bh News 2018-06:
Multiphoton FLIM with the Zeiss LSM 880 NLO
We demonstrate the performance of the
bh FASTAC (fast acquisition) FLIM system in combination with the Zeiss
LSM 880 NLO multiphoton laser scanning microscopes. The FASTAC system
uses a single fast hybrid detector the photon pulses of which are
distributed into four parallel TCSPC FLIM channels, see bh news 2018/04.
Because every new photon goes to the next TCSPC module the principle
dramatically reduces counting loss and pile up effects. Images can be
recorded at acquisition times down to the minimum frame times of the
Zeiss LSM 880. Importantly, the system makes no compromises in terms of
time resolution, time channel width, time channel number, or pixel
number. The IRF width with fast detectors is less than 25 ps FWHM, and
images are recorded with typically 1024 time channels per pixel. The
time channel width can be made as small as 0.8 ps.
It should be noted that fast acquisition is only possible if the sample
is able to feed the system with a sufficiently high photon rate. This is
the case for samples that contain high amounts of bright fluorophores.
It may not always be the case in molecular imaging experiments,
metabolic FLIM, FRET experiments, or other applications where the
fluorophores are linked to highly specific targets within the cells. It
is an advantage of the FASTAC system that it even under such
sample-limited conditions the results which are at least as good as with
the standard bh FLIM systems.
Application Note: (Click here)
Left: BPAE sample, 1024x1024 pixels,
1024 time channels, acquired in 10 seconds. Right: Convallaria sample,
512 x 512 pixels, 1024 time channels, acquired in 4 seconds
bh News 2018-05:
Time-Correlated Single Photon Counting Applications’ among Springer’s
15% of best performing books
‘Advanced Time-Correlated Single
Photon Counting Applications’, published Springer in 2015, has become
one the the best performing Springer books of the past three years.
6,792 chapter downloads in 2015
8,024 chapter downloads in 2016
12,565 chapter downloads in 2017
Total number of downloads: 27,451
Congratulations to the authors and the editor!
bh News 2018-04:
FLIM System with 25ps IRF Width
We present a fast-acquisition FLIM system comprising a single detector,
four parallel TCSPC channels, and a device that distributes the photon
pulses into the four recording channels. 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 hybrid
detector is shorter than 25 ps (FWHM). The system is virtually free of
pile-up effects and has drastically reduced counting loss. FLIM data can
be recorded at acquisition times down to the fastest frame times of the
commonly used galvanometer scanners. Fast recording does not compromise
the time resolution; the data can be recorded with the TCSPC-typical
number of time-channels numbers of up to 1024 or even 4096. Pixel
numbers can be increased to 2048 x 2048 pixels. The system is therefore
equally suitable for fast FLIM and precision FLIM applications. Fig.1
show a FLIM image of a convallaria sample recorded within an acquisition
time of 100 ms. The pixel number is 128 x 128, the number of time
channels is 1024. A precision FLIM image of a BPAE sample is shown in
Fig. 2. It was recorded within a acquisition time of 10 seconds. The
data format is 1024 x 1024 pixels, with 1024 time channels per pixel.
For more information please see application note ‘Fast-Acquition TCSPC
FLIM system with sub-25 ps IRF width’. (Click here)
Fig. 1: Convallaria
sample, 128 x 128 pixels, 1024 time channels, acquisition time 100 ms.
FLIM image, decay curve in 5x5 pixel area, and decay curve over entire
BPAE sample, 1024 x 1024 pixels, 1024 time channels,
acquisition time 10 s. FLIM image, decay curve in 10x10 pixel area, and
decay curve over entire image.
bh News 2018-03:
with New User Interface
Starting from software version 5.3, bh SPCImage FLIM
data analysis software comes with a new user interface. It takes
advantage of the new wide-screen monitor formats to better display bh
‘Megapixel’ lifetime images. The SPCImage panel can be configured by the
user; two examples are shown below.
To switch to the new user interface, please open
SPCImage without loading data, click into ‘Options’, ‘Preferences’, and
select ‘Wide Screen Adapted’, ‘Image Size variable’.
For other new
features of SPCImage, please see ‘bh TCSPC Handbook’, 7th edition, page
733, Handbook ‘DCS-120 Confocal and Multiphoton FLIM systems’, 7th
edition, page 281, or Handbook ‘Modular FLIM Systems for Zeiss LSM 710 /
780 / 880 Family Laser Scanning Microscopes’, 7th edition, page 225.
Please contact bh if you want printed copies.
bh News 2018-02:
Acquisition Software Controls Ti:Sa Lasers and AOM
Starting from software version 9.76,
the bh SPCM data acquisition software controls Coherent and Spectra
Physics Titanium-Sapphire lasers. The control software includes also the
bh AOM-100 Acousto-Optical Modulator. With the AOM, reproducible
intensity control and laser modulation for beam blanking in scanning
applications and for simultaneous FLIM / PLIM is obtained. The software
automatically adjusts the AOM control frequency to the selected laser
wavelength and thus avoids beam shift with the wavelength. For details,
please see bh TCSPC Handbook, 7th edition, page 730 or Handbook of
DCS-120 Confocal and Multiphoton FLIM systems, 7th edition, page 189.
Images: Upper: Laser and AOM control panel, NADH FLIM of live cells, recorded
by bh DCS-120MP system and ultra-fast HPM-100-06 detector. Lower: Decay
curve in selected pixel. Please note extremely fast IRF.
bh News 2017-11:
system records X-Y mosaics
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
bh News 2017-10:
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
bh News 2017-05:
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:
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
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:
Combines Time-Domain FLIM Analysis with Phasor Plot
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.
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
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:
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
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.
for data sheet of
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
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 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
for application note)
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.
for application note)
New SPC-160PCIE TCSPC/FLIM module has PCI Express
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).
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
Convallaria sample. Left: 128x128 pixels, 10 images per second. Right:
256x256 pixels, 2 image per second. Lifetime range 1ns (red) to 3ns
for full application note.
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
sheet BDS-SM: (click here)
Extended data sheet BDS-MM: (click
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
Simultaneous recording of fluorescence and phosphorescence decay (left),
simultaneous FLIM/PLIM (right).
BH News 2016-05
bh - Abberior Combination Records STED FLIM at
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.
image please see application note, click
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.
phosphorescence and fluorescence lifetime imaging by multi-dimensional
TCSPC and multi-pulse excitation, Application note, Please click
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.
BH News 2016-02
multi-wavelength detector is 6 times more sensitive than predecessor
with conventional cathode
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
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.
Handbook: Click here
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
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.
to download application note
BH News 2015-12
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.
to download application note
download DCS-120 Handbook
BH News 2015-11
bh TCSPC System Records FLIM with Piezo Stage
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
Photo of PZ‑FLIM-110 piezo scanning FLIM system
[Here] to download application note
[Here] to download
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.
IRF, FWHM = 17.8 ps. Right: Two subsequent recordings, blue and red,
timing drift is <1 ps
[Here] to download application note
information please see bh
TCSPC Handbook, 6th edition, updated Sept. 2015, page 149 (link)
BH News 2015-09
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
information please see bh TCSPC Handbook, 6th
edition, updated Sept. 2015, page 350 (link)
BH News 2015-08
bh TCSPC software runs under
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.
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
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
decay functions and FCS curves detected in the two channels of the
BiG‑2 detector. Atto 425, excitation by 405 nm ps diode laser.
[Here] to download application note
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
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.
here to download full-resolution images )
For details please see:
Confocal Scanning FLIM Systems - An Overview
Scanning FLIM Systems, User Handbook, 6th Edition, 2015
BH News 2015-04
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.
here to open application note )
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
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.
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
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)
TCSPC Fibre-Probe System with an Exchangeable Tip. Application note
(Click here to open
IFP-201 Implantable Fibre Probe for in vivo Fluorescence
Decay Measurements. Data sheet (Photo tip)
(Click here for details)
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
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BH News 2014-05
NIR FLIM with the Zeiss LSM 7MP OPO System
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
to see image
at high resolution)
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BH News 2014-01
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.
here to open datasheet)
here to open handbook)
BH News 2013-09
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.
to see image
at high resolution)
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BH News 2013-05
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.
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.
to see image
at high resolution)
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