Hpm 100 40

Manufactured by Becker & Hickl

Sourced in Germany

The HPM-100-40 is a photomultiplier module developed by Becker & Hickl. It is designed for single photon counting and time-resolved measurements. The module includes a photomultiplier tube, high voltage power supply, and signal processing electronics.

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Lab products found in correlation

Spc 150, by Becker & Hickl (9 mentions) Spc 152, by Becker & Hickl (4 mentions) Spcimage software, by Becker & Hickl (3 mentions) A1 mp, by Nikon (2 mentions) Wd 2 mm, by Nikon (2 mentions) Spcimage 6, by Becker & Hickl (2 mentions) Deepsee hp, by Spectra-Physics (2 mentions) Lsm 780 scanning unit, by Becker & Hickl (2 mentions) Spcimage version 3, by Becker & Hickl (2 mentions) Ff535 sdi01, by IDEX Corporation (2 mentions) Ff01 512 sp 25, by IDEX Corporation (2 mentions) Dcs 120, by Becker & Hickl (2 mentions) Lsm 880, by Zeiss (2 mentions) Fluorobrite dmem, by Thermo Fisher Scientific (2 mentions) Lsm510 meta nlo, by Zeiss (1 mentions) Spc 830, by Becker & Hickl (1 mentions) Bdl smn 375, by Becker & Hickl (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Acriflavine hydrochloride, by Merck Group (1 mentions) Zinpyr 1, by Cayman Chemical (1 mentions)

Close spelling variants of this product

HPM-100–40-C (4 citations) HPM-100 (3 citations)

25 protocols using hpm 100 40

Fluorescence Lifetime Imaging of Membrane Fluidity

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For microscopy, a small aliquot of the Laurdan-stained bacteria was gently pipetted onto a glass slides for microscopy, which had been coated with an agar-pad. Fluorescence Lifetime Imaging with pulsed, two-photon excitation was performed on a laser scanning fluorescence microscope (LSM880, Zeiss, Jena, Germany) equipped with a ×20 water immersion objective (NA 1.0, WD 2.1 mm; Zeiss, Jena). Laurdan was excited with 780 nm (120 fs pulses with 80 MHz repetition frequency) and its fluorescence was observed after passing through a broad band-pass filter centered at 445 nm (FWHM 90 nm; Omega Optical, Brattleboro, VT, USA) covering the blue part of the Laurdan emission spectrum, whose fluorescence decay is sensitive to the membrane fluidity. Fluorescence photons were detected with a GaAsP hybrid photodetector (HPM-100-40, Becker & Hickl, Berlin, Germany). TCSPC electronics (SPC-152; Becker & Hickl, Berlin, Germany) and acquisition software were used for FLIM^{69 ,70 (link)}. Fluorescence lifetime images were generated using SPCImage 8.4 (Becker & Hickl, Berlin, Germany). Fluorescence decays were fitted with bi-exponential functions^{71 (link)–73 (link)} and the average fluorescence lifetime (τ_ave):

τ_{ave} = (a_{1} \cdot τ_{1} + a_{2} \cdot τ_{2}) / (a_{1} + a_{2})

was used to represent the Laurdan fluorescence decays satisfactorily (τ_i = lifetime of the ith exponential component; a_i = respective amplitude).

Tharmasothirajan A., Melcr J., Linney J., Gensch T., Krumbach K., Ernst K.M., Brasnett C., Poggi P., Pitt A.R., Goddard A.D., Chatgilialoglu A., Marrink S.J, & Marienhagen J. (2023). Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis. Nature Communications, 14, 5619.

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Two-Photon FLIM Imaging of Cellular Metabolism

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FLIM measurements were performed on a Nikon microscope using two-photon excitation from a Ti:Sapphire pulsed laser (M-squared lasers) with an 80-MHz repetition rate and ~150-fs pulse width, a galvanometer scanner, TCSPC module (SPC-150, Becker & Hickl) and a hybrid single-photon counting detector (HPM-100–40, Becker & Hickl). The wavelength of excitation was set to 750 nm for NADH and 845 nm for FAD, with powers of 45mW and 75mW, respectively (measured after the objective). Optical bandpass filters were positioned in front of the detector—460/50nm for NADH and 550/88nm for FAD (Chroma Technology). Imaging was performed with a 20X Nikon objective with 0.75 numerical aperture (NA). The size of the equipment was similar to a typical microscope (with approximately 3×5 feet base area).
At each time point, NADH and FAD images were acquired at three different focal planes within the oocytes, separate by a distance of 7µm, with an integration time of 60 seconds for (11–12 integrated scans of the field of view) each plane. Pixel size was 0.8µm/pixel, and scanner dwell time was 5µs/pixel. NADH/FAD complete acquisitions were orchestrated with a combination of custom software written in Labview and Becker Hickl acquisition software. In total, each complete metabolic measurement took approximately 7 minutes to acquire.

Sanchez T., Wang T., Pedro M.V., Zhang M., Esencan E., Sakkas D., Needleman D, & Seli E. (2018). Metabolic imaging using Fluorescence Lifetime Imaging Microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes. Fertility and sterility, 110(7), 1387-1397.

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Time-Resolved Fluorescence Lifetime Imaging

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Time-domain FLIM was performed with a multiphoton microscopy system, based on a Zeiss LSM 510 Meta NLO equipped with a Ti: Sapphire Chameleon-XR pulsed laser (Coherent). Time-resolved detection was afforded by the addition at a non-descanned output of a fast hybrid photomultiplier (HPM-100-40) and SPC-830 time-correlated single-photon counting (TCSPC) electronics (Becker & Hickl). For EGFP excitation, laser power at 900 nm was adjusted to give average photon counting rates of the order 10⁴–10⁵ photons s⁻¹ (0.0001–0.001 photons/excitation event) and with peak rates approaching 10⁶ photons s⁻¹, below the maximum counting rate afforded by the TCSPC electronics to avoid pulse pile-up. FLIM acquisitions were performed on living mitotic cells (2 min acquisitions for single micrographs; 20 s acquisitions for the movie). Images were taken with a Zeiss 63X/1.4 oil Plan-Apochromat objective.
For the movie and foci trajectory study, images were aligned with ImageJ 1.44 software, using “StackReg” plugin (“Rigid body” option).
Analysis of the fluorescent transients was performed with SPCImage (Becker & Hickl) or TRI2 (Paul Barber, University of Oxford, UK).

Loukil A., Izard F., Georgieva M., Mashayekhan S., Blanchard J.M., Parmeggiani A, & Peter M. (2016). Foci of cyclin A2 interact with actin and RhoA in mitosis. Scientific Reports, 6, 27215.

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Two-Photon FLIM Imaging of pH-Lemon Fluorescence

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Fluoresence lifetime
imaging (FLIM) was performed on an
upright fluorescence microscope (A1MP; Nikon, Amsterdam, The Netherlands)
equipped with a water immersion objective (25×; NA1.1; WD 2 mm;
Nikon). Two-photon excitation of the pH-Lemon donor mT2 was achieved
by a train of 100 fs light pulses (λ_exc = 880 nm;
80 MHz; Mai Tai DeepSee HP, Newport Spectra Physics; Irvine, CA).
Fluorescence was detected with a GaAsP hybrid photodetector (HPM-100–40;
Becker & Hickl, Berlin, Germany) after passing a bandpass filter
at 445 ± 45 nm (445BP90, Omega Optical, Brattleboro, VT, USA).
Fluorescence intensity decays were generated in every pixel of the
image using multidimensional time-correlated single photon counting
(TCSPC) employing TCSPC electronics (SPC-152; Becker & Hickl).
FLIM images were generated using SPCImage 6.1 (Becker & Hickl)
by plotting the amplitude weighted average fluorescence lifetime tau_ave
as color-coded value. tau_mean was obtained from iterative least-squares
minimizing based fitting routine of a biexponential fitting function
which was reconvoluted with the instrument response function to describe
the time course of the pixel fluorescence intensity decays properly.

Burgstaller S., Bischof H., Gensch T., Stryeck S., Gottschalk B., Ramadani-Muja J., Eroglu E., Rost R., Balfanz S., Baumann A., Waldeck-Weiermair M., Hay J.C., Madl T., Graier W.F, & Malli R. (2019). pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments. ACS Sensors, 4(4), 883-891.

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Phosphorescence Lifetime Measurements of PIr1-PIr3

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Phosphorescence lifetimes of the PIr1–PIr3 probes in solutions were measured using a confocal macro-FLIM/PLIM system (Becker & Hickl GmbH, Berlin, Germany) equipped with the hybrid detector HPM-100-40 and a single-photon counting card SPC-150 [9 (link),58 (link)]. Solutions of the probes were placed either in Eppendorf tubes (aerated solutions, 50 µM) or in sealed glass tubes (degassed solutions, 50 µM). Polymer probes PIr1–PIr3 in a concentration of 10 µM were dissolved in PBS or in phenol red-free DMEM FluoroBrite (Thermo Fisher Scientific, Waltham, MA, USA) with or without the addition of 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and imaged at room temperature.
Phosphorescence was excited with a picosecond diode laser at 375 nm (BDL-SMN-375, Becker & Hickl GmbH, Germany) and detected in the range of 607–682 nm (bandwidth filter HQ640/75, Chroma, Boston, MA, USA).
The PLIM data were processed using SPCImage 8.5 software (Becker & Hickl GmbH, Berlin, Germany). The phosphorescence decay curves were fitted with a single-exponential decay model with an average goodness of fit < 1.2. The average number of photons per curve was >5000. Image collection time was 120 s.

Parshina Y.P., Komarova A.D., Bochkarev L.N., Kovylina T.A., Plekhanov A.A., Klapshina L.G., Konev A.N., Mozherov A.M., Shchechkin I.D., Sirotkina M.A., Shcheslavskiy V.I, & Shirmanova M.V. (2022). Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor. International Journal of Molecular Sciences, 23(18), 10263.

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Multimodal Imaging of Zinc Homeostasis

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Full-thickness skin set aside for en face imaging was stained with acriflavine hydrochloride (Sigma-Aldrich, Castle Hill, Australia) 100 µM in water overnight. Skin cryosections were stained with 10 µM Zinpyr-1 (Cayman Chemical Co., Ann Arbor, MI, USA).
For multiphoton microscopy (MPM), the 488 nm argon laser was used to excite Zinpyr-1, while the 800 nm tuneable titanium-sapphire Mai-Tai was used to excite ZnPT. Emission was collected from the descanned line under a filter (520–560 nm) for single-photon excitation and a non-descanned filter (395–420 nm) for two-photon excitation. A 40X water immersion objective was used.
Fluorescence lifetime imaging microscopy (FLIM) took place along the non-descanned line of the Zeiss LSM710 microscope, fitted with two bh GaAsP hybrid detectors HPM100-40 and two TCSPC modules SPC-152 (Becker & Hickl GmbH, Berlin, Germany). The samples were excited at 740 and 800 nm. Emission was collected using a 405/10 and 540/20 nm bandpass filter (Semrock Inc., Rochester, NY, USA). Images were captured over 5 min. For en face imaging, acriflavine-stained skin was imaged from surface, 40 and 90 µm using the same FLIM parameters as for Zinpyr-1.
Zin-pyr-1 cryosections were first imaged by MPM using the tile-scan function to capture an entire follicle. FLIM images were then acquired along the length of the follicle for detection of ZnPT (λ_exc = 740 nm).

Mangion S.E., Sandiford L., Mohammed Y., Roberts M.S, & Holmes A.M. (2022). Multi-Modal Imaging to Assess the Follicular Delivery of Zinc Pyrithione. Pharmaceutics, 14(5), 1076.

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Two-Photon Microscopy for Axial Imaging

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Samples were measured on a home-built two-photon microscope based on an Axiovert 200 microscope (Zeiss, Thornwood, NY) interfaced with a Ti:Sapphire laser (Tsunami, Spectra Physics, Mountain View, CA) with an excitation wavelength of 1000 nm and a power of ~1 mW. The fluorescence was collected with a 63x C-Apochromat water immersion objective lens (NA = 1.2, Zeiss) and registered by a photodetector (HPM-100-40, Becker & Hickl, Berlin, Germany) connected to a photon counting acquisition card (ISS, Champaign, IL), which recorded data with a frequency of 20 kHz. A dichroic mirror (Chroma Technology, Rockingham, VT) served to separate excitation and emission light. The z-scan was performed by moving the stage (PZ2000 piezo stage, ASI, Eugene, OR) along the direction of the beam path [6 (link)]. The stage was driven by a voltage signal from an arbitrary waveform generator (33250A, Agilient Technologies, Santa Clara, CA). The signal waveform was a linear ramp function with a frequency of 0.1 Hz and a peak-to-peak amplitude of 0.8 V, which corresponds to 8.04 μm of axial travel. The z-scan intensity profile was sampled at 20 kHz.

Hur K.H, & Mueller J.D. (2015). Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli. PLoS ONE, 10(6), e0130063.

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Fundus Autofluorescence Lifetime Measurement

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The FLT of the fundus autofluorescence was measured with the FLIO, using a system provided by Heidelberg Engineering (Heidelberg, Germany). FLIO utilizes a picosecond (70 ps)-pulsed excitation laser (473 nm) with a repetition rate of 80 MHz to excite fluorophores, and the FLT measurement is based on the to time-correlated single photon counting (TCSPC) technique. Two highly sensitive hybrid detectors (HPM-100-40; Becker & Hickl, Berlin, Germany) register the detected emission photons in a short spectral channel (SSC: 498–560 nm) and a long spectral channel (LSC: 560–720 nm) that are connected to the TCSPC module (SPC-150, Becker & Hickl) for photon counting. Infrared laser (815 nm) eye tracking system compensates for eye movements during measurement. Image acquisition took place in a darkened room until 1000 photons per pixel were detected at the fovea centralis in both channels.

Sonntag S.R., Kreikenbohm M., Böhmerle G., Stagge J., Grisanti S, & Miura Y. (2023). Impact of cigarette smoking on fluorescence lifetime of ocular fundus. Scientific Reports, 13, 11484.

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Two-Photon Microscopy for FCS Analysis

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FCS measurements were performed on a Nikon Eclipse Ti microscope using two-photon excitation from a Ti:Sapphire pulsed laser (Mai-Tai; Spectral-Physics) with 80-MHz repetition rate and ∼70-fs pulse width at 920-nm wavelength. The excitation light was expanded and collimated to fully utilize the numerical aperture of the water immersion objective (CFI Apo 40 × WI, NA 1.25; Nikon) that focuses the light into the sample. The intensity of excitation light was modulated to 3 mW at the objective by a combination of half-wave plate and polarizing beam splitter (Thorlab). Cells were grown to 80–90% confluency on glass coverslips with #1.5 thickness, 25 mm diameter, and poly-d-lysine coating (Neuvitro). During imaging, we used a home-built temperature-controlled chamber to maintain cells at 37°C in an imaging medium, FluoroBrite DMEM (Life Technologies) supplemented with 10 mM HEPES and 2 mM l-glutamine, covered with mineral oil. A hybrid detector (HPM-100-40; Becker & Hickl) with 510/42 bandpass filter (Chroma) and TCSPC module (SPC-150; Becker & Hickl) were used to collect GFP fluorescence and calculate the normalized autocorrelation function of the fluorescence intensity fluctuations.

Hanley M.L., Yoo T.Y., Sonnett M., Needleman D.J, & Mitchison T.J. (2017). Chromosomal passenger complex hydrodynamics suggests chaperoning of the inactive state by nucleoplasmin/nucleophosmin. Molecular Biology of the Cell, 28(11), 1444-1456.

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Fluorescence Lifetime Imaging Microscopy Setup

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Fluorescence lifetime images were acquired using an inverted Zeiss (Axio Observer.Z1) LSM 780 microscope equipped with a 32 channel QUASAR GaAsP spectral array detector. In this setup the emitted photons were routed through the direct coupling (DC) confocal port of the Zeiss LSM 780 scanning unit and detected by a Becker & Hickl HPM‐100‐40 hybrid photomultiplier tube (PMT). The data were recorded by a Becker & Hickl Simple‐Tau 152 system (SPC‐150 TCSPC FLIM module) with the instrument recording software SPCM version 9.42 in the FIFO image mode using 256 time channels (Becker & Hickl GmbH, Berlin, Germany). For all acquisitions a Plan‐Apochromat 40×/1.3 Oil DIC objective lens was used, and the pinhole was set to 20.2 μm. For excitation at 405 nm a Laser diode 405 nm CW/PS with a repetition rate of 50 were utilized, whereas a pulsed tunable In Tune laser with a repetition rate of 40 MHz were used for excitation at 565 nm. Data was analyzed in SPCImage version 3.9.4 (Becker & Hickl GmbH, Berlin, Germany). Typically, decays fitted to a bi‐exponential decay and the associated life‐times and weights were used to calculate an intensity averaged life‐time for plots and comparison.^[27]

Gustafsson C., Shirani H., Leira P., Rehn D.R., Linares M., Nilsson K.P., Norman P, & Lindgren M. (2020). Deciphering the Electronic Transitions of Thiophene‐Based Donor‐Acceptor‐Donor Pentameric Ligands Utilized for Multimodal Fluorescence Microscopy of Protein Aggregates. Chemphyschem, 22(3), 323-335.

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