Wolfgang Becker - Academia.edu (original) (raw)
Papers by Wolfgang Becker
Megapixel FLIM
SPIE Proceedings, 2014
ABSTRACT TCSPC (Time-Correlated Single-Photon Counting) FLIM data with megapixel resolution can b... more ABSTRACT TCSPC (Time-Correlated Single-Photon Counting) FLIM data with megapixel resolution can be recorded by using bh TCSPC modules in combination with new 64 bit data acquisition software. The large memory space available in the 64 bit environment allows new FLIM procedures to be used. We demonstrate the performance for applications that require imaging of a large number of cells in a single field of view, for multi-wavelength FLIM, for spatial mosaic imaging, and for recording transient changes in the fluorescence decay after a stimulation of the sample. Image quality was further improved by integrating a parallel counter channel that bypasses the timing electronics of the TCSPC module. Photon numbers from this counter are not affected by dead-time effects. Lifetime images are built up by using intensity data from the parallel counter and fluorescence decay data from the TCSPC electronics.
Molecular, morphological and physiological heterogeneity is the inherent property of cells, which... more Molecular, morphological and physiological heterogeneity is the inherent property of cells, which governs differences in their response to external influence. The tumor cells metabolic heterogeneity is of a special interest due to its clinical relevance to the tumor progression and therapeutic outcomes. Rapid, sensitive and non-invasive assessment of metabolic heterogeneity of cells is of a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters (FDPs) of endogenous fluorophores, such as NAD(P)H. To achieve the accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this report, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity has been demonstrated. This has been achieved...
A global fit assumes that one or several of the lifetimes of a multi-exponential decay are consta... more A global fit assumes that one or several of the lifetimes of a multi-exponential decay are constant in all pixels of a FLIM image. By using this a priori knowledge, the decay parameters can be obtained at a better
accuracy than by a multi-exponential fit with all decay parameters floating. However, a global fit is enormously computation-intensive. Processing times with standard CPU processing are therefore unacceptably long. We
present a Global Fit algorithm based on GPU processing that reduces the calculation time from previously hours to a few minutes and less.
We demonstrate a technique that combines metabolic imaging by NAD(P)H with pH imaging by BCECF (2... more We demonstrate a technique that combines metabolic imaging by NAD(P)H with pH imaging by BCECF (2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein). For excitation we used a femtosecond fibre laser
with an emission wavelength of 785 nm. Two-photon excitation with this laser excites both NAD(P)H and BCECF at reasonable efficiency. The fluorescence of the two compounds was split spectrally on the emission
side and recorded in two parallel TCSPC FLIM channels. Analysis by bh SPCImage delivers one image for the metabolic state and another for the pH of the tissue.
We demonstrate Z stack imaging of a fly (Musca domestica) with 289 Z planes of 2.5 μm Z step widt... more We demonstrate Z stack imaging of a fly (Musca domestica) with 289 Z planes of 2.5 μm Z step width, 1024 x 1024 pixels in X and Y, and 1024
channels in time by using existing functions of the Becker & Hickl DCS-120 confocal FLIM system. Doubleexponential decay analysis was performed by the batch processing function of Becker & Hickl SPCImage NG. 3D reconstructions on the basis of the SPCImage results were performed by ImageJ / FIJI.
We demonstrate two-photon FLIM with a FemtoFibre smart 780 laser of Toptica Photonics AG in combi... more We demonstrate two-photon FLIM with a FemtoFibre smart 780 laser of Toptica Photonics AG in combination with a Becker & Hickl DCS-120 MP TCSPC / FLIM system. The laser delivers femtosecond pulses at 783 nm, 80 MHz repetition rate, and 125 mW average power. We show that the laser power is sufficient to obtain highresolution images in all the commonly performed FLIM applications. We demonstrate the performance of the system for label-free (autofluorescence) imaging of tissue, label-free imaging of cells, FLIM of ultrafast fluorescence decay in biological material, and FLIM of fluorophore-labelled cells and tissues.
Optics Letters, 2023
We present a laser scanning system for macroscopic samples that records fully resolved decay curv... more We present a laser scanning system for macroscopic samples that records fully resolved decay curves in individual pixels, resolves the images in 16 wavelength channels, and records simultaneously at several laser wavelengths. By using confocal detection, the system delivers images that are virtually free of lateral scattering and out-of-focus haze. Image formats can be up to 256 × 256 pixels and up to 1024 time channels. We demonstrate the performance of the system both on model experiments with fluorescent micro-beads and on the tumor model in the living mice.
Multimodal Optical Diagnostics of Cancer, 2020
Frontiers in Oncology, 2021
Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival... more Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients’ glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glio...
Medical Photonics, 2015
Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several feat... more Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional TCSPC is based on the excitation of the sample by a high repetition rate laser and the detection of single photons of the fluorescence signal. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye.
Hybrid detectors and multichannel-plate (MCP) PMTs achieve a timing resolution (IRF width) of les... more 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 SPC-150N discriminators and the extremely low timing jitter of the timing electronics of the SPC-150NX modules.
We present an ultrafast TCSPC setup consisting of a bh SPC-150NX TCSPC module and a SCONTEL super... more We present an ultrafast TCSPC setup consisting of a bh SPC-150NX TCSPC module and a SCONTEL superconducting NbN detector. The combination 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 is about 7.9 ps.
The bh DCS-120 MP system is able to record single decay functions at extremely high precision and... more The bh DCS-120 MP system is able to record single decay functions at extremely high precision and time resolution. We used the system to record decay functions of NADH and FAD with an IRF width of 19 ps. Fluorescence decay functions were obtained for NADH and FAD in aqueous solution, and in a diluted solution of citric acid at pH = 4. The decay curves are multi-exponential, with decay components as fast as 115 ps for NADH and 59 ps for FAD. The curves measured at pH = 7 and pH = 4 are significantly different, and they are different from decay curves recorded in cells. The procedures described can be used to supplement FLIM experiments with precision decay parameters of the fluorophores involved. Similar measurement can be performed with other bh FLIM systems, especially if these are equipped with bh's ultra-fast HPM-100-06 detectors.
This application note describes a fluorescence-lifetime detection system based on a fibreoptical ... more This application note describes a fluorescence-lifetime detection system based on a fibreoptical probe with an exchangeable tip. The excitation light is delivered to the tip via a single-mode fibre, the emission light is transferred to the detector by a multi-mode fibre. The electronic part of the system consist of a bh BDL-SMN picosecond diode laser, a bh PMH-100 hybrid detector or MW-FLIM GaAsP multi-wavelength detector, and a Simple-Tau 150 TCSPC system. The system features high sensitivity and short acquisition time. Clean fluorescence decay curves from a 10-7 mol/l fluorescein solution were recorded within an acquisition time of 0.5 seconds, time-series of autofluorescence decay curves were recorded at a speed of 100 ms per step.
We describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak... more We describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak count rate on the order of 5010 6 s-1. By using a polychromator for spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live plants.
The use of a wide range of excitation wavelengths in a confocal laser scanning system leads to a ... more The use of a wide range of excitation wavelengths in a confocal laser scanning system leads to a number of design problems. The most critical one is connected to the main dichroic beamsplitter that separates to fluorescence signals from the excitation beam. For use with several lasers the beamsplitter must either be switchable or tuneable, or a multiband dichroic must be used. The result is either alignment instability, or spectral gaps in the fluorescence detection channels. We developed a version of the DCS-120 confocal FLIM scanner that bypasses most of these problems by using a wideband beamsplitter. The design allows the user to switch lasers without compromising alignment stability. The sensitivity of the system is sufficient to record autofluorescence images of single cells.
The DCS-120 system uses excitation by ps diode lasers or femtosecond titanium-sapphire lasers, fa... more The DCS-120 system uses excitation by ps diode lasers or femtosecond titanium-sapphire lasers, fast scanning by galvanometer mirrors, confocal detection, and FLIM by bh’s multidimensional TCSPC technique to record fluorescence lifetime images at high temporal resolution, high spatial resolution, and high sensitivity [3]. The DCS 120 system is available with inverted microscopes of Nikon, Zeiss, and Olympus. It can also be used to convert an existing conventional microscope into a fully functional confocal or multiphoton laser scanning microscope with TCSPC detection. Due to its fast beam scanning and its high sensitivity the DCS-120 system is compatible with live-cell imaging. DCS-120 functions include simultaneous recording of FLIM or steady-state fluorescence images simultaneously in two fully parallel wavelength channels, laser wavelength multiplexing, time-series FLIM, time-series recording, Z stack FLIM, phosphorescence lifetime imaging (PLIM), fluorescence lifetime-transient scanning (FLITS) and FCS recording. Applications focus on lifetime variations by interactions of fluorophores with their molecular environment. Typical applications are ion concentration measurement, FRET experiments, metabolic imaging, and plant physiology.
Proceedings of SPIE, 2018
Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be res... more Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.
We describe LIDAR with a bh SPC-QC-104 TCSPC module and a bh BDS-SM 640-nm picosecond diode laser... more We describe LIDAR with a bh SPC-QC-104 TCSPC module and a bh BDS-SM 640-nm picosecond diode laser. The beam of the laser was directed to a distant target, photons scattered back from the target were collected by a Meade LX90 20-cm telescope, detected by a bh PMC-150-20 PMT module, and recorded by the TCSPC device. Using a laser pulse repetition rate of 250 kHz and no more than 10 µW of average power we obtained a clean backscattering signal from a tree 160 meters away. By increasing the pulse repetition rate to 50 MHz, we were able to detect fluorescence decay curves from the chlorophyll in the leaves.
We demonstrate fluorescence-lifetime based imaging of membrane potentials in cells with a voltage... more We demonstrate fluorescence-lifetime based imaging of membrane potentials in cells with a voltage sensitive dye. We estimate the number of photons per pixel required for a given standard deviation of the membrane potential, show ways of improving the accuracy and compare the predictions with real measurement data. In the experiments described, we achieved a standard deviation of the membrane voltage on the order of a few mV.
Megapixel FLIM
SPIE Proceedings, 2014
ABSTRACT TCSPC (Time-Correlated Single-Photon Counting) FLIM data with megapixel resolution can b... more ABSTRACT TCSPC (Time-Correlated Single-Photon Counting) FLIM data with megapixel resolution can be recorded by using bh TCSPC modules in combination with new 64 bit data acquisition software. The large memory space available in the 64 bit environment allows new FLIM procedures to be used. We demonstrate the performance for applications that require imaging of a large number of cells in a single field of view, for multi-wavelength FLIM, for spatial mosaic imaging, and for recording transient changes in the fluorescence decay after a stimulation of the sample. Image quality was further improved by integrating a parallel counter channel that bypasses the timing electronics of the TCSPC module. Photon numbers from this counter are not affected by dead-time effects. Lifetime images are built up by using intensity data from the parallel counter and fluorescence decay data from the TCSPC electronics.
Molecular, morphological and physiological heterogeneity is the inherent property of cells, which... more Molecular, morphological and physiological heterogeneity is the inherent property of cells, which governs differences in their response to external influence. The tumor cells metabolic heterogeneity is of a special interest due to its clinical relevance to the tumor progression and therapeutic outcomes. Rapid, sensitive and non-invasive assessment of metabolic heterogeneity of cells is of a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters (FDPs) of endogenous fluorophores, such as NAD(P)H. To achieve the accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this report, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity has been demonstrated. This has been achieved...
A global fit assumes that one or several of the lifetimes of a multi-exponential decay are consta... more A global fit assumes that one or several of the lifetimes of a multi-exponential decay are constant in all pixels of a FLIM image. By using this a priori knowledge, the decay parameters can be obtained at a better
accuracy than by a multi-exponential fit with all decay parameters floating. However, a global fit is enormously computation-intensive. Processing times with standard CPU processing are therefore unacceptably long. We
present a Global Fit algorithm based on GPU processing that reduces the calculation time from previously hours to a few minutes and less.
We demonstrate a technique that combines metabolic imaging by NAD(P)H with pH imaging by BCECF (2... more We demonstrate a technique that combines metabolic imaging by NAD(P)H with pH imaging by BCECF (2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein). For excitation we used a femtosecond fibre laser
with an emission wavelength of 785 nm. Two-photon excitation with this laser excites both NAD(P)H and BCECF at reasonable efficiency. The fluorescence of the two compounds was split spectrally on the emission
side and recorded in two parallel TCSPC FLIM channels. Analysis by bh SPCImage delivers one image for the metabolic state and another for the pH of the tissue.
We demonstrate Z stack imaging of a fly (Musca domestica) with 289 Z planes of 2.5 μm Z step widt... more We demonstrate Z stack imaging of a fly (Musca domestica) with 289 Z planes of 2.5 μm Z step width, 1024 x 1024 pixels in X and Y, and 1024
channels in time by using existing functions of the Becker & Hickl DCS-120 confocal FLIM system. Doubleexponential decay analysis was performed by the batch processing function of Becker & Hickl SPCImage NG. 3D reconstructions on the basis of the SPCImage results were performed by ImageJ / FIJI.
We demonstrate two-photon FLIM with a FemtoFibre smart 780 laser of Toptica Photonics AG in combi... more We demonstrate two-photon FLIM with a FemtoFibre smart 780 laser of Toptica Photonics AG in combination with a Becker & Hickl DCS-120 MP TCSPC / FLIM system. The laser delivers femtosecond pulses at 783 nm, 80 MHz repetition rate, and 125 mW average power. We show that the laser power is sufficient to obtain highresolution images in all the commonly performed FLIM applications. We demonstrate the performance of the system for label-free (autofluorescence) imaging of tissue, label-free imaging of cells, FLIM of ultrafast fluorescence decay in biological material, and FLIM of fluorophore-labelled cells and tissues.
Optics Letters, 2023
We present a laser scanning system for macroscopic samples that records fully resolved decay curv... more We present a laser scanning system for macroscopic samples that records fully resolved decay curves in individual pixels, resolves the images in 16 wavelength channels, and records simultaneously at several laser wavelengths. By using confocal detection, the system delivers images that are virtually free of lateral scattering and out-of-focus haze. Image formats can be up to 256 × 256 pixels and up to 1024 time channels. We demonstrate the performance of the system both on model experiments with fluorescent micro-beads and on the tumor model in the living mice.
Multimodal Optical Diagnostics of Cancer, 2020
Frontiers in Oncology, 2021
Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival... more Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients’ glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glio...
Medical Photonics, 2015
Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several feat... more Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional TCSPC is based on the excitation of the sample by a high repetition rate laser and the detection of single photons of the fluorescence signal. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye.
Hybrid detectors and multichannel-plate (MCP) PMTs achieve a timing resolution (IRF width) of les... more 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 SPC-150N discriminators and the extremely low timing jitter of the timing electronics of the SPC-150NX modules.
We present an ultrafast TCSPC setup consisting of a bh SPC-150NX TCSPC module and a SCONTEL super... more We present an ultrafast TCSPC setup consisting of a bh SPC-150NX TCSPC module and a SCONTEL superconducting NbN detector. The combination 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 is about 7.9 ps.
The bh DCS-120 MP system is able to record single decay functions at extremely high precision and... more The bh DCS-120 MP system is able to record single decay functions at extremely high precision and time resolution. We used the system to record decay functions of NADH and FAD with an IRF width of 19 ps. Fluorescence decay functions were obtained for NADH and FAD in aqueous solution, and in a diluted solution of citric acid at pH = 4. The decay curves are multi-exponential, with decay components as fast as 115 ps for NADH and 59 ps for FAD. The curves measured at pH = 7 and pH = 4 are significantly different, and they are different from decay curves recorded in cells. The procedures described can be used to supplement FLIM experiments with precision decay parameters of the fluorophores involved. Similar measurement can be performed with other bh FLIM systems, especially if these are equipped with bh's ultra-fast HPM-100-06 detectors.
This application note describes a fluorescence-lifetime detection system based on a fibreoptical ... more This application note describes a fluorescence-lifetime detection system based on a fibreoptical probe with an exchangeable tip. The excitation light is delivered to the tip via a single-mode fibre, the emission light is transferred to the detector by a multi-mode fibre. The electronic part of the system consist of a bh BDL-SMN picosecond diode laser, a bh PMH-100 hybrid detector or MW-FLIM GaAsP multi-wavelength detector, and a Simple-Tau 150 TCSPC system. The system features high sensitivity and short acquisition time. Clean fluorescence decay curves from a 10-7 mol/l fluorescein solution were recorded within an acquisition time of 0.5 seconds, time-series of autofluorescence decay curves were recorded at a speed of 100 ms per step.
We describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak... more We describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak count rate on the order of 5010 6 s-1. By using a polychromator for spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live plants.
The use of a wide range of excitation wavelengths in a confocal laser scanning system leads to a ... more The use of a wide range of excitation wavelengths in a confocal laser scanning system leads to a number of design problems. The most critical one is connected to the main dichroic beamsplitter that separates to fluorescence signals from the excitation beam. For use with several lasers the beamsplitter must either be switchable or tuneable, or a multiband dichroic must be used. The result is either alignment instability, or spectral gaps in the fluorescence detection channels. We developed a version of the DCS-120 confocal FLIM scanner that bypasses most of these problems by using a wideband beamsplitter. The design allows the user to switch lasers without compromising alignment stability. The sensitivity of the system is sufficient to record autofluorescence images of single cells.
The DCS-120 system uses excitation by ps diode lasers or femtosecond titanium-sapphire lasers, fa... more The DCS-120 system uses excitation by ps diode lasers or femtosecond titanium-sapphire lasers, fast scanning by galvanometer mirrors, confocal detection, and FLIM by bh’s multidimensional TCSPC technique to record fluorescence lifetime images at high temporal resolution, high spatial resolution, and high sensitivity [3]. The DCS 120 system is available with inverted microscopes of Nikon, Zeiss, and Olympus. It can also be used to convert an existing conventional microscope into a fully functional confocal or multiphoton laser scanning microscope with TCSPC detection. Due to its fast beam scanning and its high sensitivity the DCS-120 system is compatible with live-cell imaging. DCS-120 functions include simultaneous recording of FLIM or steady-state fluorescence images simultaneously in two fully parallel wavelength channels, laser wavelength multiplexing, time-series FLIM, time-series recording, Z stack FLIM, phosphorescence lifetime imaging (PLIM), fluorescence lifetime-transient scanning (FLITS) and FCS recording. Applications focus on lifetime variations by interactions of fluorophores with their molecular environment. Typical applications are ion concentration measurement, FRET experiments, metabolic imaging, and plant physiology.
Proceedings of SPIE, 2018
Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be res... more Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.
We describe LIDAR with a bh SPC-QC-104 TCSPC module and a bh BDS-SM 640-nm picosecond diode laser... more We describe LIDAR with a bh SPC-QC-104 TCSPC module and a bh BDS-SM 640-nm picosecond diode laser. The beam of the laser was directed to a distant target, photons scattered back from the target were collected by a Meade LX90 20-cm telescope, detected by a bh PMC-150-20 PMT module, and recorded by the TCSPC device. Using a laser pulse repetition rate of 250 kHz and no more than 10 µW of average power we obtained a clean backscattering signal from a tree 160 meters away. By increasing the pulse repetition rate to 50 MHz, we were able to detect fluorescence decay curves from the chlorophyll in the leaves.
We demonstrate fluorescence-lifetime based imaging of membrane potentials in cells with a voltage... more We demonstrate fluorescence-lifetime based imaging of membrane potentials in cells with a voltage sensitive dye. We estimate the number of photons per pixel required for a given standard deviation of the membrane potential, show ways of improving the accuracy and compare the predictions with real measurement data. In the experiments described, we achieved a standard deviation of the membrane voltage on the order of a few mV.
Lecture on Fluorescence-Lifetime Imaging Ophthalmoscopy (FLIO) held April 23, 2021 by Wolfgang Be... more Lecture on Fluorescence-Lifetime Imaging Ophthalmoscopy (FLIO) held April 23, 2021 by Wolfgang Becker. The lecture explains what fluorescence decay functions tell us, how FLIO data are recorded, the way from the raw data to the images, the challenges of FLIO data analysis, solutions to long-existing problems, and new analysis approaches. The last section is on the analysis of FLIO data with SPCImage NG.
Lecture on Fluorescence Liftetime Imagaing Ophthalmoscopy – Understanding FLIO data recording – R... more Lecture on Fluorescence Liftetime Imagaing Ophthalmoscopy
– Understanding FLIO data recording
– Recoording the best possible data
– Understanding the data analysis
– Problems of FLIO data anaylsis
– The shifted-component Model
– Separating the fundus from the lens
– Model-parameter definitions in SPCImage
Bigger and Better Photons: The Road to Great FLIM Results, 2021
These pages are an attempt to help existing and future users of the bh FLIM technique obtain the ... more These pages are an attempt to help existing and future users of the bh FLIM technique obtain the best possible results from their FLIM experiments. The first part of the brochure explains the principle of TCSPC FLIM, and gives an impression of the photon distributions recorded. It shows that the signal-to-noise ratio of the measured lifetimes depends, in first order, on the number of photons recorded. The following sections focus on optimising the photon number without increasing the photostress imposed to the sample. We discuss the influence of excitation power, acquisition time, collection efficiency, numerical aperture, focusing precision, alignment accuracy, and detector efficiency. The next section concentrates on photon efficiency. It considers TCSPC timing parameters, counting background, number of pixels, the influence of the instrument-response function, and the challenges of multi-exponential decay functions. The last section is dedicated to data analysis. All conclusions made in this brochure are demonstrated on real measurement data recorded under realistic conditions.
This book is more than a description of the Becker & Hickl DCS-120 FLIM system. It has been writt... more This book is more than a description of the Becker & Hickl DCS-120 FLIM system. It has been written to bridge the gap between the capabilities of state-of-the art FLIM techniques and biological applications. After an introduction in the general capabilities of the system it describes the electronical and optical principles of FLIM data acquisition, basic and advanced techniques and procedures, and system configurations for different applications. The book shows how the DCS system is able to perform experiments which are entirely beyond the capabilities of other FLIM systems: FLIM of fast physiological processes, excitation multiplexed FLIM, multi-wavelength FLIM, FLIM from moving objects, FLIM with resolution of ultra-fast fluorescence decay, or simultaneous FLIM / PLIM. A large chapter is dedicated to FLIM applications in biology. It covers the measurement of local environment parameters, protein interaction experiments by FRET, autofluorescence FLIM, metabolic FLIM, and metabolic FLIM in combination with pO2 measurement. Analysis of FLIM data is described in a special chapter on bh's SPCImage NG data analysis software. The book ends with a list of 560 references of publications related to FLIM and FLIM applications.
Advanced Time-Correlated Single Photon Counting Techniques
Time-correlated single photon counting (TCSPC) is a remarkable technique for recording low-level ... more Time-correlated single photon counting (TCSPC) is a remarkable technique for recording low-level light signals with extremely high precision and picosecond-time resolution. TCSPC has developed from an intrinsically time-consuming and one-dimensional technique into a fast, multi-dimensional technique to record light signals. So this reference and text describes how advanced TCSPC techniques work and demonstrates their application to time-resolved laser scanning microscopy, single molecule spectroscopy, photon correlation experiments, and diffuse optical tomography of biological tissue. It gives practical hints about constructing suitable optical systems, choosing and using detectors, detector safety, preamplifiers, and using the control features and optimising the operating conditions of TCSPC devices. Advanced TCSPC Techniques is an indispensable tool for everyone in research and development who is confronted with the task of recording low-intensity light signals in the picosecond and nanosecond range.
Advanced Time-Correlated Single Photon Counting Applications
This book is an attempt to bridge the gap between the instrumental principles of multi-dimensiona... more This book is an attempt to bridge the gap between the instrumental principles of multi-dimensional time-correlated single photon counting (TCSPC) and typical applications of the technique. Written by an originator of the technique and by more than 75 high-end users, it covers the basic principles of the technique, its interaction with optical imaging methods, and its application to a wide range of experimental tasks in life sciences and clinical research. The book is recommended for all users of time-resolved detection techniques in biology, bio-chemistry, spectroscopy of live systems, live cell microscopy, clinical imaging, spectroscopy of single molecules, and other applications that require the detection of low-level light signals at single-photon sensitivity and picosecond time resolution.
ISBS 2021 Digital congress on biophysics and imaging of the skin, 2021
The fluorescence of the coenzymes NAD(P)H and flavins (FAD, FMN) can be used to evaluate cell met... more The fluorescence of the coenzymes NAD(P)H and flavins (FAD, FMN) can be used to evaluate cell metabolism. Of interest is the metabolism of intratissue cells of pathological skin compared to healthy skin and the impact of cosmetics.
Intratissue NAD(P)H in its free and bound form can be detected by Multiphoton FLIM Tomography with a femtosecond laser excitation wavelength of 800 nm or shorter. Intratissue flavins / flavoproteins can be also excited at a longer NIR wavelength such as 880 nm.
We report on a tomograph MPTflexTM with a two-wavelength fiber fs laser emitting two laser beams at 780 nm and 880 nm in combination with a low-noise two-detection-channel hybrid PMT detector for multiplex time-correlated single photon counting (TCSPC). Using an acousto- optical (AOM) modulator, the beams are switched permanently. The tomograph provides simultaneously four high-resolution autofluorescence images within a 6 s scan of 0.3 x 0.3 mm2 tissue. Image 1 depicts NAD(P)H when excited with 780 nm light. Image 2 depicts collagen as SHG-signal of the 880 nm beam. Image 3 provides a 780nm excited NAD(P)H + flavin image, and image 4 the 880 nm excited flavin. All 4 images can be depicted as false color images where the color represents the sub- ns/nanosecond autofluorescence lifetime. Information on the cells metabolism can be obtained by NAD(P)H/flavin ratios as well as by the ratio free to bound coenzyme.