The 2-photon calcium imaging setup was identical to a previously published design15 (link). Two-photon illumination was achieved with a Ti:Sapphire laser (Chameleon Vision II, Coherent) operating at 920nm. Fluorescence was acquired using a 40× 0.8 NA objective (LUMPLFLN40X/W, Olympus) and GaAsP PMTs (H10770PA-40, Hamamatsu) after passing through a dichroic (FF670-SDi01, Semrock), an IR filter (FF01–720sp, Semrock), reflected by a second dichroic (FF562-Di03, Semrock) and passing through a final bandpass filter (FF01–520/60, Semrock). The PMT output signal was amplified (Variable High Speed Current Amplifier; #59–179, Edmund Optics) and digitized (PXIe-7961R FlexRIO, National Instrument). The microscope was controlled by ScanImage (Vidrio Technologies) software using additional analog output units (PXIe-6341, National Instruments) for the laser power control and the scanners control. Double-distilled water was used as the immersion medium for the objective. Average beam power measured at the front of the objective was 60–160 mW. The region between the objective and imaging site was shielded from external sources of light using a black rubber tube. Horizontal scans of the laser were achieved using a resonant galvanometer (Thorlabs). Typical fields of view measured approximately 500×500 μm, and data was acquired at 30Hz.
Ff562 di03
Manufactured by IDEX Corporation
The FF562-Di03 is a lab equipment product manufactured by IDEX Corporation. It serves as a flow meter that measures and monitors the flow rate of liquid or gas through a system. The device utilizes digital technology to provide accurate and reliable flow measurements.
Automatically generated - may contain errors
Lab products found in correlation
B scope, by Thorlabs (2 mentions)
Ff03 525 50, by IDEX Corporation (2 mentions)
Ff01 607 70, by IDEX Corporation (2 mentions)
H10770pa 40, by Hamamatsu Photonics (1 mentions)
Resonant galvanometer, by Thorlabs (1 mentions)
Ff01 720 sp, by IDEX Corporation (1 mentions)
Pxie 6341, by National Instruments (1 mentions)
Ff670 sdi01, by IDEX Corporation (1 mentions)
Lumplfln 40xw, by Olympus (1 mentions)
Insight ds laser, by Spectra-Physics (1 mentions)
Xlplan n 25, by Olympus (1 mentions)
H7422pa 40, by Hamamatsu Photonics (1 mentions)
5 protocols using ff562 di03
1
Two-Photon Calcium Imaging Setup
2
Two-Photon Microscopy of Awake Mice
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2PM was performed on awake head-fixed mice, using a resonant scanner-based Bergamo B-scope (Thorlabs) with an InSight® DS + ™ laser (Spectra-Physics) with a 25x CFI plan apochromatic long working distance objective (Nikon, NA 1.10). GCaMP imaging was achieved at an excitation wavelength of 910 nm. Emission light was split by a 562 nm dichroic mirror (FF562-Di03, Semrock) and band-pass filtered by 525/50 nm (FF03, Semrock) and 607/70 nm (FF01, Semrock) into separate GaAsP PMTs. Images were acquired using ThorImage software at 30 Hz with a temporal averaging of 6 images to a final sampling rate of 5 Hz at a 512 ×512 pixel, 16-bit resolution.
3
Two-Photon Imaging in Anaesthetized Mice
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Two-photon imaging was performed with urethane-anaesthetized adult mice (as above), using a resonant scanner-based B-Scope (Thorlabs) with a Chameleon Vision 2 laser (Coherent, wavelength 920 nm) and an Olympus objective lens (XLPlan N × 25). The B-Scope is equipped with a reverse dichroic mirror (ZT405/488/561/680-1100rpc, Chroma) and the emission light was separated by using a dichroic mirror (FF562-Di03, Semrock), with band-pass filters FF03-525/50 and FF01-607/70 (both from Semrock) for the green and red channels, respectively. Images were acquired using the ThorImage software with a frame rate of 30 Hz.
4
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.
5
Two-Photon Imaging of Anesthetized Mice
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Two-photon imaging was performed on urethane-anesthetized adult mice (as above) using a resonant scanner-based B-Scope (Thorlabs) with a Chameleon Vision 2 laser (coherent, wavelength 920 nm) and an Olympus objective lens (XLPlan N 25×) as described before18 ,26 (link). The B-Scope is equipped with a reverse dichroic mirror (ZT405/488/561/680-1100rpc, Chroma) and the emission light was separated by a dichroic mirror (FF562-Di03, Semrock) with bandpass filters FF03-525/50 and FF01-607/70 (both from Semrock) for the green and red channels, respectively. Images were acquired using the ThorImage software at a frame rate of 30 Hz.
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