Downloads
Resources available for download include 3-photon data sets and software tools.
Data set: near-simultaneous 2- and 3-photon excitation
Takasaki et al. (2020) generated near-simultaneous 2- and 3-photon
fluorescence movies to compare imaging modalities.
Movies are available for download from the CRCNS data sharing site.
GCaMP6s fluorescence.
910 and 1300 nm excitation.
400 x 200 µm field of view.
~3.6 Hz frame rate.
220-650 µm below the pial surface
of mouse primary visual cortex.
Downloads
Resources available for download include 3-photon data sets and software tools.
Data set: near-simultaneous 2- and 3-photon excitation
Takasaki et al. (2020) generated near-simultaneous 2- and 3-photon
fluorescence movies to compare imaging modalities.
Movies are available for download from the CRCNS data sharing site.
GCaMP6s fluorescence.
910 and 1300 nm excitation.
400 x 200 µm field of view.
~3.6 Hz frame rate.
220-650 µm below the pial surface
of mouse primary visual cortex.
Downloads
Resources available for download include 3-photon data sets and software tools.
Data set: near-simultaneous 2- and 3-photon excitation
Takasaki et al. (2020) generated near-simultaneous 2- and 3-photon
fluorescence movies to compare imaging modalities.
Movies are available for download from the CRCNS data sharing site.
GCaMP6s fluorescence.
910 and 1300 nm excitation.
400 x 200 µm field of view.
~3.6 Hz frame rate.
220-650 µm below the pial surface
of mouse primary visual cortex.
Downloads
Resources available for download include 3-photon data sets and software tools.
Data set: near-simultaneous 2- and 3-photon excitation
Takasaki et al. (2020) generated near-simultaneous 2- and 3-photon
fluorescence movies to compare imaging modalities.
Movies are available for download from the CRCNS data sharing site.
GCaMP6s fluorescence.
910 and 1300 nm excitation.
400 x 200 µm field of view.
~3.6 Hz frame rate.
220-650 µm below the pial surface
of mouse primary visual cortex.
Downloads
Resources available for download include 3-photon data sets and software tools.
Data set: near-simultaneous 2- and 3-photon excitation
Takasaki et al. (2020) generated near-simultaneous 2- and 3-photon
fluorescence movies to compare imaging modalities.
Movies are available for download from the CRCNS data sharing site.
GCaMP6s fluorescence.
910 and 1300 nm excitation.
400 x 200 µm field of view.
~3.6 Hz frame rate.
220-650 µm below the pial surface
of mouse primary visual cortex.
Downloads
Resources available for download include 3-photon data sets and software tools.
Data set: near-simultaneous 2- and 3-photon excitation
Takasaki et al. (2020) generated near-simultaneous 2- and 3-photon
fluorescence movies to compare imaging modalities.
Movies are available for download from the CRCNS data sharing site.
GCaMP6s fluorescence.
910 and 1300 nm excitation.
400 x 200 µm field of view.
~3.6 Hz frame rate.
220-650 µm below the pial surface
of mouse primary visual cortex.
The resource for 3-photon excitation
Imaging speed
Imaging pixel and frame rates are typically lower for 3- than for 2-photon excitation.
In multiphoton excitation, one pulse drives more excitation than the equivalent power distributed across multiple pulses, in the absence of fluorophore saturation (Prevedel et al., 2016; Weisenburger et al., 2019). One pulse per pixel also maximizes the acquisition rate.
Pulses per pixel across a 256 pixel line with a 4 kHz resonant galvanometer.
The rate of 3-photon image acquisition is fundamentally limited by the repetition rate of the laser. Lasers suitable for 3-photon excitation commonly have repetition rates in the 1-4 MHz range, therefore permitting acquisition from one to four million pixels per second at one pulse per pixel. For a 512 x 512 pixel image, a 1 MHz laser therefore supports 1,000,000 / (512 x 512) = 3.81 images per second with a linear galvanometer.
Faster image rates can be acquired with resonant galvanometers. With resonant galvanometers, the speed at which the laser is swept across the sample changes across the scan field, resulting in fewer pulses per pixel in the center of the field of view than toward the edges. For a 256 x 256 image acquired with a 4 kHz resonant galvanometer, 4 MHz is the minimum pulse rate required for at least one pulse at every pixel. 4 MHz lasers are available, permitting 3-photon excitation at ~32 images per second.