Demultiplexing of fluorescence emission is performed by time resolving the excitation contributions of each of the four multiplexed beams with fast photon-counting electronics. The fluorescence signal detected is an interwoven stream of photons excited by all four excitation beams. That signal can be time gated with 3.3 ns increments to effectively isolate the signal contributions from each beam and consequently isolate the signal contribution in space as well. Emission demultiplexing is performed electronically via TCSPC analysis. Fluorescence signals are detected by a cooled GaAsP photomultiplier tube with 5-mm square active area (H7422PA-40, Hamamatsu) in non-descanned configuration. The current output from the PMT is amplified through a 2 GHz cutoff bandwidth preamplifier (HFAC-26, Becker and Hickl GmbH) and sent into a photon-counting board (SPC-150, Becker and Hickl GmbH) to be counted and correlated to the 76-MHz reference clock of the laser oscillator. Given the electronics setup our fundamental timing resolution is on the order of the instrument response frequency, which was measured to be 230 ps full-width at half-maximum (Supplementary Fig. 12 ).
H7422pa 40
Manufactured by Hamamatsu Photonics
The H7422PA-40 is a photomultiplier tube (PMT) module produced by Hamamatsu Photonics. It is designed to convert light signals into electrical signals. The H7422PA-40 has a spectral response range of 185 to 650 nanometers and a maximum supply voltage of 1100 volts.
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Lab products found in correlation
Spc 150n, by Becker & Hickl (1 mentions)
Tia60, by Thorlabs (1 mentions)
Galvanometer, by Thorlabs (1 mentions)
Maitai, by Spectra-Physics (1 mentions)
Uplsapo60x, by Olympus (1 mentions)
Ff562 di03, by IDEX Corporation (1 mentions)
Ff01 520 70, by IDEX Corporation (1 mentions)
Pci 6110, by National Instruments (1 mentions)
Xlumplfln20xw, by Olympus (1 mentions)
Sr570, by Stanford Research Systems (1 mentions)
7 protocols using h7422pa 40
1
Fluorescence Emission Demultiplexing and Timing
2
Multiphoton Fluorescence Lifetime Imaging of Cellular Metabolism
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Fluorescence lifetime images of NAD(P)H and FAD in cells were captured by a customized multiphoton fluorescence lifetime microscope (Marianas, 3i) equipped with a time-correlated single photon counting (TCSPC) electronics module (SPC-150N, Becker & Hickl). A stage-top incubator (okolab) was set to 37°C, 5% CO2, and 85% relative humidity to maintain a physiological environment while imaging. NAD(P)H and FAD in macrophages were excited by a tunable Ti: sapphire femtosecond laser (COHERENT, Chameleon Ultra II) at 750 nm (∼27 mW) and 890 nm (∼35 mW) respectively. The fluorescence lifetime images of NAD(P)H and FAD were obtained sequentially by photomultiplier tube (PMT) detectors (HAMAMATSU, H7422PA-40) and isolated by a 447/60 nm bandpass filter and a 560/88 nm bandpass filter, respectively. For each imaging dish, both NAD(P)H and FAD fluorescence lifetime images were captured in at least five random positions, and each fluorescence lifetime image (256 × 256 pixels) was acquired with a pixel dwell time of 50 μs and 5 frame repeats. The NAD(P)H and FAD fluorescence lifetime images in the cancer cell metabolic inhibitor experiment were previously collected by AJ Walsh and L. Hu with the same imaging system, and the details of autofluorescence imaging are covered in Hu et al. (Hu et al., 2022 ).
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Two-Photon Imaging System Setup
4
Femtosecond Laser Trapping and Lifetime Quantification
5
Two-photon Bleaching of GECIs
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Two-photon bleaching measurements of green and red GECIs were acquired from isolated aqueous droplets of protein using a resonant-galvo scanning 2-photon microscope (MPM-200, Thorlabs, Newton, NJ). The microscope was equipped with a 40x 1.15 NA water immersion objective (Nikon), a primary dichroic (680–1600 nm longpass filter, Thorlabs), and a secondary dichroic (FF562-Di03, Semrock) with green (530/43, Semrock) and red (605/70, Chroma, Bellows Falls, VT) filters each followed by GaAsP PMTs (H7422PA-40, Hamamatsu). Laser excitation was at 1000 nm or 1070 nm for red GECIs, and 940nm for GCaMP6s. To completely bleach the protein droplets (thickness, 5 μm) repetitive z-stacks were taken. Beam scan area was 160 μm x 160 μm, and the scan rate was 8 frames/sec. Light intensity was kept constant across measurements (Figure 2—figure supplement 2 ). However, due to the use of different wavelengths, the laser spot size, and therefore the intensity profile, was slightly different across these measurements.
6
Two-Photon Imaging of GCaMP6s Fluorescence
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Imaging was performed on a custom two-photon microscope controlled using ScanImage 3.8 (45 (link)). A Ti-Sapphire laser (Chameleon Ultra II, Coherent) tuned to 920 nm was used to excite GCaMP6s. The laser was focussed at the sample using a 20× 1.0–numerical aperture (NA) objective lens (XLUMPLFLN20XW, Olympus), downstream of 50- and 300-mm focal length scan and tube lenses (AC-300-050B and AC-508-300-B, Thorlabs). Scanning was achieved using 3-mm aperture scan mirrors (6215H, Cambridge Technology). Fluorescence emission from GCaMP was band-pass–filtered (FF01-520/70, Semrock) and detected with a GaAsP PMT (H7422PA-40, Hamamatsu). Output from the PMT was amplified using a preamplifier (SR570, Stanford Research Systems) and acquired using the same data acquisition card used to drive the scan mirrors (PCI-6110, National Instruments). Data acquisition from the microscope was triggered by a TTL pulse generated at the beginning of each trial by an Arduino connected to a separate computer running the behavior protocol. A continuous time series of two-photon images was acquired for every trial of the experimental protocol. Multitrial protocols had an ITI of 30 s. Imaging was discontinued for 24 s of the 30-s interval.
7
Two-Photon Imaging with Galvo-Resonant Scanner
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The imaging path is based on an 8-kHz galvo-resonant commercial 2P design (Bergamo I Series, Thorlabs, Newton, NJ, United States) seeded by a Ti:Sapphire source (Chameleon Ultra II, Coherent). For imaging GCaMP6, the source is tuned at 920 nm and a water dipping Nikon CFI75 LWD 16X W objective is used for all the acquisitions. The fluorescence collection path includes a 705 nm long-pass main dichroic, an IR blocking filter and a 495 nm long-pass dichroic mirrors transmitting the fluorescence light toward a high sensitivity GaAsP PMT detector (H7422PA-40, Hamamatsu) equipped with EM525/50 emission filter. The PMT signal is sampled at 400 MHz by a fast digitizer board (Alazar Tech Board) and the images, acquired at 30 frames per second, stored in the PC in a .raw file format. The commercial system includes a control unit (MCU) which provides the analog waveforms driving the galvo-resonant scanner and other TTL synchronization signals. In particular, a 30 Hz TTL signal, named Frame Sync, marks the beginning of the scanning along the slow axis (Y), i.e., the beginning of the image acquisition and is used to synchronize the ETL scanning.
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