Two-Photon Fluorescence Imaging Protocol

Two-photon fluorescence measurements were obtained with a modified movable objective microscope (MOM) (Sutter instruments, Novator, CA) and made using an Olympus ×60, 1.00 NA, LUMPlanFLN objective (Olympus America, Melville, NY) for single-cell resolution imaging (field of view, FOV: 203 × 203 µm) or a Nikon ×16, 0.80 NA, N16XLWD-PF objective (Nikon, Tokyo, Japan) for large FOV (850 × 850 um) imaging. Two-photon excitation was evoked with an ultrafast pulsed laser (Chameleon Ultra II; Coherent) tuned to 920 nm to image Cal520, GCaMP6s, and tdTomato. Laser power was set between 6.5 and 12 mW for imaging of Cal520 and tdTomato expression. The microscope system was controlled by the ScanImage software (https://www.scanimage.org/). Scan parameters were [pixels/line × lines/frame (frame rate in Hz)]: [256 × 256 (1.48 Hz)], at 2 ms/line. This MOM was equipped with a through-the-objective light stimulation and two detection channels for fluorescence imaging.

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Voufo C., Chen A.Q., Smith B.E., Yan R., Feller M.B, & Tiriac A. (2023). Circuit mechanisms underlying embryonic retinal waves. eLife, 12, e81983.

Publication 2023

Cell Chameleon Fluorescence Frame Light stimulation Microscope Scan Tdtomato

Corresponding Organization :

Other organizations : University of California, Berkeley

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Variable analysis

independent variables

Laser power (set between 6.5 and 12 mW)

dependent variables

Fluorescence imaging of Cal520, GCaMP6s, and tdTomato expression

control variables

Objective used for single-cell resolution imaging (Olympus ×60, 1.00 NA, LUMPlanFLN)
Objective used for large field of view imaging (Nikon ×16, 0.80 NA, N16XLWD-PF)
Two-photon excitation wavelength (920 nm)
Scan parameters (256 × 256 pixels, 1.48 Hz frame rate, 2 ms/line)

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