Becker & Hickl GmbH

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October 2018

 

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.

 

 

 

 

FLIM Systems for Laser Scanning Microscopes

 

General Features

Principle

The FLIM systems are based on bh's multi-dimensional time-correlated single photon counting (TCSPC) process in combination with confocal or multiphoton scanning by a high-frequency pulsed laser beam. 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 of its detection. The recording process builds up a photon distribution over these parameters. The result is an array of pixels, each containing a full fluorescence decay curve in a large number of time channels [20, 26, 27].

Fig. 1: bh's multi-dimensional TCSPC FLIM process probes the sample by randomly emitted photons

The principle shown in Fig. 1 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. The principle of multi-wavelength FLIM is shown in Fig. 2.

Fig. 2: 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.

The technique works at even the fastest scan rates available in laser scanning microscopes, combines near-ideal photon efficiency, excellent time resolution, excellent timing stability, fast recording speed, multi-wavelength capability, and resolution of multi-excellent decay functions into their components with optical sectioning capability and suppression of lateral scattering [23, 26]. The systems are live-cell and live-tissue compatible, able to record fast physiological effects in the sample, record spatial mosaics and Z stacks of FLIM images, and to simultaneously record fluorescence and phosphorescence lifetime images.

The bh FLIM systems are using 64-bit data acquisition software [27]. As a result, images with extremely high spatial and temporal resolution can be recorded. Images can be large as 2048 x 2048 pixels with 256 time channels per pixel, or 1024 x 1024 pixels with 1024 time channels. Such images cover the full field of view of even the best microscope lenses at diffraction-limited resolution. Multiwavelength FLIM is possible with 16 wavelength intervals and up to 512 x 512 pixels and 256 time channels.

Data Acquisition Hardware

The bh FLIM system contain one or several (usually two) TCSPC FLIM modules, a detector controller, and, if the bh DCS-120 scan head is used, a scan controller module. The modules can be operated inside a PC, or in an extension box connected to a laptop computer, see Fig. 3.

                        

Fig. 3: Left: PC-based FLIM system, shown with DCS-120 scan head, BDL-SMC picosecond diode laser, and HPM-100 hybrid detectors. Middle: Simple-Tau 152 dual-channel FLIM system, containing tw0 SPC-150 TCSPC FLIM cards and a DCC-100 detector controller. Right: Simple-Tau II system, containing two SPC-160pcie TCSPC FLIM cards and a DCC-100pcie detector controller.

bh FLIM systems are compatible with almost any high-frequency pulsed excitation source. These can be Ti:Sa lasers of multiphoton microscopes, super-continuum lasers, or pulsed fibre lasers. For FLIM with scanning microscopes that have no pulsed excitation source bh deliver a wide range of picosecond diode lasers.

Most bh FLIM systems are using the bh HPM-100 hybrid detectors [22]. However, the system also work with single-photon avalanche diodes (SPADs), with InGaAs SPADs [3], with conventional PMTs [4], with MCP PMTs [19], and even with superconducting NbN detectors [7]. bh TCSPC FLIM hardware delivers unsurpassed time resolution and timing stability. Time-channel widths down to 405 fs are feasible, and the electronic jitter is less than 3 ps rms. With ultra-fast detectors fluorescence lifetimes in the 20 ps range can be resolved. Please see [27] for details.

 

Data Acquisition Software

The bh FLIM systems use bh SPCM data acquisition software [27]. 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.

The main panel of the SPCM data acquisition software is configurable by the user. Four configurations for FLIM systems are shown in Fig. 4. 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. Frequently used operation modes and user interface configurations are selected from a panel of predefined setups.

  

  

Fig. 4: 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 Analysis

All bh FLIM systems use bh SPCImage data analysis software. 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.

The main panel of the SPCImage data analysis is shown in Fig. 5. It shows a lifetime image caculated from the decay data in the pixels (left), a lifetime distribution over the pixels of a region of interest (upper middle), 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. Please see [1, 2] or [27] for a detailed description of FLIM data analysis.



Fig. 5: Main panel of the SPCImage data analysis

Since 2018 SPCImage combines time-domain analysis with the phasor plot [29]. Pixels with different decay profiles are represented as different clusters of phasors in the phasor plot. Cells of different lifetimes therefore form separate clusters of phasors marked with different colours in the phasor plot, see Fig. 6, right.

     

Fig. 6: SPCIMage, lifetime image and 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.

Pixels with similar phasor signature can be combined, and the combined data be used for high-accuracy multi-exponential decay analysis. Please see chapters ‘SPCImage Data Analysis’ in [1, 13, 27].

FLIM Functions in Brief

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. 7.

Fig. 7: Changing between different instrument configurations: The software switches from a FLIM configuration into an FCS configuration by a simple mouse click

Interactive Scan Control

Any change in the scan area of the microscope immediately becomes effective in the recorded images.

Fig. 8: Interactive scanner control for external microscope software. Example for Zeiss LSM 780/880.

For systems using the GVD-120 (such as the bh DCS 120 system) the control of the scanner is integrated in the SPCM data acquisition software. 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.

Fig. 9: Interactive scanner control for systems using the bh GVD-120 scan controller module

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 bh FLIM systems have a fast preview function. The preview function displays images in intervals of 1 second and faster. Both intensity and lifetime images can be displayed. The preview function can be combined with fast online-FLIM display, please see Fig. 23, page 15.



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

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 [27]. There is no intensity or lifetime crosstalk. Even if one channel overloads the other channel is still able to produce correct data. More parallel channels can be added if necessary, please see [27].

        

Fig. 11: 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.

Megapixel FLIM Images

With 64 bit SPCM software pixel numbers can be increased to 2048 x 2048 pixels, with a temporal resolution of 256 time channels. Two such images can be recorded simultaneously in different wavelength channels.

Fig. 12: BPAE cells, recorded with a spatial resolution of 2048 x 2048 pixels. 256 time channels per pixel.

 

 

Multiphoton NDD FLIM: Clear Images from Deep Tissue Layers

bh FLIM systems for multiphoton microscopes are compatible with non-descanned detection (NDD). With non-descanned detection, fluorescence photons scattered on the way out of the sample are detected efficiently and assigned to the correct pixels of the image. The result is that bright and clear images are obtained from deep tissue layers. An example is shown in Fig. 13.

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Fig. 13: 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.

Ultra-High Time Resolution: FLIM with <20ps IRF width

In combination with the ultra-fast HPM-100-06 and -07 detectors, bh multiphoton FLIM systems system achieve an instrument response function (IRF) of less than 20 ps FWHM [8]. 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 imaging. In the past few years the field has been rapidly expanding [27]. Metabolic FLIM reqires separation of the decay components bound and unbound NADH. Typical NADH FLIM images of the amplitude-weighted lifetime and of the amplitudes and liftimes of the fast and slow decay component are shown in Fig. 14 and Fig. 15. Please see [10] for details.

   

Fig. 14: 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. Time-channel width 10 ps.

  

Fig. 15: 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.

Multi-Spectral FLIM

bh FLIM systems are able to record simultaneously in 16 wavelength channels. The images are recorded by an extended multi-dimensional TCSPC process which uses the wavelength of the photons as a coordinate of the photon distribution [21, 27]. An example is shown in Fig. 16.

Fig. 16: Multi-wavelength FLIM, 16 images with 512 x 512 pixels and 256 time channels were recorded simultaneously. bh DCS‑120 confocal scanner, bh MW-FLIM GaAsP 16-channel detector, Zeiss Axio Observer microscope.

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. The individual images in the 16 wavelength channels are recorded at a resolution of up to 512x512 pixels and 256 time channels.

Fig. 17 and Fig. 18 demonstrate the true resolution of the data. Images from two wavelength channels, 502 nm and 565 nm, were selected form the data shown Fig. 16, 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 that normally used for single-wavelength FLIM.

           

Fig. 17: Two images from the array shown in Fig. 16, displayed in larger scale and with individually adjusted lifetime range. The images have 512 x 512 pixels and 256 time channels.

         

Fig. 18: 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.

 

Multiphoton Multispectral NDD FLIM

bh’s MW FLIM is the world’s first simultaneously detecting multiphoton multispectral NDD FLIM system [21]. It uses a special optical interface that connects the NDD ports of multiphoton microscopes to the input slit of the detector [1, 2, 27]. A typical result is shown in Fig. 19.



Fig. 19: Multiphoton Multispectral NDD FLIM. Plant tissue, lifetime images and decay curves in selected pixels and wavelength channels. Recorded with LSM 710 NLO and bh MW FLIM detector

 

Z Stack Recording by Record-and-Save Procedure

The bh FLIm systems are able to record Z stacks of FLIM images [1, 27]. For each Z plane, a FLIM image is scanned and acquired for a specific ‘collection time’. Then the data are saved in a file, the microscope steps to the next plane, and the next image is acquired. The procedure continues for a specified number of Z planes. A Z stack of autofluorescence images taken at a water flee is shown in Fig. 20.

Fig. 20: Z stack recording, part of a water flee, autofluorescence. Images 256x256 pixels, 256 time channels.

Another way of recording Z stacks is by Mosaic FLIM. In that case, the images of the individual planes are recorded in subsequent elements of a FLIM data mosaic. Please see ‘Z Stack Mosaic FLIM’, page 15.

 

Lateral 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 [27]. The individual images within this array can be for different displacement of the sample (lateral mosaic), different depth within the sample (z-stack mosaic), of for different times after a stimulation of the sample (temporal mosaic). Lateral mosaic FLIM combines favourably with the Tile Imaging capability of the Zeiss LSM 710/780/880 and similar procedures in other microscopes. An example is shown in Fig. 21. 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 higher numerical aperture can be used, resulting in higher detection efficiency and higher spatial resolution.

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Fig. 21: 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 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 image planes the FLIM system records the data into consecutive elements of a FLIM mosaic. The advantage over the traditional record-and-save procedure (page 13) is that no time has to be reserved for save operations, and that the entire array can be analysed in a single data analysis run.

Fig. 22: 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.

Fast Online FLIM

The bh TCSPC/FLIM systems record and display fluorescence lifetime images at a rate of up to 10 images per second [11, 14]. The function is normally used to select interesting cells within a larger sample for subsequent high-accuracy FLIM acquisition. In FLIM experiments with longer acquisition time it helps the user evaluate the signal-to-noise ratio of the data and decide whether enough photons have been recorded to reveal the expected lifetime effects in the sample.

Fig. 23: Fast online FLIM. Intensity image (left) and lifetime image (right). Images 128 x 128 pixels, recorded at a speed of 5 images per second.

Time-Series FLIM by Record-and-Save Procedure

Time-series FLIM is available for all system versions, and all detectors [1, 2, 27]. Time series as fast as 2 images per second can be obtained. A time series taken at a moss leaf is shown in Fig. 24. Time-series FLIM at higher speed can be performed by temporal mosaic FLIM, see Fig. 25 and Fig. 26. Time-series FLIM can be combined with online-FLIM display, please see section above.

Fig. 24: Time-series FLIM, 1 image per second. Chloroplasts in a leaf, the fluorescence lifetime of the chlorophyll decreases with the time of exposure.

Time-Series Recording by Temporal Mosaic FLIM

Mosaic FLIM can be used to record FLIM time series. The recording principle is the same as for lateral mosaic FLIM, except for the fact that the sample is not moved between the individual recordings. The result is thus a mosaic of FLIM images for consecutive times after the start of an experiment. An example is shown in Fig. 25.

Fig. 25: 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.

Faster than Fast FLIM: Temporal Mosaic FLIM with Triggered Accumulation

The advantage of Mosaic FLIM is that no time has to be reserved for save operations between the recording of the individual images. 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 many laser scanning microscopes frame times of less than 50 milliseconds can be achieved [28]. This brings the transient-time resolution down to the range where physiological effects in live samples occur. A typical application is the recording of  Ca2+ transients in neurons. An example is shown in Fig. 26.

Fig. 26: 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.

FLITS: Fluorescence Lifetime-Transient Scanning

FLITS records transient 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 [24].

      

Fig. 27: 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.

The bh FASTAC Fast-Acquisition FLIM System

The bh Fast-Acquition FLIM system uses four parallel TCSPC channels and a device that distributes the photon pulses of a single detector into the four recording channels [15, 16, 17]. The system features an electrical IRF width of less than 7 ps (FWHM), and a time channel width down to 820 fs. The optical time resolution with an HPM-100-06 or -07 hybrid detector is shorter than 25 ps (FWHM). The system is virtually free of pile-up effects. FLIM data can be recorded at acquisition times down to the fastest frame times of the commonly used galvanometer scanners. The data are recorded with the TCSPC-typical number of time-channels of up to 4096, and with pixel numbers from 128 x 128 to 2048 x 2048 pixels. The system is therefore equally suitable for fast FLIM and precision FLIM applications.

         

Fig. 28: FASTAC FLIM. Left: 256x256 pixels, acquisition time 0.5 s. Insert: Decay data in 10x10 pixel area. Right: IRF



Fig. 29: High-accuracy FLIM image, recorded in 10 seconds. 1024 x 1024 pixels, 1025 time channels. FASTAC FLIM system with Zeiss LSM 880 NLO multiphoton microscope.

Excitation Wavelength Multiplexing

By multiplexing several ps diode lasers images can be obtained quasi-simultaneously for different excitation wavelength [27]. With the two detection channels of the bh systems, images for three or four combinations of excitation and emission wavelength are obtained. An example is shown in Fig. 30.

Fig. 30: 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.

Near-Infrared FLIM

Scattering coefficients in biological tissue in the near-infrared region are lower than in the visible. Therefore, FLIM with near-infrared dyes is a second way to obtain images from deep layers of biological tissues. Different than for multiphoton FLIM, where only the excitation is in the NIR, both the excitation and the emission are in the near infrared. Therefore, deep-tissue imaging is possible even with one-photon excitation and confocal detection. Moreover, many near-infrared dyes display large lifetime variations with the local molecular environment and are thus potential molecular markers. Near-infrared FLIM can be performed by one-photon excitation with ps diode lasers, by one-photon excitation with Ti:Sapphire lasers, or two-photon excitation by an OPO [5, 25]. Please see Fig. 31, Fig. 32 and Fig. 33.

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Fig. 31: Near-Infrared FLIM with ps diode laser, bh DCS-120 system. Pig skin sample stained with 3,3’-diethylthiatricarbocyanine, detection wavelength from 780 nm to 900 nm.

        

Fig. 32: Pig skin samples stained with 3,3’-diethylthiatricarbocyanine. Zeiss LSM 780 NLO system, one-photon excitation by Ti:Sa laser at 780nm, confocal detection at 800nm to 900nm

Fig. 33: Pig skin stained with Indocyanin Green. Zeiss LSM 780 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).

FLIM / PLIM: Simultaneous Fluorescence and Phosphorescence Lifetime Imaging

Phosphorescence and fluorescence lifetime images are recorded simultaneously by bh’s proprietary FLIM/PLIM technique. The technique is based on modulating a ps diode laser synchronously with the pixel clock of the scanner. FLIM is recorded during the ‘On’ time, PLIM during the ‘Off’ time of the laser [9, 27, 28, 32]. The SPCM software delivers separate images for the fluorescence and the phosphorescence which are then analysed with SPCImage FLIM/PLIM analysis software.

Currently, there is increasing interest in PLIM for background-free recording and, especially, for oxygen sensing. In these applications, the bh technique delivers a far better sensitivity than PLIM techniques based on single-pulse excitation. The real advantage of the bh FLIM/PLIM technique is, however, that FLIM and PLIM are obtained simultaneously. It is thus possible to record metabolic information via FLIM of the NADH and FAD fluorescence, and simultaneously map the oxygen concentration via PLIM [30, 32]. The number of publications in this area is literally exploding, please see FLIM/PLIM chapters in [1], [2] or [27]. An example is shown in Fig. 34.



Fig. 34: Yeast cells stained with (2,2’-bipyridyl) dichlororuthenium (II) hexahydrate. FLIM and PLIM image, decay curves in selected spots.

FLIM of Macroscopic Objects

With the bh DCS-120 MACRO version objects as large as 15 mm can be scanned [2]. Image obtained with the DCS-120 MACRO is shown in Fig. 35 and Fig. 36.

       

Fig. 35: FLIM of a macroscopic object. Resolution 2048 x2048 pixels, 256 time channels. Left: Original image. Right: digital zoon into recorded FLIM image, showing the excellent resolution of the data.

                    

Fig. 36: Tumor in a live mouse. NADH FLIM image (left) and decay curves inside and outside tumor (right).

Scanning of Well Plates

With an optional motor stage, both the DCS-120 and the DCS MACRO system can be used to scan well plates. An example is shown in Fig. 36.

             

Fig. 37: Well plate scanned with DCS-120 MACRO. Lifetime image and decay functions in wells 4 and 5, lower row.

STED FLIM

TCSPC FLIM can be combined with STED [27]. The combination of a STED microscope of Abberior Instruments (Göttingen, Germany) with the bh Simple-Tau 150/154 TCSPC FLIM system records FLIM data at a spatial resolution of better than 40 nm. The image format can be as large as 2048 x 2048 pixels, with 256 time channels per pixel. An image area of 40 x 40 micrometers can thus be covered with 20 nm pixel size, fully satisfying the Nyquist criterion. With smaller numbers of time channels even larger pixel numbers are possible. The system especially benefits from Windows 64 bit technology used both in the Abberior and in the bh data acquisition software, from the combined processing power of two parallel system computers, and the high data throughput of up to four parallel TCSPC FLIM channels. The system achieves peak count rates in excess of 5 MHz per FLIM channel, resulting in unprecedented signal-to-noise ratio and short acquisition time.

      

Fig. 38: STED FLIM with Abberior Intruments STED microscope. 2048 x 2048 pixels. Single cell, stained with tubulin-binding dye, recorded at a resolution of 20 nm per pixel. Decay curve in selected pixel shown on the right. The initial peak is undepleted fluorescence. It is gated off in the intensity data of image shown on the left.

 

Clinical FLIM

Clinical FLIM applications use the fact that pathological processes induce changes in the molecular environment or in the conformation of endogenous fluorophores. These, in turn, cause detectable changes in the fluorescence decay profiles. bh FLIM has been introduced into clinical instruments for opthalmology and dermatology. Developments for other applications are in progress. Please see [26] or [27] for an overview and for technical details. The first clinical instruments are on the market. FLIM images recorded with the FLIO Fluorescence Fifetime Ophthalmoscope of Heidelberg Engineering and with the MPT Flex multiphoton skin tomography system of Jenlab are shown in Fig. 37.

                                                           

Fig. 39: Left: FLIM of a human retina, recorded in vivo with Heidelberg Engineering FLIO ophthalmoscope. Right: Multiphoton FLIM of human skin, recorded in vivo with Jenlab MPT FLEX multiphoton tomography system.

FCS

The bh GaAsP hybrid detectors of the bh FLIM systems deliver highly efficient FCS [1, 22, 27].  Because the detectors are free of afterpulsing there is no afterpulsing peak in the autocorrelation data. Thus, accurate diffusion times and molecule parameters are obtained from a single detector. Compared to cross-correlation of split signals, correlation of single-detector signals yields a four-fold increase in correlation efficiency. The result is a substantial improvement in the SNR of FCS recordings [22, 27]. FCS is be obtained both with confocal systems and with multiphoton NDD systems. Gated FCS is obtained by hardware gating the photon times within the TCSPC modules, FCCS by cross-correlating the signals of two TCSPC channels.

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Fig. 40: FCS with bh TCSPC FLIM systems, GaAsP hybrid detectors. Left to right: Confocal FCS with ps diode laser,  two-photon NDD FCS, cross correlation of photons recorded in different detection channels.

 

bh FLIM Systems for Various Microscopes

DCS-120 Confocal Scanning FLIM Systems

       

 

FLIM with up to 2048 x 2048 pixels

Complete Confocal Laser Scanning FLIM microscopes

FLIM upgrade for existing conventional microscopes

Includes scanner with fast galvanometer mirrors

Two fully parallel confocal detection channels

One or two BDL-SMC or BDL-SMN picosecond diode lasers

Laser wavelengths 375, 405, 440, 473, 488, 510, 640, 685, 785 nm

Channel separation by dichroic or polarising beamsplitters

Individually selectable pinholes, individually selectable filters

GaAsP hybrid detectors for visible range, GaAs hybrid detectors for NIR range

16-channel multi-wavelength GaAsP detector module

Z-stack FLIM acquisition with Zeiss Axio Observer Z1

Simultaneous fluorescence and phosphorescence lifetime imaging (PLIM)

Optional motor stage, control integrated in instrument software

Spatial and temporal mosaic FLIM

Ultra-fast recording of time series

Fluorescence lifetime-transient scanning (FLITS)

Wideband (WB) version, compatible with tuneable lasers

FASTAC FLIM version available

Please see [2] for details

 

 

DCS-120 MP Multiphoton FLIM Systems

       

Multiphoton version of DCS-120 scanning system

Excitation by fs Ti:Sa laser or fs fibre laser

Laser intensity and wavelength control integrated in SPCM data acquisition software

PLIM laser modulation by DCS-120 scan controller and AOM, control functions integrated in instrument software

Clear Images from deep tissue layers

Excellent spatial and temporal resolution

Full field of view of microscope lens scanned

Images up to 2048 x 2048 pixels, 256 time channels

Two non-descanned detection channels, plus two optional confocal channels

bh HPM-100-40 GaAsP hybrid detectors

Optional HPM-100-06 detectors for ultra-high time resolution

Optional 16-channel multi-wavelength GaAsP detector module

Z-stack FLIM acquisition with Zeiss Axio Observer Z1

Simultaneous fluorescence and phosphorescence lifetime imaging (PLIM)

Optional motor stage, control integrated in instrument software

Spatial and temporal mosaic FLIM

Ultra-fast recording of time series

Fluorescence lifetime-transient scanning (FLITS)

FASTAC FLIM system available

Please see [2] for details

 

 

DCS-120 Macro System

     

FLIM of macroscopic objects

Scan field up to 15 mm diameter

FLIM with up to 2048 x 2048 pixels

Scanning by fast galvanometer mirrors

Two fully confocal detection channels

One or two BDL-SMC or BDL-SMN picosecond diode lasers

Laser wavelengths 375, 405, 440, 473, 488, 510, 640, 685, 785 nm

Tuneable excitation by super-continuum laser with AOTF

One or two confocal detection channels, parallel acquisition

Channel separation by dichroic or polarising beamsplitters

Individually selectable pinholes, individually selectable filters

GaAsP hybrid detectors for visible range, GaAs hybrid detectors for NIR range

16-channel multi-wavelength GaAsP detector module

Simultaneous fluorescence and phosphorescence lifetime imaging (PLIM)

Optional motor stage, control integrated in instrument software

Spatial and temporal mosaic FLIM

Ultra-fast recording of time series

Fluorescence lifetime-transient scanning (FLITS)

Wideband (WB) version, compatible with tuneable lasers

FASTAC FLIM system available

Please see [2] for details

 

 

 

 

FLIM Systems for Zeiss LSM 710 / 780 / 880 Microscopes

LSM 710 / 780 / 880 NLO, LSM 7MP Multiphoton Microscopes

LSM 710, LSM 780, LSM 880 Confocal Microscopes

FLIM with up to 2048 x 2048 pixels

Multiphoton FLIM, PLIM, multispectral FLIM, FCS

Confocal FLIM, PLIM, multispectral FLIM, FCS

FLIM with bh HPM hybrid detectors or Zeiss BIG-2 detectors

Fast preview mode, both for intensity and lifetime

Mosaic FLIM

Z Stack FLIM

Fast Time-series FLIM

Acquisition by 1, 2, 3 or 4 parallel TCSPC FLIM channels

Detection by bh HPM-100  hybrid detectors or Zeiss BIG 2 detector

Simultaneous fluorescence and phosphorescence lifetime imaging (PLIM)

Fluorescence lifetime-transient scanning (FLITS)

Spatial and temporal mosaic FLIM

Ultrafast time-series recording by temporal mosaic FLIM function

Confocal NIR FLIM up to 900 nm detection wavelength

Two-Photon OPO FLIM up to 900nm detection wavelength

FASTAC FLIM system available

Please see [1] for details

 

Still available: FLIM Systems for Zeiss LSM 510 NLO Multiphoton Microscopes

           

FLIM with up to 2048 x 2048 pixels

Multiphoton excitation with non-descanned detection

Detectors connected to Zeiss NDD switch box

Single-wavelength NDD FLIM

Dual-wavelength NDD FLIM

Multi-spectral NDD FLIM

Fast preview mode

Mosaic FLIM

Z Stack FLIM

Fast time-series FLIM

HPM‑100‑40 hybrid detectors

One or two parallel SPC‑150 TCSPC channels

PC-based systems or Simple-Tau TCSPC systems

Portable to LSM 710, 780, 880 microscopes

Software-Integrated FLIM for Nikon A1+ Confocals

 

 

Integrated in Nikon’s NIS-Elements Instrument Software

Excitation by bh BDS-SM ps diode lasers

Detection by bh HPM-100-40 GaAsP hybrid detectors

Two fully parallel SPC-150N TCSPC FLIM channels

Data analysis by bh SPCImage

Fast acquisition, high optical resolution, high efficiency, high time resolution

Please see [18] for details

 

 

 

 

Non-descanned FLIM Systems for Nikon A1 MP Multiphoton Microscopes

         

 

64-bit megapixel FLIM technology

One FLIM channel or two parallel FLIM channels

High-efficiency PMH-100 hybrid detectors

Non-descanned detection for deep-tissue imaging

Multi-spectral FLIM with 16-channel GaAsP detector

ROI and Zoom functions of A1 available

Works at any scan rate

Megapixel FLIM

Fluorescence lifetime-transient scanning (FLITS)

Ultra-fast time series by temporal mosaic FLIM

FASTAC FLIM system available

 

 

FLIM Systems for Sutter Instrument MOM Microscopes

                    

Up to four parallel FLIM channels

Multiphoton excitation by Ti:Sa laser

Non-descanned detection for deep-tissue imaging

Overload protection of FLIM detectors

Up to 1024 x 1024 pixels, 1024 time channels

High efficiency

Fast acquisition

SPCM Online FLIM function available

Simultaneous FLIM / PLIM

FASTAC FLIM system available

Please see [12] for details.

 

 

Non-Descanned FLIM Systems for Leica SP5 MP,  SP8 MP Microscopes

           

 

Non-descanned detection via Leica RLD port

1 detector coupled directly to RLD port

2 detectors via external beamsplitter

Simple-Tau 150 or 152 TCSPC systems

Acquisition in 1 or 2 parallel TCSPC FLIM channels

bh HPM‑100‑40 GaAsP hybrid detectors or Leica HYD detectors

Optional HPM-100-20 ultra-fast hybrid detectors

Multi-spectral FLIM with 16-channel GaAsP detector

Works at any scan rate of SP microscope

No nonlinearity by Leica sinusoidal scan

Fast acquisition, fast preview mode

Megapixel FLIM, 2048 x 2048 pixels

Fluorescence lifetime-transient scanning (FLITS) and temporal mosaic FLIM available

Ultra-fast time series by temporal mosaic FLIM

Simultaneous FLIM / PLIM

FASTAC FLIM system available

Please see [6] for details.

Non-descanned  FLIM Systems for Olympus Multiphoton Microscopes

              

 

Multiphoton FV systems with inverted microscopes

High efficiency by non-descanned FLIM detection

Deep-tissue imaging capability

HPM-100-40 GaAsP hybrid detectors

Optional HPM-100-06 hybrid detectors for ultra-high time resolution

Optional 16-channel multi-spectral GaAsP detector

Full overload protection of FLIM detectors

ROI and Zoom functions of available

Works at any scan rate

Fluorescence lifetime-transient scanning (FLITS) and temporal mosaic FLIM available

FASTAC FLIM system available

 

 

 

PZ-FLIM-110 Stage-Scanning FLIM System

  

 

Sample scanning by piezo scan stage

Excitation by BDL or BDS series ps diode lasers

Confocal detection

HPM-100-40 GaAsP  hybrid detector

Optional HPM-100-06 hybrid detetcor for ultra-high time resolution

Optional PML-SPEC GaAsP multi-spectral detector

Excellent contrast and resolution

Fully controlled by bh SPCM TCSPC/FLIM data acquisition software

Compact electronics, integrated in bh Simple Tau system

Megapixel FLIM technology - images up to 2048 x 2048 pixels

Lateral (x-y) an vertical (z) scanning

Simultaneous FLIM / PLIM

Please see [27] for details.

 

FLIM for NSOM Systems

For NSOM systems of Nanonics, MD-NDT and others

Combines atomic-force and fluorescence lifetime information

High sensitivity by HPM‑100-40 GaAsP hybrid detectors

Optional HPM-100-06 detectors for ultra-hihg time resolution

Fluorescence and phosphorescence lifetime imaging

Single-point transient-lifetime recording

Please see bh TCSPC Handbook [27]or contact bh.

 

 

FLIM Systems for Clinical Imaging

                

FLIM systems for ophthalmology

FLIM systems for dermatology

FLIM systems for tissue imaging

FLIM through endoscopes

Time-resolved NIRS and fNIRS Imaging

Online FLIM at rates of up to 10 images per second

Please see bh TCSPC Handbook [27] or contact bh

FLIM for other Scanning Systems

              

Left: FLIM recorded with Lucid Vivascope, ultra-fast polygon scanner. Right: STED FLIM recorded with STED microscope of Abberior Systems, Goettingen

 

bh FLIM systems can be configured for almost any conceivable laser scanning system. They work with galvanometer scanners, polygon scanners, resonance scanners, and motor-driven and piezo-driven scan stages.

Please see bh TCSPC Handbook [27] or contact bh.

 

References

1.      Becker & Hickl GmbH, Modular FLIM systems for Zeiss LSM 710/780/880 family laser scanning microscopes. User handbook. 7th edition (2017), available on www.becker-hickl.com, please contact bh for printed copies

2.      Becker & Hickl GmbH, DCS-120 Confocal and Multiphoton FLIM Systems, user handbook, 7th edition (2017). Available on www.becker-hickl.com, please contact bh for printed copies

3.      Becker & Hickl GmbH, 80 ps FHWM Instrument Response with ID230 InGaAs SPAD and SPC 150 TCSPC Module. Application note, www.becker-hickl.com

4.      Becker & Hickl GmbH, Zeiss BiG 2 GaAsP Detector is Compatible with bh FLIM Systems. Application note, www.becker-hickl.com

5.      Becker & Hickl GmbH, Multiphoton NDD FLIM at NIR Detection Wavelengths with the Zeiss LSM 7MP and OPO Excitation. Application note, www.becker-hickl.com

6.      Becker & Hickl GmbH, Multiphoton FLIM with the Leica HyD RLD Detectors. Application note, www.becker-hickl.com

7.      Becker & Hickl GmbH, World Record in TCSPC Time Resolution: Combination of bh SPC-150NX with SCONTEL NbN Detector yields 17.8 ps FWHM. Application note, www.becker-hickl.com

8.      Becker & Hickl GmbH, Sub-20ps IRF Width from Hybrid Detectors and MCP-PMTs. Application note, available on www.becker-hickl.com

9.      Becker & Hickl GmbH, Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation. Application note, www.becker-hickl.com

10.    Becker & Hickl GmbH, Ultra-fast HPM detectors improve NADH FLIM. Application note, www.becker-hickl.com

11.    Becker & Hickl GmbH, SPCM Software Runs Online-FLIM at 10 Images per Second. Application note, available on www.becker-hickl.com

12.    Becker & Hickl GmbH, bh TCSPC Systems Record FLIM with Sutter MOM Microscopes. Application note, www.becker-hickl.com.

13.    Becker & Hickl GmbH, New SPCImage Version Combines Time-Domain Analysis with Phasor Plot. Application note, available on www.becker-hickl.com

14.    Becker & Hickl GmbH, New SPCM Version 9.80 Comes With New Software Functions. Application note, available on www.becker-hickl.com

15.    Becker & Hickl GmbH, Fast-Acquisition Multiphoton FLIM with the Zeiss LSM 880 NLO. Application note, available on www.becker-hickl.com

16.    Becker & Hickl GmbH, Fast-Acquisition TCSPC FLIM System with sub-25 ps IRF Width. Application note, available on www.becker-hickl.com

17.    Becker & Hickl GmbH, Fast-Acquisition TCSPC FLIM: What are the Options? Application note, available on www.becker-hickl.com

18.    Becker & Hickl GmbH, Software-Integrated FLIM for Nikon A1+ Confocals. Application note, available on www.becker-hickl.com

19.    W. Becker, A. Bergmann, M.A. Hink, K. König, K. Benndorf, C. Biskup, Fluorescence lifetime imaging by time-correlated single photon counting, Micr. Res. Techn. 63, 58-66 (2004)

20.    W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005

21.    W. Becker, A. Bergmann, C. Biskup, Multi-Spectral Fluorescence Lifetime Imaging by TCSPC. Micr. Res. Tech. 70, 403-409 (2007)

22.    Becker, W., Su, B., Weisshart, K. & Holub, O. (2011) FLIM and FCS Detection in Laser-Scanning Microscopes: Increased Efficiency by GaAsP Hybrid Detectors. Micr. Res. Tech. 74, 804-811

23.    W. Becker, Fluorescence Lifetime Imaging - Techniques and Applications. J. Microsc. 247 (2) (2012)

24.    W. Becker, V. Shcheslavkiy, S. Frere, I. Slutsky, Spatially Resolved Recording of Transient Fluorescence-Lifetime Effects by Line-Scanning TCSPC. Microsc. Res. Techn. 77, 216-224 (2014)

25.    Wolfgang Becker, Vladislav Shcheslavskiy, Fluorescence lifetime imaging with near-infrared dyes. Photon Lasers Med 2015; 4(1): 73–83

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

27.    W. Becker, The bh TCSPC handbook. 7th edition. Becker & Hickl GmbH (2017), available on www.becker-hickl.com, please contact bh for printed copies.

28.    W. Becker, V. Shcheslavskiy, A. Rück, Simultaneous phosphorescence and fluorescence lifetime imaging by multi-dimensional TCSPC and multi-pulse excitation. In: R. I. Dmitriev (ed.), Multi-parameteric live cell microscopy of 3D tissue models. Springer (2017)

29.    W. Becker, S. Frere, I. Slutsky, Recording Ca++ Transients in Neurons by TCSPC FLIM. In: F.-J. Kao, G. Keiser, A. Gogoi, (eds.), Advanced optical methods of brain imaging. Springer (2019)

30.    M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, The phasor approach to fluorescence lifetime imaging analysis, Biophys J 94, L14-L16 (2008)

31.    S. Kalinina, V. Shcheslavskiy, W. Becker, J. Breymayer, P. Schäfer, A. Rück, Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping. J. Biophotonics 9(8):800-811 (2016)

32.    H. Kurokawa, H. Ito, M. Inoue, K. Tabata, Y. Sato, K. Yamagata, S. Kizaka-Kondoh, T. Kadonosono, S. Yano, M. Inoue & T. Kamachi, High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime, Scientific Reports 5, 1-13 (2015)

33.    V. I. Shcheslavskiy, A. Neubauer, R. Bukowiecki, F. Dinter, W. Becker, Combined fluorescence and phosphorescence lifetime imaging. Appl. Phys. Lett. 108, 091111-1 to -5 (2016)

Specifications

                                                                                                            General Principle

Lifetime measurement                                                                                     time-domain

Excitation                                                                                            high-frequency pulsed lasers

Buildup of lifetime images                                           Single-photon detection by multi-dimensional TCSPC [27]

                                                                                     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 [27]

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 [27] for details

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

Number of parallel TCSPC / FLIM channels                                                        up to 4

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

Principle                                                                                            Advanced TAC/ADC principle

Electrical time resolution, IRF width, SPC-150, SPC-160                       2.3 ps rms / 6.8 ps fwhm

Minimum time channel width, SPC-150, SPC-160                                                813 fs

Electrical time resolution, IRF width, SPC-150NX                                 1.6 ps rms / 3.5 ps fwhm

Minimum time channel width, SPC-150NX                                                          405 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 [27]                                                    64                   256                 1024                4096

 

                                                                           Data Acquisition Software, please see [27] for details

Operating system                                                                            Windows 7 or Windows 10, 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 [1, 2] or [27]

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

                                                                                                              shifted-component model

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 [1] for details

Picosecond Diode Lasers                                                                 bh BDS-SM or BDL-SMC lasers

Number of lasers                                                                                                  max 4

Number of lasers simultaneously operated (multiplexed)                                         2

Available wavelengths                                     375nm, 405nm, 445nm, 473nm, 488nm, 515nm, 640nm, 685nm, 785nm

Mode of operation                                                                                 picosecond pulses or CW

Pulse width, typical                                                                                          30 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.

 

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 370nm to 785nm

 

Synchronisation / Modulation of Lasers

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

                                                                                                    DCS-120: Integrated in scan controller

                                                                                   Other microscopes: multiplexing requires bh DDG-210 card

Interleaved excitation                                                                      Sync of diode laser to diode laser

Laser Modulation for PLIM                                      Integrated in DCS system, otherwise requires bh DDG-210 card

 

                                                                                         Excitation Sources, Multi-Photon Excitation

Femtosecond NIR Lasers                         any femtosecond Ti:Sa laser, Ti:Sa-pumped OPO, or fs fibre laser

Wavelength                                                                                                 700nm to 1000nm

Repetition rate                                                                                                 40 to 80 MHz

Laser Modulation for PLIM                     integrated in DCS MP systems, otherwise requires bh DDG-210 card and AOM

 

                                                                                                                         Detectors

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

Spectral Range                                                                                                300 to 710nm

Peak quantum efficiency                                                                                   40 to 50%

IRF width, FWHM                                                                                          110 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

 

Ultra-Fast Hybrid Detectors                                                           bh HPM-100-06 hybrid detector

Spectral Range                                                                                               300 to 600 nm

Peak quantum efficiency                                                                                       20 %

IRF width (with Ti:Sa laser of fs fibre laser)                                                         <20 ps

Detector area                                                                                                         3mm

Background count rate, thermal                                                         300 to 1000 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 with bh diode laser                                                                         120 to 180 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 GaAsP FLIM assembly

Spectral range                                                                                                 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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Becker & Hickl GmbH

Nunsdorfer Ring  7-9

12277 Berlin, Berlin

Tel.  +49 / 30 / 787 56 32, Fax. +49 / 30 / 787 57 34

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