simultaneous fluorescence and phosphorescence lifetime imaging (FLIM/PLIM), fluorescence lifetime-transient scanning (FLITS) and FCS recording. Typical applications are molecular imaging, ion concentration measurement, pH measurement, protein-interaction experiments by FRET techniques, metabolic imaging, and plant physiology.

The DCS-120 systems and their applications are described in a 430-page handbook [2], available on www.becker-hickl.com. As supplementary literature we recommend the bh TCSPC Handbook [1]. On 1058 pages, the TCSPC Handbook covers the basic principles of multidimensional TCSPC, priciples and use of light sources and detectors, optical principles, and applications of TCSPC / FLIM in life sciences.

Principle of the DCS-120 FLIM Systems

The DCS-120 systems use a combination of bhs multidimensional time-correlated single-photon counting (TCSPC) process [1, 2, 3] with confocal or multiphoton laser scanning. In other words, the systems use a combination of the most accurate and most sensitive principles to record in space and in time. Laser scanning yields depth resolution, optical sectioning capability, and suppression of longitudinally and laterally scattered light. The result is high-contrast recording of images from a clearly defined focal plane. Multi-dimensional TCSPC yields ultimate time-resolution and sensitivity, high dynamic range, recording of the full decay functions in a large number of time channels, and the capability to split multi-exponential fluorescence decay functions in their exponential components.

The basic principle of the DCS-120 systems is shown in Fig. 1. The sample is repetitively scanned by a high-repetition rate pulsed laser beam. Single photons of the fluorescence signal are detected, and each photon is characterised by its time in the laser pulse period and the coordinates of the laser spot in the scanning area in the moment its detection. The recording process builds up a photon distribution over these parameters. The photon distribution can be interpreted as an array of pixels, each containing a full fluorescence decay curve in a large number of time channels. In other words, the result is a perfect FLIM image, see Fig. 2.

Fig. 1: Basic Principle of TCSPC FLIM

The recording process is independent of the scan rate of the microscope. The signal-to-noise ratio depends only on the total acquisition time and the photon rate available from the sample. This gives the recording process additional flexibility. On the one hand, recording of FLIM within surprisingly short acquisition time can be performed. On the other hand, high accuracy FLIM data can be recorded by simply increasing the acquisition time.

The technique can further be extended by including additional parameters in the photon distribution. These can be the depth of the focus in the sample, the wavelength of the photons, the time after a stimulation of the sample, or the time within the period of an additional modulation of the laser. These techniques are used to record Z stacks or lateral mosaics of FLIM images, multi-wavelength FLIM images, images of physiological effects occurring in the sample, or to record simultaneously fluorescence and phosphorescence lifetime images.

Fig. 2: FLIM Image recorded with the DCS-120. Fluorescence decay curves in selected pixels are shown on the right. Each pixel contains a full florescence decay curve in a large number of time channels, enough for multi-exponential decay analysis.

The DCS-120 system has been introduced in 2007. Since then, it has been upgraded constantly with improved scanner optics, new detectors, new lasers, new TCSPC / FLIM modules, new recording functions, and new data acquisition and data analysis software.

Two of the early DCS-120 confocal versions are shown in Fig. 3. A DCS-120 system with an inverted microscope is shown on the left, a system with an upright microscope on the right.

Fig. 3: DCS‑120 Confocal Scanning FLIM system. Left: Inverted version with Zeiss Axio Observer. Right: Upright version with Zeiss Axio Examiner

DCS-120 Versions

DCS-120 and DCS-120 CMI Confocal Systems

Recent DCS-120 versions are shown in Fig. 4 through Fig. 14. Fig. 4 shows the basic confocal DCS‑120 system. It has two BDS-SM ps diode lasers, two detection channels with HPM‑100‑40 or HPM-100-06 hybrid detectors, two SPC-180NX or one SPC-QC-104 TCSPC/FLIM modules, and an upgraded optical scanner module. The new scanner has improved optical and mechanical stability, improved transmission, electronic pinhole alignment, and a super-stable main dichroic beamsplitter which can be replaced by the user.

An upscaled version of the basic DCS-120 system is the DCS-120 CMI Molecular Imaging System. It uses the bh LHB-104 'Laser Hub' to deliver the excitation light. The Laser Hub contains four lasers of different wavelength, and is able to multiplex them for simultaneous FLIM of fluorophores of different excitation spectra. The DCS-120 CMI is shown in Fig. 5.

Fig. 4: DCS‑120 Confocal Scanning FLIM systems. Left: Basic version with two lasers and two detection channels. Right: DCS-120 CMI Molecular Imaging Confocal FLIM System. Four lasers and up to four parallel detection channels

Fig. 5: DCS-120 CMI Molecular Imaging Confocal FLIM System. Four lasers and up to four parallel detection channels

The optical principles of the DCS-120 and the DCS-120 CMI are shown in Fig. 6 and Fig. 7. In the DCS-120 the beams of two lasers are delivered separately into the scan head. In the DCS-120 CMI the beams of the four lasers of the bh Laser Hub are delivered via a single optical fibre.

The beams pass the main dichroic beamsplitter, and are deflected by two fast-moving galvanometer mirrors. The scan lens projects the beams down the microscope light path into the back aperture of the microscope lens. As the system is scanning, the angle of the beams is changing, and the illuminated spot in the sample is raster-scanning over the image area.

The fluorescence light from the sample is collected by the microscope lens and again reflected by the galvanometer mirrors. This compensates the angular motion of the beam so that the reflected beam becomes stationary. It is separated from the excitation by the main dichroic beamsplitter and split in two wavelength intervals by the secondary beamsplitter. It then passes a pinhole in a plane conjugated with the image plane in the sample. Only light from this plane is focused in the pinholes, and only light from the image plane can pass the pinholes. The light passing the pinholes is cleaned up by filters and, finally, fed to the detectors.

Fig. 6: DCS-120 Basic confocal System with two lasers and two detection channels

Fig. 7: Principle of the DCS-120 CMI Confocal Molecular Imaging System. The system has four laser wavelengths and two detection channels.

DCS-120 MP and DCS-120 DMP Multiphoton Versions

Multiphoton versions of the DCS-120 are shown in Fig. 8 and Fig. 9. Fig. 8 shows the basic DCS-120 MP multiphoton system. It uses a 785 nm Toptica femtosecond fibre laser (either a Femto Fibre Pro or a Femto Fibre Smart) for excitation. With these fibre lasers, the system is not only the least expensive multiphoton FLIM microscope in the world, it is also the one easiest to use. The system is ready for use immediately after turn-on, the sample is positioned visually through the eyepieces, and fine focusing is done in the Preview Mode of the SPCM software. The excitation wavelength is ideal for label-free imaging, using fluorescence of NAD(P)H, FAD, carotenoids, porhyrines, or melanin. It also works for metabolic FLIM with NADH, and for molecular imaging with most of the commonly used exogenous fluorescence probes. Another outstanding feature is time resolution. Because the laser pulse is much shorter than the detector response, the system reaches less than 20 ps fwhm (or <10 ps rms) IRF width with the fast HPM-100-06 hybrid detectors. With an IRF width this short, the DCS-120 MP is able to detect fluorescence lifetimes down to 10 ps.

For users who feel uneasy with a single excitation wavelength the DCS-120 MP can also be delivered with a Ti:Sa laser. Ti:Sa-based systems are available either with a Coherent or Spectra Physics lasers. Ti:Sa systems usually also contain an acousto-optical modulator (AOM). With these extensions the DCS-MP rises into the league of the 'big' multiphoton microscopes. Unavoidable, the price rises correspondingly.

Fig. 8: DCS-120 MP system. Excitation by femtosecond fibre laser of 785 nm.

Fig. 9 shows the DCS-120 DMP Dual Multiphoton Excitation system. The DCS‑120 DMP uses two synchronised femtosecond Toptica Femto Fibre Ultra lasers of 780 nm and 920 nm. In the DCS-120 DMP, the lasers can be multiplexed for simultaneous FLIM of NAD(P)H and FAD. With this capability, the DCS‑120 DMP is the ideal multiphoton metabolic FLIM system. It can also be used for metabolic FLIM with NAD(P)H and FAD in combination with exogenous fluorescence probes or oxygen measurement by PLIM. Also here, the time resolution with the ultra-fast HPM-100-06 detectors is better than 20 ps fwhm. As the DCS-120 MP, it resolves fluorescence lifetimes down to 10 ps.

Fig. 9: DCS-120 DMP system. Two femtosecond fibre lasers of 785 and 880 nm.

The optical principle of the DCS-MP and the DCS-DMP is shown in Fig. 10 and Fig. 11. The systems use the same scanner optics as the confocal systems. However, there is an important difference in the way the fluorescence light is detected. Two-photon excitation occurs only in a thin layer around the focal plane of the microscope lens. Therefore, no pinhole is needed to suppress the detection of out-of focus fluorescence. Consequently, there is no need to send the fluorescence light all the way back through the scanner. Instead, the fluorescence is split from the excitation light directly behind the microscope lens, and directly send to the detectors. The proncople is calles 'Non-Deescanned Detection', or 'NDD'. The result is that even photons scattered on the way out of the sample have a chance to reach the detectors. The fact that scattered photons are detected does not impair the image quality - the data acquisition system automatically assigns them to the x-y position of the laser focus, not to the position where they left the sample. The result is high image quality, high contrast, and high detection efficiency from deep sample layers.

Fig. 10: Optical principle of the DCS-120 MP system

Fig. 11: Optical principle of the DCS-120 DMP system

DCS-120 Super MPC Combined Multiphoton / Confocal System

Fig. 12 shows another unique FLIM system of the DCS family, the DCS-120 Super MPC. The system uses a Ti:Sa laser or an OPO for tuneable multiphoton excitation. In addition, the SHG of the Ti:Sa laser is available for tuneable excitation in the visible range. Multiphoton FLIM is performed via a non-descanned beam path, one-photon excitation via a confocal beam path. The DCS-120 Super MPC can be used for deep-tissue FLIM and single-cell imaging as well. Because the excitation pulses are derived from the pulses of the Ti:Sa laser the time resolution of the DCS-120 Super MPC system is extremely high. The system is able to resolve lifetimes down to 10 ps even for excitation in the visible range. No other FLIM system has these capabilities. The principle of the DCS-Super-MPC system is shown in Fig. 13.

Fig. 12: DCS-120 Super MPC system. Multiphoton FLIM by Ti:Sa laser combined with tuneable confocal FLIM via SHG of Ti:Sa laser

Fig. 13: Principle of DCS-Super-MPC Multiphoton / Confocal System

DCS-120 MACRO System

The DCS‑120 MACRO system records images of macroscopic objects directly in the image plane of the scanner. FLIM images of objects as large as 15 mm can be scanned directly, larger images by mosaic FLIM in combination with a motorised sample stage. Like the basic DCS system, the DCS-120 MACRO uses two ps diode lasers for excitation and two HPM-100 hybrid detectors for detection. With lasers of 375 nm and 405 nm or 445 nm, the system is perfectly suitable for NADH/FAD metabolic imaging by laser multiplexing. Main applications are inspection of excised tissue during tumor surgery, imaging of whole small organisms, and quality control of solar cells.

Fig. 14: DCS-120 MACRO System: Right: Optical principle

DCS-120 Systems with Incubator Box

DCS-120 systems can be delivered with an incubator box. The box helps maintain controlled environmental conditions during the measurement of live cells. The box also keeps off environment light from the detection beam path. A DCS-120 DMP system with an incubator box is shown in Fig. 15.

Fig. 15: DCS-120 system with an incubator box. DCS-120 DMP dual-excitation multiphoton system with a NIKON Eclipse Ti2 microscope.

Overview on DCS‑120 FLIM Features

Fully Integrated Systems

The DCS‑120 systems are 'fully integrated systems', meaning they are controlled by one piece of software running on one computer. Scanner control, laser control, detector control, and control of single measurements or measurement sequences are implemented in the SPCM data acquisition software. As an example, Fig. 16 shows the SPCM main panel with the scanner control panel. The configuration allows the user to select the image format, scan rate, and scan area, configure the multiplexing of the lasers, start the preview, and start and stop the measurement. Imaging parameters can be changed at any time, even during a measurement.

Fig. 16: SPCM Version 9.92 data acquisition software with integrated scanner control

Direct Interaction with SPCIMage NG Data Analysis

FLIM data can be sent directly to SPCIMage NG data analysis, without prior saving them into a file and importing this file into SPCImage.

Fig. 17: Interaction with SPCImage NG Data analysis

Precision Confocal FLIM

FLIM images recorded by the DCS-120 systems feature diffraction-limited spatial resolution, ultra-high temporal resolution, suppression of out-of focus fluorescence, suppression of longitudinal and lateral scattering, optical sectioning capability, near-ideal sensitivity and photon efficiency, and low background. An example is shown in Fig. 18.

Fig. 18: FLIM image recorded by the DCS-120 basic confocal system. Decay curves shown on the right.

Deep-Tissue Multiphoton FLIM

Fig. 19 show a FLIM image of a pig skin sample, excited at 785 nm and detected from 510 to 600 nm. The image was recorded with 1024 x 1024 pixels and 1024 time channels.

Fig. 19: Pig skin, autofluorescence, 2p excitation at 785 nm, detection 510 to 600 nm. 1024 x 1024 pixels, 1024 time channels. DCS-120 MP system with Toptica Femto Fibre Smart laser.

Megapixel FLIM in Two Parallel Channels

With 64 bit SPCM software pixel numbers can be increased to 2048 x 2048 pixels, with a temporal resolution of 256 time channels. The DCS-120 system is able to simultaneously record two high-resolution images in different wavelength or polarisation channels, see Fig. 20 and Fig. 21.

Fig. 20: BPAE sample (Invitrogen) scanned with 2048 x 2048 pixels. Green channel, 485 to 560 nm

Recording is performed in two fully parallel TCSPC channels, avoiding any electronic lifetime or intensity crosstalk. Even if one channel should saturate the other is still producing correct data.

The capability to record images of large pixel numbers is beneficial for a wide range of FLIM applications. One example is tissue imaging where the samples are large, and the images are containing a wealth of detail. It is also useful when a large number of cells have to be investigated and the FLIM results be compared. Megapixel FLIM records images of many cells simultaneously, and under exactly 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.

Fig. 21: BPAE sample (Invitrogen), scanned with 2048 x 2048 pixels. Red channel, 560 to 650 nm

Sub-20 ps Time Resolution

The DCS-120 multiphoton systems can be equipped with ultra-high speed HPM‑100‑06 and ‑07 hybrid detectors. The time resolution (IRF width) with these detectors is less than 20 ps, full width at half maximum [26]. Lifetimes in the range of 5 to 10 ps can be detected by these systems, even if they are part of a multi-exponential decay. Applications are FLIM of carotenoids, melanins, and other natural short-lifetime fluorophores. An example is shown in Fig. 22.

Fig. 22: Spores of Paxillus involutus, time-channel width 300 fs, 4096 time channels, observation-time interval 1.25 ns. Left: Image of the fast decay component, t1, colour scale 0 to 25 ps. Right: Decay curve (blue), IRF (green), and decay parameters at the cursor position. DCS-120 MP system with HPM-100-06 detector.

Other applications of high time resolution are in the field of metabolic FLIM, which requires separation of the decay components from bound and unbound NADH and/or FAD. High time resolution greatly improves the accuracy at which these parameters are obtained [27]. Fig. 23 shows NAD(P)H FLIM recorded with a DCS-120 MP multiphoton system. It shows an image of the mean lifetime, tm, and an image of the amplitude ratio, a1/a2. The system IRF, and a decay curve from the cursor position are shown at the bottom.

Fig. 23: Image of the mean lifetime, tm, and image of the amplitude ratio, a1/a2. Lower row: system IRF and decay curve at the cursor position. DCS-120 MPC system with HPM-100-06 detectors, two-photon excitation.

Fast Acquisition

The DCS-120 uses fast beam scanning by galvanometer mirrors. A complete frame is scanned within a time from 100 ms to a about one second, with pixel dwell times down to one microsecond. With its fast scanner and its multi-dimensional TCSPC process the DCS systems achieve surprisingly short acquisition times. In most cases the acquisition speed is not limited by the recording process but by the ability of the sample to withstand high excitation power. For highly fluorescent samples excellent FLIM images can be obtained within a second or less. An example is shown in Fig. 24.

Fig. 24: Enchytraeus Albidus, Autofluorescence, Acquisition Time 1 second, Image Area 1mm x 1mm. Online-lifetime display of SPCM software. Right: decay curve in selected spot of the image.

Express FLIM

Recording a typical TCSPC image within a short period of time requires an enormous data transfer rate from the TCSPC module to the computer. The data transfer problem increases if a longer sequence of images is to be recorded, and if several TCSPC channels are operated at high count rate simultaneously. bh have solved the problem by a technique called 'Express FLIM'. Express FLIM does not transfer data into the computer photon by photon. Instead, the hardware of the TCSPC module combines the information of all photons within a given pixel into a just two numbers. One is the first moment of the decay curve, the other the number of photons within the pixel. Both numbers are transferred to the computer at the end of each pixel. Even for fast scanning, the required data transfer rate can easily be achieved. The result is a lifetime image that contains first-moment values in the individual pixels. Express FLIM is available for all DCS systems containing the SPC-QC-104 module. An example is shown in Fig. 25.

Fig. 25: Express-FLIM of a live Enchytraeus albidus. Autofluorescence, four subsequent images from a 5-frames/second sequence. DCS-120 system with SPC-QC-104. Excitation pulse rate 80 MHz, average photon rate about 10×10⁶ s^-1.

Fast Preview Function

When FLIM is applied to live samples the time and the excitation dose needed for sample positioning, focusing, laser power adjustment, and region-of-interest selection has to be 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. 26. Scan parameters can be changed during the preview, without stopping the scan.

Fig. 26: SPCM software in fast preview mode, display rate one image per second.

Online Display of Lifetime Images and Decay Curves

The SPCM software is able to display lifetime images and decay curves online during the measurement. Online display of lifetime data helps the user evaluate the quality of the data recorded and, if necessary, continue in a different region of the sample, with different zoom of the scanner, or with different pinhole size or laser power. An example of online display is shown in Fig. 27.

Fig. 27: Online display of lifetime image and decay curves.

Ultra-High Efficiency

The hybrid detectors of the DCS‑120 combine ultra-high sensitivity with low background and short IRF width. The combination of these features makes it easy to detect fluorescence from endogenous fluorophores in single cells. Advanced data analysis by MLE procedures can be used to split the decay curves in the pixels into several decay components and determine the lifetimes and amplitudes of the individual decay components, see Fig. 28. This capability is the basis of advanced applications, like metabolic imaging or calibration-free FRET imaging.

Fig. 28: Autofluorescence lifetime images of NADH in single cells. Lifetime image of mean lifetime of double exponential decay (left) and image of amplitude of fast decay component, a1 ( right). SPCImage NG data analysis, MLE fit.

Wide Range of Excitation Wavelengths

The DCS-120 confocal system can be used with a wide range of excitation wavelengths. Available diode-laser wavelengths range from 375 nm for excitation of NADH to 785 nm for excitation of NIR dyes. An NADH (autofluorescence) image is shown in Fig. 29, an image of a pig skin sample incubated with 3,3-diethylthiatricarbocyanine in Fig. 30.

Fig. 29: UV-Excitation FLIM. NADH image of cells, excitation 370 nm, detection 420 to 475 nm.

Fig. 30: Near-Infrared FLIM. Pig skin sample stained with 3,3-diethylthiatricarbocyanine, detection wavelength, excitation 690 nm, detection wavelength from 780 nm to 900 nm.

High Image Contrast up to the Highest Count Rates

Images taken with conventional TCSPC FLIM at high count rate often suffer from low intensity contrast. The reason is not the pile-up effect, as commonly believed, but intensity nonlinearity by the dead time of the TCSPC process. DCS-120 systems using the SPC-180 or SPC-QC-104 modules do not show this effect. The SPC-180 takes the intensity information from a fast parallel counter with almost no dead time, the SPC-QC-104 avoids dead time by a fast time-conversion method. An image taken at an average count rate of 5.5 MHz is shown in Fig. 31. Peak count rate is about 10 MHz. No loss in contrast by dead time effects is visible.

Fig. 31: Image taken at an average count rate of 5.5 MHz. Peak count rate is about 10 MHz. No loss in contrast by dead time effects is visible. DCS-120 with SPC-180 NX module, data analysis by SPCImage NG.

Photon-Counting Intensity Images

A frequently asked question is whether the DCS system can record conventional intensity images. Of course it can - the number of photons, and thus the intensity is part of the FLIM information contained in every pixel of a lifetime image. Recent DCS systems using SPC-180 or SPC-QC-104 TCSPC modules even deliver the intensity without dead-time-induced nonlinearity. Intensity images can be displayed side by side with lifetime images, see Fig. 32.

Fig. 32: Lifetime image (left) and intensity image (right), simultaneously displayed by SPCM. SPC-180N, lifetime-intensity mode.

Multiphoton FLIM with Fibre Laser

Standard DCS-120 MP and DCS-DMP multiphoton systems use femtosecond fibre lasers [11] as excitation sources. The standard wavelength for the DCS-120 MP is 780 nm, making the system perfectly suitable for NADH imaging in cells and tissue.

Fig. 33: FLIM of pig skin, NADH image, DCS-120 MP system with fs fibre laser. Two-photon excitation, non-descanned detection.

Aa frequent concern is that a fs fibre laser is not tuneable, and, therefore may not be able to excite the fluorophore to be used in a particular experiment. It turns out, however, that the fibre laser excites most of the commonly used exogenous fluorophores at reasonable efficiency. This makes the DCS-120 MP an good choice for general 2p FLIM imaging with visible-range fluorophores, and the cheapest multiphoton laser scanning microscope on the market.

Fig. 34: Mouse kidney sample labelled with Alexa 488, Alexa 568, and DAPI. DCS-120 Fibre, Images simultaneously detected in two spectral channels. Image format 1024 x 1024 pixels, 1024 time channels. Excitation power 3 mW in the sample plane, count rate about 2×10⁶ s^-1 in each channel.

Multiphoton FLIM with Ti:Sa Laser

The DCS-120 Super MPC system uses a Ti:Sa laser for excitation, and the DCS-120 MP can is available with a Ti:Sa laser instead of the standard fibre laser. Laser control is embedded in the DCS data acquisition software, see 'Software', page 53. FLIM images of pig skin in different depth of the tissue are shown in Fig. 35.

Fig. 35: Pig skin, NADH autofluorescence, image in different depth in the sample. Amplitude-weighted lifetime of triple-exponential decay model

DCS-120 MACRO: FLIM of cm-Size Objects

The DCS-120 MACRO system records lifetime images of objects as large as 20 mm in diameter. The system does not use a microscope. Instead, the object is placed directly in the primary image plane of the scanner. For autofluorescence applications the DCS MACRO scan head is available with a scan lens of especially high UV transmission. An example of a MACRO FLIM image is shown in Fig. 36.

Fig. 36: High-resolution MACRO FLIM image. 2048 x 2048 pixels, 256 time channels per pixel.

With two lasers of 375 nm and 405 or 445 nm, the DCS-120 MACRO is an excellent system for inspection of excised tumor tissue and tumors in small animals by metabolic FLIM. An example is shown in Fig. 37. The colour represents the metabolic indicator, a1. Decay functions are shown on the right.

Fig. 37: Lifetime Image of a tumor in a mouse. NAD(P)H image, image size 15 x 15 mm, recorded by DCS‑120 Macro system. Colour represents the metabolic indicator, a1. Decay functions of tumor and surrounding tissue shown on the right. Data courtesy of Marina Shirmanova and Vladislav Shcheslavskiy, University Nishni Novgorod.

Fig. 38 shows a similar image of a freshly excised rat brain with glioblastoma. The acquisition time for both images was about 100 seconds, the data were analysed by the MLE fit of SPCImage NG.

Fig. 38: Rat brain with glioblastoma. Autofluorescence, NADH image, excitation 375 nm, detection 430 to 475 nm. Data courtesy of Marina Shirmanova and Vladislav Shcheslavskiy, University Nishni Novgorod.

FCS

With its time-tag, or, more precisely, parameter-tag mode the DCS-120 confocal system delivers highly efficient FCS. Because the hybrid detectors are free of afterpulsing there is no afterpulsing peak in autocorrelation data [1]. It is not necessary to suppress the afterpulsing peak by cross-correlation, resulting in a two-fold increase of the signal-to-noise ratio [1]. An example of FCS is shown in Fig. 39.

Fig. 39: Single molecules diffusing through the laser focus. Decay curve, FCS curve, intensity trace. Raman light suppressed by time-gating. Online fit with FCS procedures of SPCM data acquisition software.

Detection of Nanoparticles

The parameter-tag mode of the DCS system can also be used for single-particle detection. An example for the diffusion of fluorescent nanoparticles through the laser focus in shown in Fig. 40.

Fig. 40: Fluorescent nanoparticles drifting through the laser focus. Intensity trace.

The individual photon bursts can be further analysed by bh 'SPCDynamics' software, see Fig. 41. SPCDynamics displays fluorescence decay curves integrated over the bursts in a selected time interval, a phasor plot of the decay data of the bursts within a selected time interval, and decay curves and fluorescence lifetimes of individually selected bursts.

Fig. 41: Analysing photon bursts from single particles or single molecules by bh 'SPCDynamics' software

Recording of Single Decay Curves

The DCS-120 system can record single decay curves from fluorophore solutions or from selected spots in a two-dimensional sample. Curves are obtained either in the 'Single' mode of SPCM, or from summing up the decay data from a pixel area within a FLIM image. This makes a separate lifetime spectrometer for fluorophore characterisation unnecessary. Moreover: A multiphoton system with ultra-fast detectors beats any lifetime spectrometer in time resolution. An example is shown in Fig. 42.

Fig. 42: Fluorescence decay curve recorded with a DCS-120 MP.

Advanced DCS‑120 Functions

Mosaic FLIM

Mosaic FLIM records a large number of consecutive images into a single FLIM data array. The individual images within this array can represent the elements of a tile scan (x-y mosaic), images in different depth in the sample (z-stack mosaic), or images for different times after a stimulation of the sample (temporal mosaic). An example of an x-y mosaic is shown in Fig. 43. 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.

Fig. 43: Mosaic FLIM of a BPAE sample. The mosaic has 4x4 elements, each element has 512x512 pixels with 256 time channels. The entire mosaic has 2048 x 2048 pixels, each pixel holding 256 time channels. DCS‑120 MP multiphoton system with motorised sample stage.

Temporal Mosaic FLIM: Recording of Dynamic Effects

SPCM 64-bit software versions later than 2014 have a Mosaic Imaging function implemented. For time-series recording, subsequent frames of the scan are recorded into subsequent elements of the mosaic. The sequence can be repeated and accumulated [1]. The time per mosaic element can be as short as a single frame, which can be less than 100 ms. Another advantage is that the entire array can be analysed in a single SPCImage data analysis run. Fig. 44 shows the change of the lifetime of chlorophyll in plant tissue with the time of illumination.

Fig. 44: Time series of chloroplasts in a leaf recorded by Mosaic Imaging. 64 mosaic elements, each 128x128 pixels, 256 time channels. Scan time per element 1s. Experiment time from lower left to upper right. Amplitude-weighted lifetime of double-exponential decay.

Temporal Mosaic FLIM with Triggered Accumulation

When dynamic effects in the fluorescence behaviour of an object are to be recorded the speed is limited by the decrease of the photon numbers in the pixels. The DCS system solves the problem by 'Triggered Accumulation'. A dynamic effect in the measurement object is induced periodically, and the start of the mosaic recording is synchronised with the stimulation. The photon number in the pixels then only depends on the total acquisition time (the number of stimulation periods), and not on the speed of the mosaic recording. As a result, a very fast image sequence can be obtained without the need of exceedingly high photon rate. In fact, triggered accumulation FLIM can be faster than any 'fast FLIM' technique and still be live-cell friendly. An example is shown below.

Fig. 45, Left: Calcium transient in cultured neurons, temporal mosaic imaging, 40 ms per image. Image elements 64x64 pixels. bh SPC-150 FLIM system with SPCM software, attached to a Zeiss LSM 7MP microscope.

FLIM of Moving Objects

The recording of fluorescence-lifetime images of live cells or organisms is often impaired by motion in the sample. Nevertheless, the DCS system is able to obtain precision fluorescence-lifetime data from such objects. The technique is based on temporal-mosaic recording and image segmentation by the phasor plot of the bh SPCImage NG data analysis software. A cluster of phasors is selected in the phasor space, identifying pixels of a given decay signature in the FLIM mosaic. These pixels are back-annotated in the mosaic, selecting parts of the objects irrespectively of their location in the individual images. The decay data of the pixels within the selected areas are summed up. The result is a single decay curve with extremely high photon number which can be analysed at high precision [6, 5].

Fig. 46: Precision lifetime analysis on a moving object. A water flee is imaged by temporal mosaic FLIM (left), the phasor range of a structure of interest is selected, and fluorescence-decay analysis is performed on the decay data of the combined pixels within the phasor range.

FLITS: Fluorescence Lifetime-Transient Scanning

FLITS records dynamic effects in the fluorescence lifetime of a sample along a one-dimensional scan. The technique is based on building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses, and the experiment time after a stimulation of the sample. 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 [1, 15].

Fig. 47: FLITS of chloroplasts in a grass blade, change of fluorescence lifetime after start of illumination. Left: Non-photochemical transient, transient resolution 60 ms. Right: Photochemical transient. Triggered accumulation, transient resolution 1 ms.

Z Stack recording

Z stack recording is achieved by controlling the Z drive of the microscope, usually a Zeiss Axio Observer or Axio Examiner, synchronously with the acquisition of an image sequence. The DCS system has two procedures to record Z stacks. One is based on Mosaic FLIM: The data of subsequent planes are recorded in a large FLIM Mosaic. An example of a Mosaic-FLIM Z stack is shown in Fig. 48.

Fig. 48: FLIM Z stack of a part of a water flee. Z stack by mosaic FLIM procedure.

The advantage of Mosaic Z stack recording is that the images of all Z planes are recorded in a single, large FLIM data set. This avoids delay by writing data in subsequent data sets, and guarantees that the data of all planes are exactly comparable. However, the maximum number of planes is limited by the available data space. Depending on the desired lateral resolution, that means that 16 to 64 Z planes can be recorded.

A virtually unlimited number of Z planes can be recorded by a 'record and save' procedure. That means an image of the current plane is recorded, saved into a file, and then the focal plane is moved to the next Z plane. The procedure is repeated until the desired number of planes has been recorded. The record-and-save procedure needs time to save the data but is able to record large number of planes at high x-y resolution. An example of a high-resolution Z stack obtained from a fly, Musca domestica, is shown below. The stack contains 289 planes, each scanned with 1024 x1024 pixels and 1024 time channels. Fig. 49 shows a projection of all planes in a single FLIM image by the 'Multi-File View' of SPCM.

Fig. 49: Vertical projection of all 289 planes of the z stack into a single FLIM image. Planes added by Multi-File View of SPCM, image displayed by Online-Lifetime function of SPCM. Single-exponential lifetime by first-moment analysis.

Fig. 50 shows the same data, analysed by SPCImage and combined into a 3D representation by Image J. All 289 planes were processed with a double-exponential model by the batch-processing function of SPCImage NG, and the resulting 289 tm images written into bmp files by the batch-export function. The bmp files were imported into Image J, which then constructed the 3D representation. For details please see [10].

Fig. 50: Decay analysis by SPCImage NG and 3D reconstruction by ImageJ. Colour represents mean lifetime, tm, of double-exponential decay, lifetime range red to blue = 0 to 1250 ps.

Multi-Wavelength FLIM

With the bh multispectral FLIM detectors the DCS‑120 records FLIM simultaneously in 16 wavelength channels [1, 3, 8, 13]. The images are recorded by a multi-dimensional TCSPC process which uses the wavelength of the photons as a coordinate of the photon distribution. There is no time gating, no wavelength scanning and, consequently, no loss of photons in this process. The system thus reaches near-ideal recording efficiency. Moreover, dynamic effects in the sample or photobleaching do not cause distortions in the spectra or decay functions. Multi-wavelength FLIM got an additional push from the new 64-bit SPCM software, and from the introduction of a highly efficient GaAsP multi-wavelength detector [1]. 64-bit software works with enormously large photon distributions, and the GaAsP detector delivers the efficiency to fill them with photons. As a result, 16 images in 16 wavelength channels can be recorded at a resolution of 512x512 pixels and 256 time channels. An example is shown in Fig. 51. For applications please see [1, 30, 40].

Fig. 51: Multi-wavelength FLIM with the bh MW-FLIM GaAsP 16-channel detector. 16 images with 512 x 512 pixels and 256 time channels were recorded simultaneously. Wavelength from upper left to lower right, 490 nm to 690 nm, 12.5 nm per image. DCS‑120 confocal scanner, Zeiss Axio Observer microscope, x20 NA=0.5 air lens.

It may be suspected that the spatial and temporal resolution of the individual images is mediocre, at best. This is, however, not the case, thanks to the large data space available in the 64-bit environment. Fig. 52 demonstrates the true spatial resolution of the data. Images from two wavelength channels, 502 nm and 565 nm, were selected from the data shown Fig. 51, and displayed at larger scale and with individually adjusted lifetime ranges. With 512x512 pixels and 256 time channels, the spatial and temporal resolution of the individual images is comparable with what previously could be reached for FLIM at a single wavelength. Decay curves for selected pixels of the images are shown in Fig. 53.

Fig. 52: Two images from the array shown in Fig. 51, displayed in larger scale and with individually adjusted lifetime range. Wavelength channels 502 nm (left) and 565 nm (right). The images have 512 x 512 pixels and 256 time channels.

Fig. 53: Decay curves at selected pixel position in the images shown above. Blue dots: Photon numbers in the time channels. Red curve: Fit with a double-exponential model.

Laser Wavelength Multiplexing

Laser multiplexing is used to record FLIM images from fluorophores that cannot be excited at the same wavelength, or the fluorescence of which cannot be discriminated by emission filters. Two, three, or four lasers can be multiplexed in time. Multiplexing is automatically synchronised with the pixels, lines or frames of the scan. The data acquisition software builds up separate images for the individual lasers. An example is shown in Fig. 54. It shows a mouse kidney section, stained with Alexa 488 WGA, Alexa 568 phalloidin, and DAPI. Two excitation wavelengths, 405 nm and 473 nm, were multiplexed. The detection wavelength intervals were 432 nm to 510 nm and 510 nm to 550 nm. Only the combinations of 405 nm with 432 nm to 510 nm and 510 nm to 550 nm, and 473 nm and 510 nm to 550 nm are shown. The fourth combination, 473 nm with 432 nm to 510 nm does not contain reasonable data because the detection interval is too close to the excitation wavelength.

Fig. 54: Excitation wavelength multiplexing, 405 nm and 473 nm. Detection wavelength 432 nm to 510 nm and 510 nm to 550 nm. Mouse kidney section, stained with Alexa 488 WGA, Alexa 568 phalloidin, and DAPI.

Metabolic FLIM with NAD(P)H and FAD

Metabolic FLIM is based on simultaneous recording of lifetime images of NAD(P)H (nicotinamide adenine (pyridine) dinucleotide) and FAD (flavin adenine dinucleotide). The DCS-120 FLIM systems use laser wavelength multiplexing to simultaneously record lifetime images of the two fluorophores. By multi-exponential decay analysis of the NADH and FAD signals, the system delivers information on the metabolic state of the cells or the tissue investigated.

Fig. 55: NADH and FAD images, recorded simultaneously by the DCS-120 metabolic-FLIM system

In metabolic FLIM, the primary information is not in the mean lifetime, tm, but in the amplitudes of the decay components, a₁ and a₂. Due to its superior photon efficiency and time resolution the DCS-120 metabolic FLIM system provides such images at unprecedented accuracy. An example is shown in Fig. 77.

Fig. 56: NADH and FAD images, showing the amplitude of the fast decay component, a₁.

Wavelength-Multiplexed Multiphoton Metabolic FLIM

The DCS-120 DMP system is able to multiplex two Toptica Femto Fibre Ultra lasers of 780 nm and 920 nm. The typical application is metabolic FLIM of tissues by simultaneous detection of NAD(P)H and FAD data. An example is shown in Fig. 57 and Fig. 58.

Fig. 57: 2p metabolic FLIM of Pig skin, t_m image in the NADH channel. DCS-120 DMP, SPCIMage NG data analysis

Fig. 58: 2p metabolic FLIM of Pig skin, t_m image in the FAD channel. DCS-120 DMP, SPCIMage NG data analysis

Simultaneous FLIM / PLIM

The DCS-120 records FLIM and PLIM by bh's simultaneous FLIM/PLIM technique. The technique is based on on/off modulation of the excitation laser, and recording of lifetime images for the photon times in the laser pulse period and in the laser modulation period [1, 25]. On/off modulation is defined by the laser control parameters. The recording process is synchronised with the modulation via the GVD-120 or -140 Scan controller.

Fig. 59: Simultaneous FLIM / PLIM, main panel of data acquisition software

High-Performance FLIM Data Analysis

Data analysis is performed by bh's legendary SPCImage NG FLIM Data analysis. SPCImage NG combines Time-Domain and Phasor analysis. It uses an MLE fit procedure, resulting in superior accuracy down to low photon numbers, and unprecedented accuracy of multi-exponential decay analysis. By running the calculations on a GPU, results are obtained within seconds. This makes it possible to analyse complex data structures, such as FLIM/PLIM data, multi-wavelength data, and metabolic-FLIM data. A Global Fit procedure is included for precision analysis of triple-exponential FRET and Metabolic-FLIM data [24]. Please see [2], chapter 'SPCImage Data Analysis Software'.

Fig. 60: SPCImage NG combines time-domain and phasor analysis

Fig. 61: Global fit, triple-exponential analysis of FAD / FMN fluorescence. Separation of bound FAD, free FAD, and FMN

Fig. 62: Double-exponential fit on 318 photons. Decay components of FAD are correctly reproduced.

Fig. 63: Calibration-free FRET analysis by double-exponential MLE fit

Fig. 64: Image segmentation by phasor plot. Precision decay analysis of a moving object.

Applications in Life Sciences

Molecular Imaging

FLIM uses the fluorescence decay function of a fluorophore as an indicator of its molecular environment. The fluorescence decay function, within reasonable limits, neither depends on the fluorophore concentration nor on the excitation power, or other instrumental details. This is a striking advantage over intensity-based imaging techniques. When fluorescence in a sample is excited (Fig. 65, left) the emission intensity (second left) depends not only on possible interaction of the fluorophore with the molecular environment but also on the fluorophore concentration, on possible absorption in the sample, on the excitation power, and on the light collection efficiency of the optics. Changes in the molecular environment can thus not be distinguished from changes in these parameters. 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. The fluorescence lifetime of a fluorophore however (Fig. 65, right), only depends on the fluorophore itself and its interaction the molecular environment.

Fig. 65: 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.

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 [1, 4, 37].

Typical FLIM applications are ion concentration measurements, probing of protein interaction via FRET, and the probing of the metabolic state and the cell viability via the fluorescence decay parameters of NADH and FAD.

Fluorescence decay functions in these applications are usually multi-exponential. The components of the decay function represent different binding states, different conformations of the fluorophore, or other biologically relevant information. Highly efficient multi-exponential fluorescence-decay analysis is therefore an integral part of the DCS-120 system [1, 2, 5, 6, 7].

Fig. 66: Real decay function of a fluorophore in biological environment (left) and composition of the curve (right).

Molecular Parameters - Derived from Fluorescence-Decay Data

Molecular environment parameters, such as local pH, ion concentrations, local viscosity or redox potential are available through TCSPC FLIM and precision decay analysis [1]. With appropriate calibration of the probe the results are quantitative, i.e. independent of the laser power, the fluorophore concentration, the parameters of the optical-system, and other instrumental details. Examples are shown below.

Fig. 67: pH in skin, detected by lifetime of BCECF

Fig. 68: Calcium concentration in barley root. Detected by lifetime of Oregon Green Bapta

Fig. 69: Local viscosity, detected by lifetime of BODIPY

Fig. 70: Redox potential, detected by lifetime of Methylen Blue

FRET - Results from a Single Donor FLIM Image

FRET (Förster Resonance Energy Transfer) is used to probe protein interaction and protein structure in biological systems. The excitation energy is absorbed by a donor. It then transfers to an acceptor, and is emitted via the emission band of the acceptor. The energy transfer occurs only if the distance between the donor and the acceptor is less than a few nm. FRET is used to obtain information about protein interaction, protein folding, and protein structure. FRET experiments by steady-state spectroscopy are difficult to calibrate. FRET is therefore performed mainly by FLIM. But also with FLIM there are problems if the measurement is based on simple single-exponential 'fluorescence lifetimes'. Single-exponential FRET is often considered a quantitative technique but in fact it is not [18].

Quantitative FRET results are only obtained by FLIM in combination with double-exponential decay analysis. The method has been developed by bh already in 2005 and has been constantly improved in the past years. In contrast to single-exponential techniques, the method delivers correct FRET efficiencies and FRET distances even for incomplete donor-acceptor linking, and without reference measurement of a donor-only sample [1, 19]. The classic FRET efficiency, the FRET efficiency of the interacting donor, the amount of interacting donor, and the donor-acceptor distance are displayed directly by SPCImage NG [5]. An example is shown in Fig. 71.

Fig. 71: FRET Measurement in life cell. Classic FRET efficiency, FRET efficiency of interacting donor, amount of interacting donor, and ratio of donor-acceptor distance to Förster radius. bh double-exponential FRET technique, DCS-120 confocal, SPCImage NG data analysis.

FRET-Based Sensors

A large group of sensors for cell parameters is based on FRET. A donor and an acceptor are attached to the ends of a amino-acid linker. The linker changes its conformation with the molecular environment, and so does the fluorescence decay of the donor. For a summary please see FRET chapter in [1]. Fig. 72 shows an open tumor in a mouse, expressing a sensor for apoptosis. The sensor consist of mKate2 (donor) and iRFP (acceptor), connected by an amino-acid linker [41].

Fig. 72: Open tumor in a mouse, expressing a FRET sensor for apoptosis. DCS-120 MACRO system, analysis by SPCImage NG

Label-Free Imaging

FLIM of Small Organisms

The wide range of excitation and detection wavelengths and the high sensitivity makes the DCS-120 an excellent system for label-free (autofluorescence) FLIM of small organisms. Fig. 73 shows an autofluorescence image of Artemia salinas, a small shrimp living in briny water. A two-photon FLIM image of Artemia salinas recorded by the DCS‑120 MP Fibre system [29] is shown in Fig. 74.

Fig. 73: Autofluorescence FLIM of Artemia salinas. Left: Amplitude-weighted lifetime, tm. Right: Metabolic parameter, a1. Basic DCS-120 confocal system with HPM‑100‑40 hybrid detectors and SPC-180N TCSPC modules, Analysis by SPCImage NG.

Fig. 74: Two-photon autofluorescence FLIM image of Artemia salinas, mean (amplitude-weighted) lifetime of double-exponential decay. Decay functions of selected areas shown on the right. DCS-120 MP system.

Metabolic Imaging by NADH FLIM

For several decades, it has been attempted to obtain metabolic information from the fluorescence lifetime of NADH (nicotinamide adenine dinucleotide). These attempts were not successful in deriving the metabolic state because the lifetimes of the NADH decay components depend also on molecular parameters other than the metabolic state. bh FLIM systems have overcome the problem by using either the amplitude ratio of the decay components (a1/a2) or the amplitude of the decay component of free NADH, a1. It turns out that a1/a2 (the metabolic ratio) or a1 (the metabolic indicator) describe the metabolic state independently of the type of the cell and its molecular environment. Tumor cells have an a1 above 0.7, normal cells an a1 below 0.7. An example is shown in Fig. 75.

Fig. 75: Metabolic FLIM, a1 image and decay curves. Upper curve: Tumor cell. Lower curve: Normal cell. The tumor cell has an a1 above 0.7. Live human bladder cells from a biopsy. Data analysis with SPCImage NG, MLE algorithm.

NADH FLIM for Clinical Diagnostics

It has been shown that NADH FLIM can be used for clinical diagnostics. FLIM data were obtained from human bladder cells excised during surgery. The patients had been diagnosed with bladder cancer or other suspicious lesions by classic endoscopy. Measurements on the excised material were performed immediately after surgery, before the material went to histology [43]. By using a normal / cancer discrimination threshold of a1 = 0.71 perfect agreement with the histology results was obtained. Please see [1, 16, 17, 43] for details.

NADH FLIM with Multiphoton Excitation and Ultra-Fast Detectors

Metabolic FLIM requires double-exponential data analysis to extract the amplitudes of the decay components of bound and unbound NADH. Separation of the components improves with the time resolution of the FLIM system [27]. The DCS-120 MP multiphoton system in combination with the ultra-fast HPM-100-06 and -07 detectors achieves a time resolution (width of the instrument-response function) of less than 20 ps FWHM [1, 26]. The fast response greatly improves the accuracy at which fast decay components can be extracted from a multi-exponential decay. Date taken with the system not only show the metabolic state of the cells reliably, they also show heterogeneity in the a1 of different mitochondria. An NADH FLIM image recorded with the DCS-120 MP and an HPM-100-06 is shown in Fig. 76. The cells were cultured from a biological cell line. These cells are derived from tumor cells, therefore a1 is larger than 0.7.

Fig. 76: Left: NADH Lifetime image, amplitude of free NADH, a1. Right: Decay curve at cursor position, 4x4 pixel area. DCS-120 MP with HPM-100-06 detector, FLIM data format 512 x 512 pixels, 1024 time channels. SPCImage NG data analysis.

Metabolic Imaging by Simultaneous FLIM of NADH and FAD

When it comes to spectroscopic measurement of the metabolic state often only the fluorescence of NADH is considered. However, also the fluorescence of FAD (flavin adenine dinucleotide) shows a dependence on the metabolic state. Like NADH, FAD exists in a bound and an unbound component. The bound / unbound ratio depends on the metabolic state. The components have different lifetimes, and can be separated by double-exponential decay analysis. The amplitudes of the decay components or the ratio of the amplitudes depend on the metabolic state [2, 1, 38, 44]. Recording FAD FLIM in combination with NADH FLIM may therefore increase the reliability of metabolic imaging. However, there is a problem: The excitation of NADH inevitably also excites FAD, and the fluorescence of FAD cannot be separated from the fluorescence of NADH by emission filtering. The DCS-120 system therefore uses excitation-wavelength multiplexing in combination with dual-channel detection [1, 16], see 'Laser Wavelength Multiplexing', page 35. An example is shown in Fig. 77.

Fig. 77: NADH and FAD images, showing the amplitude of the fast decay component, a1. Same sample as shown in Fig. 75.

Detection of FMN

A problem of using FAD for metabolic imaging is that a part of the fluorescence in the FAD emission range comes from FMN. FNM has a similar emission spectrum as FAD but a different fluorescence lifetime. FMN does not react to the metabolic state the same was as FAD. Therefore its presence can be a problem for quantitative metabolic FLIM. With the high quality of the DCS-120 data and the superior multi-exponential capabilities of SPCImage NG data analysis FAD and FMN can be distinguished in the FLIM data. Fig. 78 shows images of fractions of bound FAD (a1), free FAD (a2), and FMN (a3) in live human bladder cells.

Fig. 78: Amount of bound FAD, free FAD, and FMN in live human bladder cells. Recorded by DCS-120 basic confocal FLIM system. Global analysis by SPCImage NG.

Label-Free Imaging of Macroscopic Objects

Metabolic FLIM of macroscopic objects is possible with the DCS-120 MACRO system. It differs from the other DCS systems in that the sample is placed directly, without a microscope, in the image plane of the DCS scanner. Objects as large as 20 mm can be imaged in a single scan [1, 9]. Fig. 79 shows an NADH image of an open tumor in a mouse. Decay curves and decay parameters in selected spots are shown on the right. The metabolic parameter, a1, is 0.61 in the healthy tissue and 0.84 in the tumor. This corresponds well with a1 values from other metabolic FLIM data: a1 is <0.7 in the good tissue and >0.7 in the tumor. For applications please see [39, 42].

Fig. 79: Open tumor in a mouse, image recorded by DCS-120 MACRO system. Excitation wavelength 370 nm, detection from420nm to 480 nm. The metabolic parameter, a1, is <0.7 in the good tissue and >0.7 in the tumor. This is exactly what is to be expected from other metabolic FLIM experiments.

Ultra-Fast Fluorescence Decay in Biological Material

Due to the short pulse width of the femtosecond lasers the DCS-120 MP system delivers extremely high temporal resolution. An example is shown in Fig. 80. The image shows mushroom spores of Boletus edulis [20]. The image was recorded by a DCS-120 MP with a Femto-Fibre-Pro laser (Toptica), ultra-fast HPM-100-06 detectors, and SPC-150NX TCSPC modules. The data show clearly a decay component of 20 ps lifetime.

An interesting application of ultra-fast FLIM is lifetime imaging of Carotenoids. The lifetimes are in the lower ps range but can easily be resolved by the DCS-120 MP system with ultra-fast detectors [21]. Not only can carotenoids can be localised by their short fluorescence lifetimes, different carotenoids can also be distinguished by different lifetime. Ultra-fast fluorescence decay was also found in human hair and - interestingly - in malignant melanoma. Please see [22] and [23]. Moreover, there are indications that similar carotenoids display different lifetime in different molecular environment. Examples are shown in Fig. 81 and Fig. 82.

Fig. 80: Spores of Boletus edulis. Left to right: Image of fast decay component, t₁, of triple-exponential decay model, histogram of t₁, and decay curve in selected spot. Data from [20].

Fig. 81: Lifetime image of carrot tissue. Amplitude-weighted lifetime, tm, of triple-exponential fit. Decay curves in locations without and with b-carotene shown on the right. Triple-exponential decay analysis with SPCImage NG. The fast lifetime component in carotene-rich regions is 8.1 ps.

Fig. 82: Decay curves of lutein, astaxanthin, b-carotene, and Lypcopene. Lifetimes are in the range from 10 to 20 ps [21].

Oxygen Sensing by PLIM

Oxygen sensing is based on the quenching of the phosphorescence decay of (exogenous) phosphorescent dyes by oxygen. PLIM can be recorded by the DCS-120 system using triggered MCS recording and multi-pulse excitation [25, 1, 32, 34], see also page 38 of this brochure. Two examples are shown in Fig. 83. The figure shows PLIM images of cultured human embryonic kidney cells incubated with a palladium-based phosphorescence dye. Fig. 83, left was recorded under atmospheric oxygen partial pressure. The maximum of the lifetime distribution over the pixels (upper right) is at 75 µs. Fig. 83, right, was recorded under decreased oxygen partial pressure. As can be seen, the maximum of the lifetime distribution has shifted to 144 µs.

Fig. 83: HEK cells incubated with a palladium dye imaged under different oxygen partial pressure. Left: Atmospheric O2 pressure. Right: reduced oxygen partial pressure. Recorded by bh DCS‑120 confocal FLIM system, data analysis by bh SPCImage NG. Lifetime scale 0 (red) to 300 µs (blue). Phosphorescence lifetime at the Cursor-Position 65 µs. The maximum of the lifetime distribution over the pixels is at 75 µs.

There is currently an increasing interest in PLIM not only for oxygen sensing but also for background-free imaging and luminescence decay of inorganic compounds. In all these applications the bh technique delivers a far better sensitivity than PLIM techniques based on single-pulse excitation.

Simultaneous Sensing of Oxygen and Metabolic State

Oxygen concentration has a large influence of the metabolism of a cell [35, 36]. In fact, the normal / tumor threshold of 0.7 for the metabolic indicator, a1, strictly applies only for oxygen concentrations in the normal physiological range. Consequently, there is large interest to obtain images of the oxygen concentration and the metabolic state simultaneously. This is exactly what the bh FLIM / PLIM technique has been designed for. The technique is based on modulating a ps diode laser synchronously with the pixel clock of the scanner [1, 25]. FLIM is recorded during the On time, PLIM during the Off time of the laser. The SPCM software delivers separate images for the fluorescence and the phosphorescence which are then analysed with SPCImage NG FLIM/PLIM analysis software [5]. An example is shown in Fig. 84.

Fig. 84: Yeast cells stained with (2,2-bipyridyl) dichlororuthenium (II) hexahydrate. FLIM and PLIM image, decay curves in selected spots.

Software

64-bit Data Acquisition Software

The DCS‑120 FLIM systems use the bh SPCM 64 data acquisition software [1]. SPCM runs the data acquisition in the various operation modes of the SPC modules while controlling peripheral devices, such as detectors, lasers, scanners, or motor stages. Operation modes are available for almost any conceivable TCSPC application. There are modes for fluorescence and phosphorescence decay recording, multi-wavelength decay recording, laser-wavelength multiplexing, recording of time series, FCS and photon counting histograms, and there are modes for FLIM, multi-wavelength FLIM, Mosaic FLIM, time-series FLIM, Z stack FLIM, and simultaneous FLIM/PLIM. Since July 2019 SPCM comes with extended multi-threading capabilities, greatly improving the throughput rate even in case of complex online data and display operations. A direct link is provided for communication with SPCImage NG FLIM analysis 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 [1].

The Main Panel of SPCM is configurable by the user. Different configurations can thus be created for different applications and measurement tasks. The configurations can be stored in a Predefined Setup panel and recalled on demand by a single mouse click. A few typical configurations for FLIM systems are shown in Fig. 85. A 154-page description of the SPCM software is available in [1].

Fig. 85: Main panel of SPCM software. Configurations for dual-channel FLIM, multi-wavelength FLIM, single-channel MCARO FLIM, multi-wavelength curve mode.

Easy Change Between Instrument Configurations

Frequently used instrument configurations are stored in a Predefined Setup panel. Changing between the different configurations and user interfaces is just a matter of a single mouse click, see Fig. 86.

Fig. 86: Changing between different instrument configurations: The DCS-120 system switches from a FLIM configuration into an FCS configuration by a simple mouse click

Integrated Scanner Operation

Scanner control is fully integrated in the data acquisition software. It includes definition of the frame size, pixel numbers, scan speed, and zoom factor. A fast preview mode is provided for sample setup and focus tuning. For a detailed description please see [1] and [2].

Fig. 87: Scanner Control Panel of the DCS-120 system

Interactive Scanner Control

The zoom factor and the position of the scan area can be adjusted via the scanner control panel or via the cursors of the display window. Changes in the scan parameters are executed online, without stopping the scan. The result becomes immediately visible in the preview images.

Fig. 88: Interactive scanner control

Automatic Scanner Speed

The DCS-120 scanner control automatically selects the maximum speed of the scanner. Unless otherwise selected, the scanner thus always runs at the highest possible pixel rate, resulting in fast acquisition, minimum triplet excitation, and minimum photobleaching.

Fig. 89: Automatic selection of scan speed

Integrated Laser Control

Control of up to four lasers is implemented in the scanner control panel. It includes selection of an active laser, beam blanking during the line and frame flyback, intensity control, and laser multiplexing.

Fig. 90: Laser control part of the scan control panel

Integrated Control of Ti:Sa Laser and AOM

In DCS-120 Super MPC systems and in Ti:Sa-based DCS-120 MP systems control of the Ti:Sa Laser and the AOM is integrated in the SPCM software. The control panel is shown in Fig. 91.

Fig. 91: Ti:Sa laser and AOM control panel.

Integrated Control of Scan Stages

Control of motorised sample stages and Z drives of microscopes is integrated in the DCS operating software. Please see Fig. 92

Fig. 92: Control of motorised sample stage and Z drive of Axio Observer

SPCImage NG FLIM Data Analysis

The DCS-120 system uses SPCImage NG FLIM data analysis. SPCImage NG is a new generation of bh's legendary SPCImage TCSPC-FLIM data analysis software. It combines time-domain and frequency-domain analysis, uses a maximum-likelihood (MLE) algorithm to calculate the parameters of the decay functions in the individual pixels, and accelerates the analysis procedure by GPU processing. 1D and 2D parameter histograms are available to display the distribution of the decay parameters over the pixels of the image or over selectable ROIs. Image segmentation can be performed via the phasor plot. Pixels with similar phasor signature can be combined for high-accuracy time-domain analysis. SPCImage NG provides decay models with one, two, or three exponential components, incomplete-decay models, and shifted-component models. Another important feature is advanced IRF modelling, making it unnecessary to record IRFs for the individual FLIM data sets. For a detailed description please see [1, 2, 5], and SPCImage NG Overview Brochure [6]. A typical main panel of SPCImage NG is shown in Fig. 93.

Fig. 93: Example of SPCImage NG main panel. Combination of time-domain analysis (left and lower right) and phasor plot (upper right)

Deconvolution and Fit Procedure

SPCImage NG runs an iterative fit and de-convolution procedure on the decay data of the individual pixels of the FLIM images. In the simplest case, the result is the lifetime of the decay functions in the individual pixels. For complex decay functions the fit procedure delivers the lifetimes and amplitudes of the decay components. SPCImage then creates colour-coded images of the amplitude- or intensity-weighted lifetimes in the pixels, images of the lifetimes or amplitudes of the decay components, images of lifetime or amplitude ratios, and images of other combinations of decay parameters, such as FRET intensities, FRET distances, bound-unbound ratios, or the fluorescence-lifetime redox ratio, FLIRR. A few examples are shown in Fig. 94 and Fig. 95. For details please see [1, 2, 5].

Fig. 94: Cell with interacting proteins, labelled with a FRET donor and a FRET acceptor. Left to right: Classic FRET efficiency, fraction of interacting donor, FRET distance

Fig. 95: Metabolic FLIM. Bound-unbound ratio of NADH, Bound/unbound ratio of FAD, Fluorescence-Lifetime Redox Ratio, FLIRR.

GPU Processing

SPCImage NG uses GPU (Graphics Processor Unit) processing. GPU processing is running on NVIDIA cards and a number of other NVIDIA-compatible devices. The TCSPC data are transferred into the GPU, which then runs the de-convolution and fit procedure for a large number of pixels in parallel. This way, data processing times for large images are reduced from formerly more than 10 minutes to a few seconds.

Maximum-Likelihood Algorithm

Unlike earlier SPCImage versions, SPCImage NG uses a maximum-likelihood algorithm (or maximum-likelihood estimation, MLE) for fitting the data. In contrast to the usual least-square fit, the MLE algorithm takes into account the Poissonian distribution of the photon numbers. Compared to the least-square method, the fit accuracy is improved especially for low photon numbers, and there is no bias toward shorter lifetime as it is often observed for the least-square fit. For comparison with older data sets the weighted least-square fit and the first-moment algorithms of the previous SPCImage versions are still available in SPCImage NG.

Instrument-Response Function

SPCImage NG avoids troublesome recording of an instrument response function (IRF) for each FLIM measurement. This is achieved by modelling the IRF with a generic function. The parameters of this function are determined by fitting it to the FLIM data together with the selected decay model. The results of this procedure are so good that an accurate IRF is obtained even for decay functions containing ultra-fast components, see Fig. 96. For details please see [5].

Fig. 96: Synthetic IRF. Left: Autofluorescence of cells, ps diode laser, HPM-100-40. Right: Sample with extremely fast decay component, femtosecond fibre laser, HPM-100-06

Global Analysis

Global analysis assumes that one or several of the lifetimes of a multi-exponential decay are constant in all pixels of a FLIM image. By using this a priori knowledge, the decay parameters can be obtained at a much better accuracy than by a multi-exponential fit with all decay parameters floating. A global fit is enormously computation-intensive. SPCImage therefore uses a Global Fit algorithm based on GPU processing [24]. GPU processing reduces the calculation time from previously hours to a few minutes and less. The global fit is available in SPCImage NG starting from version 9.073.

Phasor Plot

SPCImage NG combines time-domain multi-exponential decay analysis with a phasor plot. In the phasor plot, the decay data in the individual pixels are expressed as phase and amplitude values in a polar diagram [31]. 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. 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 [5, 28]. An example is shown in Fig. 97.

Fig. 97: Combination of time-domain analysis with phasor plot. Left to right: Lifetime image, phasor plot, decay curve of combined pixels within selected phasor range

Image Segmentation

SPCImage NG provides automatic image segmentation functions via the phasor plot and 2D histograms of the decay parameters. Areas with different decay signature form separate clusters in these presentations. Interesting clusters can be selected and back-annotated in the images. The decay data of the corresponding pixels are combined into a single decay curve with extremely high photon number. Multi-exponential decay analysis on the combined data delivers precision decay parameters even if the photon numbers in the individual pixels are low. An example is shown in Fig. 98.

Fig. 98: Image segmentation on temporal-mosaic FLIM data of a live water flee

Single-Curve Analysis

SPCImage NG can be used to analyse single decay curves, The data can come from traditional cuvette experiments, or from the combined pixels of a FLIM recording. An example is shown in Fig. 99.

Fig. 99: SPCImage used for fluorescence decay analysis of single curves. NADH dissolved in water, recorded with bh DCS-120 MP FLIM system.

References

1. W. Becker, The bh TCSPC Handbook. 10th edition. Becker & Hickl GmbH (2023), available on www.becker-hickl.com, printed copies available from bh

2. Becker & Hickl GmbH, DCS-120 Confocal and Multiphoton FLIM Systems, user handbook, 10th edition 2026. www.becker-hickl.com, printed copies available from bH

3. W. Becker, Adva nced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005

4. W. Becker, Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York (2015

5. SPCImage NG next generation FLIM data analysis software. In: W. Becker, The bh TCSPC Handbook, 10th ed., (2023). Available on www.becker-hickl.com.

6. Becker & Hickl GmbH, SPCImage NG next generation FLIM data analysis software. Overview brochure, available on www.becker-hickl.com

7. W. Becker, Bigger and Better Photons: The Road to Great FLIM Results. Education brochure, available on www.becker-hickl.com.

8. W. Becker, Introduction to Multi-Dimensional TCSPC. In W. Becker (ed.) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York (2015)