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bh News 2017-11:

DCS-120 FLIM system records X-Y mosaics

With software version 9.76, the control of a motorised sample stage has been integrated in the bh SPCM TCSPC/FLIM data acquisition software. In combination with the Mosaic FLIM function of SPCM, the sample stage can be used to record arrays of FLIM images with the bh DCS-120 confocal and multiphoton FLIM systems. The FLIM system scans an image at one position of the sample, then offsets the sample by the size of the scan area, and scans a new image. The process is repeated, combining the images of the individual frames into a single, large x-y-t data set. Images covering an area of several mm diameter can be obtained with high-NA objective lenses. Compared to a single scan with a low-magnification objective lens, a higher collection efficiency, a higher 2p excitation efficiency, and a higher optical resolution is obtained.

For details please see application note, please click here to download

Left: FLIM mosaic of BPAE cell scample, 248x248 pixels, 256 time channels. Right: Zoom into data shown left


bh News 2017-10:

New PMC-150 detector has <120 ps IRF width

The PMC-150 is a cooled PMT module for TCSPC applications. It contains a fast miniature PMT along with a Peltier cooler, a high voltage generator, a GHz pulse amplifier and a current sensing circuit. Due to the high gain and bandwidth of the device a single photon yields an output pulse with an amplitude in the range of 100 to 200 mV and a pulse width of 1.5 ns.  Due to the high gain and the efficient shielding noise pickup is minimised. Therefore the PMC-150 yields high time resolution and high counting efficiency. The TCSPC instrument response function (IRF) has a width of less than 130 ps FWHM. The IRF shift with the position at the photocathide is less than 50 ps. Overload conditions are detected by sensing the PMT output current. Overload is indicated by an LED, an acoustic signal, and a logical overload signal. The PMC 150 is operated by the bh DCC-100 detector controller card. The DCC delivers the operating voltage for the PMT, the current for the Peltier cooler, controls the detector gain, and shuts down the PMT in case of overload. Compared to its predecessor, the PMC-100, the PMC-150 has a shorter IRF width and a better IRF uniformity over the active area.

Left: IRF for 1 mm spot and for full cathode illuminated. Right: Variation of IRF with position on photocathode.

For details please see data sheet , please click here to download.


bh News 2017-05:

New ultra-fast detectors improve NADH FLIM

NADH FLIM is based on the separation of the fluorescence decay components of the bound and the unbound fraction of NAD(P)H. The amplitudes and the decay times of the components are used to derive information on the metabolic state of the cells or the tissue. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using the ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the SPC-150N and SPC-150NX TCSPC modules is less than 20 ps, see bh news 2017/04. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis virtually becomes a simple curve fitting, and the decay parameters are obtained at unprecendented accuracy.
A lifetime image of the amplitude-weighted lifetime of a double-exponential fit is shown below, upper row. The decay data in a selected spot of 9x9 pixels is shown on the right. Due to the fast response of the detector-TCSPC combination the rise of the fluorescence occurs almost instantaneously. Images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH) are shown in the second row.

Images: Upper row: NADH Lifetime image, amplitude-weighted lifetime of double-exponential fit, Decay curve in selected spot, 9x9 pixel area. Lower row: 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. HPM 100 06 detector, SPC-150N TCSPC module, Zeiss LSM 880 NLO, two-photon excitation at 750 nm.

For details please see application note ‘New ultra-fast detectors improve NADH FLIM’, please click here to download.

bh News 2017-04:

Sub-20ps IRF Width from Hybrid Detectors and MCP PMTs

Hybrid detectors and multichannel-plate (MCP) PMTs achieve a timing resolution (IRF width) of less than 20 ps FWHM when operated with the new bh SPC-150NX TCSPC modules. As a test light source, we used a Toptica FemtoFErb laser with a pulse width of 100 fs. The laser beam was directed through a package of ND filters to the photocathodes of a bh HPM-100-06 and a HPM-100-07 detector (based on Hamamatsu R10467-06 and 07 tubes) and a Hamamatsu R3809U-50 MCP PMT. In all cases, an IRF width around 20 ps FWHM and below was obtained. This is considerably shorter than previously reported for these detectors. We attribute the improvement to the superior bandwidth of the discriminators and the extremely low jitter of the timing electronics of the SPC-150NX modules.

Top to bottom: IRF of HPM-100-06, HPM-100-07, R3809U. Response to 100-fs pulse, Recorded with SPC-150NX TCSPC module. Time scale 100 ps/div, 405 fs / time channel.

So far, similar IRF widths have only be obtained with superconducting NbN detectors (see bh news 2015 / 10), and, in a few cases, with MCP PMTs operated at maximum voltage and a CFD threshold that detected only a small fraction of the photon pulses. However, NbN detectors have extremely small active areas, and MCP PMTs operated at high CFD threshold deliver poor efficiency and limited count rate. No such tradeoffs were made for the tests described here: The light was spread over an area of about 5 mm2 for the hybrid detectors, and 50 mm2 for the MCP PMT. The MCP PMT was operated at 3000 V (88% of the maximum), and at a CFD threshold that suppressed no more than 50% of the photon pulses. The maximum count rate under these conditions is several MHz. For the HPM detectors a CFD threshold was used that did not suppress any photon pulses at all.
We believe that the detector / SPC combinations described here deliver the best combination of time resolution, detection area, and sensitivity currently available.
For details please see application note ‘Sub-20ps IRF Width from Hybrid Detectors and MCP PMTs’, please click here to download.



bh News 2017-03:

SPCImage Combines Time-Domain FLIM Analysis with Phasor Plot

Version 6.0 SPCImage FLIM analysis software combines time-domain decay analysis with the phasor plot. In the phasor plot, the decay data in the individual pixels are expressed as phase and amplitude values in a polar diagram. Independently of their location in the image, pixels with similar decay signature form clusters in the phasor plot.


Different clusters can be selected in the phasor plot, 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 an accuracy comparable to that of single-point decay measurements in cuvettes. Low-amplitude decay components or decay components with almost similar lifetimes can thus be identified in the data. Examples for differently selected phasor ranges of the data shown above are given in figures below.


For details please see application note ‘New SPCImage Version Combines Time-Domain Analysis with Phasor Plot’, please click here to download.


bh News 2017-02:

bh Introduce Ultra-Fast Hybrid Detector

bh have released an ultra-fast version of their HPM-100 series hybrid detectors. The new HPM-100-07 detector has an IRF width of less than 38 ps, full width at half maximum, including the pulse width of a bh BDS-SM-405nm ps diode laser. The HPM-100-07 contains a Hamamatsu R10467-07 hybrid detector tube together with a preamplifier and the generators for the tube operating voltages in a single compact housing. The principle of the hybrid detector yields excellent timing resolution, a clean TCSPC instrument response function, high detection quantum efficiency, and extremely low afterpulsing probability. The absence of afterpulsing results in a substantially increased dynamic range of TCSPC measurements.

The HPM 100-07 module is operated via the bh DCC-100 detector controller of the bh TCSPC systems. The DCC 100 provides for power supply, gain control, and overload shutdown. The HPM 100 interfaces directly to all bh SPC or Simple Tau TCSPC systems. It is available with standard C-mount adapters, fibre adapters, adapters for the bh DCS-120 confocal scanning FLIM system, and adapters for the NDD and BIG ports of the Zeiss LSM 710/780/880 NLO multiphoton laser scanning microscopes.


Please click here for data sheet of HPM-100-07. 


BH News 2016-12

Full Set of FLIM Cards with PCI Express Interface available

bh have released a full set of PCI Express cards for TCSPC FLIM system. The set consists of one or two SPC-160pcie TCSPC / FLIM modules and a DCC-100pcie detector controller. For the bh DCS-120 scanners or for customer-specific galvanometer scanners a GVD-120pcie scan controller can be added to the system.
The system works with all the commonly used confocal and multiphoton laser scanning microscopes, and with the bh DCS-120 confocal and multiphoton systems. It records single and dual-channel FLIM, FCS, multi-wavelength FLIM, Z-stack FLIM, lateral mosaic FLIM, ultra-fast time-series FLIM and, for the DCS-120 system, simultaneous FLIM/PLIM. Online FLIM is available up to an image rate of about 10 images per second. The system is using 64-bit data acquisition software. Images as large as 2048x2048 pixels and 256 time channels can be recorded. The electronic time resolution of the SPC-160pcie is 2.5 ps rms, the minimum time channel width is 813 fs.

Please click here for data sheet of SPC-160pcie.

For general information about the bh TCSPC FLIM systems and their applications in life sciences please see bh TCSPC Handboook, click here to download.


BH News 2016-11

bh TCSPC Systems Record FLIM with Sutter MOM Microscopes

The Sutter Instrument MOM microscope is a modular platform for fluorerscence imaging deep within live samples. It uses multi-photon excitation by a titanium-sapphire laser in combination with non-descanned detection. Due to its pulsed excitation source and its high modularity the MOM system can easily be combined with the bh TCSPC FLIM systems. Up to four FLIM detectors can be attached to the system. The signals are processed in up to four entirely parallel TCSPC FLIM channels. Due to the parallel system architecture, high photon count rates and short acquisition times can be achieved. Multiphoton excitation and non-descanned detection make the system especially useful for FLIM of live cells and tissues. FLIM data can be recorded with up to 1024x1024 pixels and 1024 time channels per pixel. Typical applications are metabolic imaging by recording the fluorescence of NADH and FAD, protein interaction experiments by FLIM-FRET techniques, and ion concentration measurements with environment-sensitive fluorescent dyes.


Left: Sutter MOM with two FLIM detectors. Right: FLIM of Lepeophtheirus Salmonis, two-photon excitation at 750 nm, detection at 460 nm

(Click here for application note)


BH News 2016-09

SPC Modules Record TCSPC FLIM with Bidirectional Scanning

Starting from Version 9.73 SPCM Software, the bh TCSPC / FLIM systems are able to record FLIM with bidirectional scanning. The data are recorded in the ‘FIFO Imaging’ mode. The data acquisition is synchronised with the scanning by frame clock, line clock, and pixel clock pulses from the scanner. Each first line clock pulse indicates the beginning of a forward scan, each second one the beginning of a backward scan. The recording procedure automatically reverses the data from the backward scan and compensates for the line shift caused by the dynamic behaviour of the scanner. Bidirectional recording has been implemented for all bh TCSPC modules which have the FIFO Imaging (software accumulation) mode implemented. That means the function is available for all SPC-150, SPC-150N, SPC-160 and SPC-160pcie modules, and for SPC‑830 modules manufactured later than May 2007. The structure of the data recorded is the same as for unidirectional scanning, and the same high pixel numbers and time channel numbers can be achieved. The online intensity and lifetime image display functions of the SPCM software are available, and the recorded data can be analysed by bh SPCIMage FLIM data analysis software as usual.



FLIM recorded by bidirectional scanning. Left: FLIM of Convallaria sample, 512x512 pixels. Right: BPAE cell sample, 1024 x 1024 pixels. No blurring of image details or double structures are visible, indicating a perfect match of the forward and backward scan. bh Simple-Tau 152 FLIM system, Zeiss LSM 880 in bidirectional scan mode.

(Click here for application note)



BH News 2016-08

New SPC-160PCIE TCSPC/FLIM module has PCI Express Interface

The SPC-160PCIE is a PCI Express version of the SPC-160 TCSPC FLIM module. It features excellent time resolution of 2.5 ps rms, a minimum time channel width of 813 fs, and a dead time of only 80 ns. The new module contains the full range of functions of the SPC-160: Single and multiple curve recording, histogram and parameter-tag modes, multi-wavelength recording, FCS, FLIM, simultaneous FLIM/PLIM, multi-wavelength FLIM, and spatial and temporal mosaic FLIM. In combination with bh’s 64-bit SPCM data acquisition software images as large as 2048x2048 pixels can be can be recorded at 256 time channels per pixel. An additional fast counter channel records pixel-intensity data at a dead time of <10 ns in parallel with the FLIM images. (Click here for application note).

(Please click here for data sheet).



BH News 2016-07

New SPCM Software Runs Online-FLIM at a Rate of 10 Images per Second

With version 9.72 64-bit SPCM software, bh FLIM systems record and display fluorescence lifetime images at a rate of up to 10 images per second. The data are recorded by bh’s multi-dimensional TCSPC technique in combination with confocal or multiphoton laser scanning, the images are created by first-moment analysis. The technique combines near-ideal photon efficiency with short acquisition and calculation times. It works for all SPC-150, SPC-150N, SPC-160, and SPC-830 FLIM systems that use fast scanning. The figure below shows online-FLIM images recorded with 128x128 pixels at a rate of 10 images per second (left) and with 256x256 pixels at a rate of 2 images per second (right).

FLIM of Convallaria sample. Left: 128x128 pixels, 10 images per second. Right: 256x256 pixels, 2 image per second. Lifetime range 1ns (red) to 3ns (blue).

Please click [here] for full application note.



BH News 2016-06

Small ps diode lasers deliver high power

The Becker & Hickl BDS Series picosecond diode lasers combine small size with high power. The lasers come in industry-standard housings just 40 x 40 x 110 mm in size. Electronics is contained in the laser housing, the lasers are operated from a simple +12V wall-mounted power supply. The BDS-SM single-mode version has two internal repetition rates (20 MHz and 50 MHz) and a CW mode. Power is up to 5 mW in the pulsed mode and 50 mW in the CW mode. The pulse width is from 60 ps to 200 ps, depending on the wavelength version and the power. The lasers are available with free-beam output, with a permanently installed single-mode fibre, or with single-mode fibre couplers. The BDS-MM high-power multi-mode version delivers ps pulses at an average power up to 60 mW. It has two repetition rates, 50 MHz and 20 MHz. The BDS-MM version is available with a free-beam output or with a multi-mode fibre output. All BDS series lasers can be synchronised to an external clock source, have internal power stabilisation feedback, and fast on/off (modulation) capability.


Extended data sheet BDS-SM: (click here)
Extended data sheet BDS-MM: (click here)

The BDS series lasers can be used for a wide range of time-resolved spectroscopy and imaging applications. With their single-mode fibre capabilities the SM versions are suitable especially for FLIM / PLIM applications in laser scanning microscopy. The MM versions are targeting applications like fluorescence and phosphorescence decay measurements in cuvettes, and NIRS and fNIRS measurements in biological tissue. A few examples are shown below.



Upper row: Fluorescence decay recording (left), FLIM (right)
Lower row: Simultaneous recording of fluorescence and phosphorescence decay (left), simultaneous FLIM/PLIM (right).


BH News 2016-05

bh - Abberior Combination Records STED FLIM at Megapixel Resolution

The combination of the Abberior STED system 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. The system benefits from Windows 64 bit technology used both in the Abberior and in the bh data acquisition software, from the combined processing power of the two 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.


For uncompressed image please see application note, click [Here]


BH News 2016-04

bh FLIM / PLIM technique simultaneously records pO2 and
NAD(P)H unbound/bound ratio

The Becker & Hickl FLIM systems use a combined FLIM / PLIM technique to simultaneously record images of the oxygen concentration and of the NAD(P)H unbound/bound ratio in cells and tissues. The oxygen concentration is derived from the luminescence lifetime of a phosphorescent dye, the unbound/bound ratio from the amplitudes of the double-exponential fluorescence decay of NAD(P)H. The FLIM/PLIM technique is based on scanning with a high-frequency pulsed laser that is on/off modulated at a period in the microsecond range synchronously with the pixels of the scan. The signals are recorded by bh’s multi-dimensional TCSPC process. FLIM is obtained by building up a photon distribution over the times of the photons in the laser pulse period and the scan coordinates, PLIM by building up the distribution over the times of the photons in the laser modulation period and the scan coordinates. The technique records FLIM and PLIM simultaneously, avoids a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using high pulse energy for phosphorescence excitation. Compared with techniques which use only one laser pulse to for every phosphorescence excitation cycle it reaches a far higher PLIM sensitivity, avoids pile-up problems for the FLIM recording, and is perfectly compatible with two-photon excitation.

FLIM and PLIM image of SCC-4 cells stained with (2,2’-bipyridyl) dichlororuthenium (II) hexahydrate. FLIM shown left, PLIM shown right. Zeiss LSM 780 NLO with, bh Simple-Tau 152 FLIM/PLIM system, 2-photon excitation at 750 nm. Data analysis by bh SPCImage.

Simultaneous phosphorescence and fluorescence lifetime imaging by multi-dimensional TCSPC and multi-pulse excitation, Application note, Please click [Here] to download

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 (2016)


BH News 2016-02


PML-16 GaAsP multi-wavelength detector is 6 times more sensitive than predecessor with conventional cathode

PML-16‑GaAsP devices are 16-channel TCSPC detectors with highly efficient GaAsP cathodes. Signal recording is based on bh’s multi-dimensional TCSPC process. For every photon, the detector delivers a timing pulse and the number of the channel that detected the photon. From this information, the TCSPC module builds up a photon distribution over the times of the photons in the signal period and the channel number. The results is a set of individual optical signal waveforms for the 16 channels of the detector.

The PML-SPEC-GaAsP and MW-FLIM-GaAsP devices are combinations of the PML-16‑GaAsP detectors with a polychromator. The polychromator splits the optical signal into its spectral components. These are detected by the 16 channels of the detector. The results is a set of optical waveforms for 16 wavelength channels. The PML-SPEC has a free-beam or an optical-fibre input, the MW-FLIM has a fibre-bundle input for connecting to non-descanned ports of multiphoton microscopes. All detectors connect directly to the bh TCSPC modules or the bh Simple-Tau systems, see below.

The PML-16 GaAsP, PML-SPEC GaAsP, and MW-FLIM GaAsP detectors are about 6 times more sensitive than their PML-16C predecessors with conventional cathodes. A comparison is shown in the figures below.



Multi-wavelength fluorescence decay recording. Left: PML-16C (multialkali). Right: PML-16 GaAsP (gallium arsenide phosphide). Same acquisition time and intensity scale.


Multi-wavelength FLIM, 8 of 16 channels shown. Top: MW FLIM (multialkali). Bottom: MW FLIM GaAsP. Same acquisition time, same intensity scale.

Download Handbook: Click here

Download data sheet: Click here




BH News 2016-01

80 ps FHWM with ID230 InGaAs SPAD and SPC 150 TCSPC Module

The new ID Quantique ID230 InGaAs SPAD delivers an instrument response width of 80 ps FWHM with the bh SPC-150 TCSPC module. To our knowledge, this is the fastest TCSPC response reported for InGaAs SPADs so far. Compared to its predecessor, the new ID-230 InGaAs SPAD also has a much lower dark count rate. For signals of low count rate the dark count rate can further be reduced by selecting a long detector dead time. The detector we tested had less than 300 dark counts when operated with a dead time of 40 µs. Optical signals as weak as 800 photons per second count rate could be detected at high signal-to-background ratio.


Left: ID230 with SPC-150 TCSPC module. BDL-SM-1064 nm ps diode laser, 50 ps pulse width. Right: Laser pulse recorded at low intensity. 800 counts per second, detector dead time 40 microseconds.


Please click [Here] to download application note


BH News 2015-12

DCS-120 MP System Records Multiphoton FLIM and PLIM

The DCS‑120 MP is an extended version of the bh DCS‑120 confocal scanning FLIM System. It uses multiphoton excitation by a femtosecond titanium-sapphire laser, fast galvanometer scanning, non-descanned detection, hybrid detector technology, and single-photon recording by bh’s multi-dimensional TCSPC process. An AOM is included to control the laser power and to modulate the laser for PLIM acquisition. The system records FLIM data in two fully parallel recording channels, runs Z stacks, accumulates fast FLIM time series, and records simultaneously FLIM and PLIM. All components, including the laser and the AOM, are controlled by bh’s SPCM 64 bit data acquisition software. By using bh’s 64 bit Megapixel FLIM technology, images of the full field of view of the microscope can be recorded at diffraction-limited resolution. Image formats as large as 2048 x 2048 pixels with 256 time channels per pixel are available. The DCS-120 MP system is available with inverted microscopes of Zeiss, Nikon, and Olympus. Due to its fast scan rates and its high sensitivity, the DCS-120 MP is compatible with live cell and life tissue imaging. Typical applications are measurements of local molecular environment parameters, protein interaction experiments by FRET, imaging of metabolic parameters derived from the fluorescence decay functions of endogenous fluorophores, and correlated metabolic and oxygen saturation imaging.


Left: FLIM of a Convallaria sample, 1024x1024 pixels, 256 time channels per pixel. Right: PLIM of cellulose fibre stained with Ruthenium dye.


Please click [Here] to download application note

Please click [Here] to download DCS-120 Handbook



BH News 2015-11

bh TCSPC System Records FLIM with Piezo Stage

The PZ‑FLIM-110 Piezo Scanning FLIM system uses bh’s multi-dimensional TCSPC technique in combination with a Mad City Labs piezo scanner. The scanner is controlled via a bh GVD-120 scanner control card, the FLIM data are recorded by an SPC-150 or SPC-160 TCSPC / FLIM module. Both scanning and data acquisition are controlled by 64-bit bh SPCM TCSPC software. The system is able to run X-Y scans, X-Z (vertical) scans, and to record simultaneously FLIM and PLIM data. The system is able to scan images with up to 2048 x 2048 pixels, still recording decay data with 256 time channels per pixel. At 512 x 512 pixels the decay curves can be recorded into up to 4096 time channels. FLIM data are thus obtained at excellent spatial and the temporal resolution. Scan times for 512 x 512 pixel images are on the order of 100 to 300 seconds, scan times for 2048 x 2048 pixel images can be 6 minutes and more. That means the acquisition time is normally determined by the speed of the scanner, not by the time needed to acquire a sufficient number of photons. If the slow scan speed is tolerated the PZ‑FLIM-110 is a cost-efficient alternative to a galvanometer scanner system.

Convallaria sample, 512 x 512 pixels, 1024 time channels per pixel. Scan time 500 seconds. Intensity-weighted lifetime of triple-exponential decay.

Photo of PZ‑FLIM-110 piezo scanning FLIM system


Please click [Here] to download application note

Please click [Here] to download data sheet


BH News 2015-10

World Record in TCSPC Time Resolution: Combination of bh SPC-150NX with SCONTEL NbN Detector yields 17.8 ps FWHM

We present an ultrafast TCSPC setup consisting of a bh SPC‑150NX TCSPC module and a SCONTEL superconducting NbN detector. The entire system delivers an instrument response function (IRF) with a full width at half maximum of 17.8 ps. The RMS value of the overall single-photon timing jitter was determined to be 7.9 ps. For testing the time resolution we used a dual-output AVESTA Project EFO-80 laser. The laser emits sub-ps pulses at a wavelength of 1560 nm and a repetition rate of 50 MHz. One output of the laser was used to generate a synchronisation signal for the TCSPC device via a fast photodiode, the other one was fed into the detector via an optical attenuator. The single-photon pulses from the detector were amplified by standard low-noise GHz bandwidth RF amplifiers and fed into the ‘CFD’ input of a bh SPC‑150NX TCSPC module. Compared to the commonly used SPC‑150 the SPC‑150NX has a 4 times higher discriminator bandwidth and 2 times faster TAC ranges. The minimum time channel width is 405 fs, the electrical IRF width is 3.6 ps FWHM. For the entire system, including laser, detector, reference photodiode, fibre system, and TCSPC device we obtained an IRF of 17.8 ps FWHM, see figure below, left. The timing drift of the setup was less than 1 ps over a time of 5  minutes, see below, right.


Left: System IRF, FWHM = 17.8 ps. Right: Two subsequent recordings, blue and red, timing drift is <1 ps

Please click [Here] to download application note

For more information please see bh TCSPC Handbook, 6th edition, updated Sept. 2015, page 149  (link)

and SCONTEL Superconducting Nanotechnology


BH News 2015-09

bh SPC-150 Modules Record Super-Resolution FLIM in Abberior STED FLIM Microscopes

The bh SPC-150 TCSPC FLIM modules have been integrated in the STED FLIM microscopes of Abberior Instruments GmbH, Göttingen, Germany. The system is based on Stefan Hell’s stimulated-emission-depletion technique, see ‘Nanoscopy with Focused Light (Nobel Lecture)’, Angewandte Chemie, June 2015. The system records single STED FLIM images or 3D stacks of STED FLIM images. In single images the fluorescence intensity and lifetime are mapped at a lateral resolution of <30nm. 3D FLIM stacks are recorded at a longitudinal and lateral resolution of <70nm. Four FLIM detection channels are available; three-channel STED measurements are performed with a single depletion laser. The system is able to separate the signals of several fluorophores by their fluorescence lifetimes, or to use the fluorescence lifetime as a probe function for the molecular environment. Moreover, variable post-process time-gating allows the user to tune the optical resolution of the images versus the signal level.

Mammalian cells labelled with tubulin/ Atto647N and vimentin/ Abberior STAR 635P. Left: Confocal FLIM. Right: STED FLIM

For more information please see bh TCSPC Handbook, 6th edition, updated Sept. 2015, page 350 (link)

and abberior-instruments.com/products/superresolution-microscopes .


BH News 2015-08

bh TCSPC software runs under Windows 10

The bh SPCM TCSPC 64-bit software has passed the test with Windows 10. The full functionality is available: Recording of fluorescence decay curves, sequences of decay curves, simultaneous fluorescence and phosphorescence decay recording, FCS, FLIM at megapixel image formats, time-series FLIM, multi-wavelength FLIM, spatial and temporal mosaic FLIM, Z-stack FLIM, FLITS, simultaneous FLIM/PLIM, excitation wavelength multiplexing, detector control, control of the DCS-120 confocal scanning system and the DCS-120 macro system, control of user-defined galvanometer and piezo scanners. Single-curve and FLIM decay data analysis by SPCImage runs as usual.

For more information please download The bh TCSPC Handbook, 6th edition, page 509 (link)



BH News 2015-07

DCS-120 MACRO system records FLIM of cm-size objects

The bh DCS-120 MACRO system records FLIM of centimeter-size objects. The system uses the scan head of the DCS-120 confocal scanning FLIM system, with a telecentric lens in place of the scan lens. The object is placed directly in the image plane of this lens. The maximum scan field has a diameter of 15 mm. Images are recorded at a maximum resolution of 2048 x 2028 pixels, still resolving the decay curves in the pixels with 256 time channels.

Both optical zoom (by different scan amplitude) or digital zoom (by extracting a selected area from the data of a larger field) can be applied to the images. Two ps diode lasers, with wavelengths from 375 nm to 785 nm, or a super-continuum laser with a acousto-optical filter are used for excitation. Scanning is performed by a fast galvanometer scanner. Frame times are from 40 ms to 2 seconds, depending on the resolution and the optical zoom. The photons returned from the sample are detected in two wavelength or polarisation channels by HPM-100-40 or HPM-100-50 hybrid detectors. The lifetime images are recorded by two parallel SPC-150 TCSPC-FLIM modules.

The system uses bh’s SPCM 64 bit data acquisition software and SPCImage data analysis software. It is able to record FLIM simultaneously in the two detection channels, FLIM with excitation-wavelength multiplexing, FLIM time-series, temporal mosaic FLIM of fast physiological processes, FLITS down to millisecond resolution, and FLIM simultaneously with PLIM. 16 channel multi-wavelength FLIM with the MW-FLIM GaAsP detector is available as an option.

Fig. 1: Left: Lifetime image of a wasp, recorded at 2048x2048 pixels, 256 time channels. Right: Digital zoom into the data shown left, showing spatial resolution of the data.

Fig. 2: Photo of DCS-120 MACRO FLIM system

For more information please see bh TCSPC Handbook, 6th edition (page 341) and Handbook of the DCS-120 confocal scanning FLIM systems (page 160).

BH News 2015-06

bh FLIM systems record FLIM and FCS with Zeiss BiG 2 detectors

The bh FLIM systems for the Zeiss LSM 710 / 780 / 880 laser scanning microscopes record high-efficiency FLIM and FCS with the Zeiss BiG 2 detectors. The detectors feature high efficiency, low thermal background, and low afterpulsing. They can be used for confocal FLIM with excitation by ps diode lasers, multiphoton FLIM with excitation by a Ti:Sa laser or an OPO, and for FCS. Separate images are detected in both channels of the BiG 2 detector and recorded simultaneously by the two parallel channels of the FLIM system. With bh’s recently introduced Megapixel FLIM technology data formats of up to 2048 x 2048 pixels, with 256 time channels per pixel can be used. Images can thus be recorded at diffraction-limited resolution over the full field of view of the microscope lens. FCS is recorded at high signal-to-noise ratio, without any signs of spurious signals. FCCS is obtained by cross-correlating the signals of the two detector and TCSPC channels.

Figure: FLIM with BIG 2, OPO excitation, non-descanned detection. Pig skin, stained with methylen blue.

Figure: Fluorescence decay functions and FCS curves detected in the two channels of the BiG‑2 detector. Atto 425, excitation by 405 nm ps diode laser.

Please click [Here] to download application note

Please click [Here] to download Handbook of bh FLIM systems for Zeiss LSM 710/780/880 laser scanning microscopes


BH News 2015-05

DCS-120 System Records FLIM at Megapixel Resolution

Using new 64 bit SPCM data acquisition software, the bh DCS‑120 FLIM system records images of the full field of view of the microscope lens at diffraction-limited resolution. Image formats of up to 2048 x 2048 pixels with 256 time channels per pixel can be used. Two such images are recorded simultaneously in the two parallel channels of the DCS‑120 system. Megapixel FLIM is extremely useful for tissue imaging, and when FLIM data of a large number of cells have to be compared. FLIM data of all cells in the fields of view are obtained simultaneously, and under perfectly identical experimental conditions. The results are therefore exactly comparable.

pdf  (click here to download full-resolution images )  

For details please see:

Please click [Here] to download DCS-120 Confocal Scanning FLIM Systems - An Overview

Please click [Here] to download Confocal Scanning FLIM Systems, User Handbook, 6th Edition, 2015



BH News 2015-04

bh FLIM Systems Record Calcium Transients in Live Neurons

Transient changes of the Ca2+ concentration in live neurons have been recorded by the Fluorescence Transient Lifetime Scanning (FLITS) and and the Mosaic FLIM functions of the bh TCSPC FLIM systems. FLITS is based on the build-up of a photon distribution over the distance along a line scan, the times of the photons after the laser pulse, and the times of the photons after a periodic stimulation of the sample, temporal mosaic FLIM on the buildup of a photon distribution over the coordinates of a fast repetitive x-y scan, and the photon times after the laser pulses and the stimulation pulses. For the commonly used scanners the time resolution is about 1 ms for FLITS and about 40 ms for temporal mosaic FLIM.

pdf  (click here to open application note ) 

 For details please see: The bh TCSPC Handbook, page 327 and page 336.

FLITS of Ca2+ transients in live neurons. Left: FLITS image. Right: FLIM image taken after the FLITS recording. Location of FLITS scan indicated.


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


Previous News:

BH News 2015-03

bh TCSPC Detects Mouse Behaviour

Cui et al. used a bh SPC‑150 TCSPC module in combination with a fibre-optical system to record Ca++ signals from the brain of mice performing an operant task. The fluorescence of a Ca++ sensor was excited and detected via fibre-optics implanted in the head of the mouse. Concurrent activation of SPNs from both pathways in one hemisphere preceded the initiation of contraversive movements and predicted the occurance of specific movements within 500 ms.

Please see:

G. Cui, S.B.Jun, X. Jin, M.D. Pham, S.S. Vogel, D.M. Lovinger, R.M. Costa, Concurrent activation of strial direct and indirect pathways during action initiation. Nature 494, 238-242 (2013) http://www.ncbi.nlm.nih.gov/pubmed/23354054

G. Cui, S.B.Jun, X. Jin, G. Luo, M.D. Pham, D.M. Lovinger, S.S. Vogel,  R.M. Costa, Deep brain optical measurement of cell type-specific neural activity in behaving mice. Nature Protocols, 9(6) 1213-1228 (2014)  http://www.ncbi.nlm.nih.gov/pubmed/24784819


TCSPC Fibre-Probe System with an Exchangeable Tip. Application note (Photo System) (Click here to open application note)

IFP-201 Implantable Fibre Probe for in vivo Fluorescence Decay Measurements. Data sheet (Photo tip) (Click here for details)

BH News 2014-10

Megapixel FLIM - the New 64 bit SPCM Software

Becker & Hickl have recently introduced version 9.60 of their SPCM TCSPC data acquisition software. SPCM version 9.60 not only runs on 64‑bit computers, it is a real 64-bit application. It thus takes full advantage of the capabilities of 64-bit Windows. The most significant one is that a large amount of memory can be addressed. As a result, FLIM data can be recorded with unprecedented numbers of pixels and time channels. Moreover, the large memory space allows multi-dimensional FLIM procedures to be used without compromising spatial resolution. Multi-spectral FLIM can be recorded at unprecedented spatial resolution, the image area can be increased by spatial mosaic recording, Z stacks can be efficiently acquired without the need of intermediate data save actions, and fast time series of FLIM data can be accumulated.

Image: FLIM Z stack of pig skin stained with a near-infrared dye

pdf  (click here to open application note)


BH News 2014-05

Multiphoton NIR FLIM with the Zeiss LSM 7MP OPO System

We demonstrate multiphoton NDD FLIM of tissue samples stained with near-infrared dyes. For the experiments we used a Zeiss LSM 7MP multiphoton microscope with a Coherent Chameleon OPO (optical parametric oscillator) as an excitation source. The excitation wavelengths range from 1000 nm to 1300 nm. The fluorescence was detected by an HPM‑100-50 NIR hybrid detector attached to the NDD (non-descanned detection) port of the microscope; the FLIM data were recorded by a standard bh TCSPC FLIM system. We demonstrate the performance of the system for tissue samples stained with Methylene Blue, Indocyanin Green (ICG), and 3,3’-Diethylthiatricarbocyanine (DTTCC). All three dyes could be efficiently excited at wavelengths from 1200 nm to 1300 nm. The dyes showed remarkable variability in their fluorescence lifetimes. The lifetimes clearly depended on the tissue structures the dyes were located in.

Image: Pig skin stained with 3,3’-Diethylthiatricarbocyanine. bh Simple-Tau 152 TCSPC FLIM system.

high (click here to see image at high resolution)

pdf  (click here to open application note)



BH News 2014-01          BDL-SMN Laser

New BDL-SMN Picosecond Diode Lasers

The new BDL-SMN picosecond diode laser familiy features high optical power, short pulse width, circular beam profile, high coupling efficiency into single-mode fibres, and extraodinarily high stabitity. The optical power is stabilised by a regulation loop both in the the picosecond and in the CW mode. The lasers are available for all the typical laser diode wavelengths from 375 nm to 1064 nm.

pdf  (click here to open datasheet)

pdf  (click here to open handbook)



BH News 2013-09

Multiphoton FLIM with the Leica HyD RLD Detectors

Leica have recently introduced hybrid detectors for the non-descanned (RLD) ports of their SP5 and SP8 multiphoton laser scanning microscopes. We have tested these detectors for FLIM with the bh TCSPC modules. We describe the TCSPC parameter setup and operating conditions for the detectors, and demonstrate the performance for typical samples.

BPAE cells, 512x512 pixels, 256 time channels. bh SPC-830 TCSPC FLIM module.

high (click here to see image at high resolution)

 pdf  (click here to open application note)


BH News 2013-05 

TCSPC at Wavelengths from 900 nm to 1700 nm

We describe picosecond time-resolved optical signal recording in the spectral range from 900 nm to 1700 nm. The system consists of an id Quantique id220 InGaAs SPAD, a bh SPC‑150 TCSPC device, and a bh BDS‑SM 1064 nm ps diode laser. In contrast to earlier InGaAs SPADs the id220 works in a continuous (asynchronous) mode. The id 220 / SPC‑150 combination can be operated at a pulse repetition rate in the 10 to 100 MHz range. As a result, there is virtually no pile-up distortion, and advanced multi-dimensional TCSPC modes are applicable. The width of the temporal IRF (Instrument Response Function) is about 230 ps, including laser pulse width and pulse dispersion in the optics. We demonstrate the application of the system to the recording of time-of-flight distributions in turbid media, for fluorescence decay measurement, and for fluorescence lifetime imaging (FLIM) in combination with fast galvanometer scanning.

Image: Cellulose fibres, stained with IR1061. Excitation 1064 nm, detection 1100nm to 1400nm. 512x512 pixels, 256 time channels per pixel. Colour represents fluorescence lifetime, lifetime range from 0 to 80 ps.

 high (click here to see image at high resolution)

 pdf  (click here to open application note)





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