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
Pulse duration and dispersion
3-photon pulses typically exit the laser at ~50 fs FWHM. Group Velocity Dispersion introduced by the microscope optics and sample inevitably broaden the pulse, reducing the excitation probability. At 1300 nm, glass prism compressors are commonly used to compensate for dispersion in the microscope optics and sample, with single- and two-prism designs being common, effective and simple to construct and align.
Swamp Optics have a series of excellent tutorials introducing the measurement of pulse duration and prism compressors. Swamp Optics also provide commercial autocorrelators and compressors.
Single-prism compressor: design
In the single-prism compressor design of Akturk et al. (2006), the beam passes through the prism four times. The figure below illustrates the beam path through a slight variant on the design of Akturk et al. (2006), constructed with a single N-SF11 prism and two roof prism mirrors (Takasaki et al., 2019).
A single-prism compressor for 1300 nm dispersion compensation. (A) Schematic of 4 passes the laser beam makes through the prism, each depicted in a different colour. Passes 1 and 2 are shown separately from passes 3 and 4, for clarity. The roof prism mirrors shift the beam slightly in one dimension. RP1 shifts the beam in the horizontal plane. RP2 shifts the beam in the vertical plane, towards the table by several millimeters. RP2 returns the beam along an equivalent path back through the prism. On pass 4 the beam reverses the route of pass 1, albeit at a slightly different height above the table. Likewise, pass 3 reverses the route of pass 2. (B) Side-view of the prism, illustrating the relative positions of the beams on the prism and also the height of the D-mirror used to pick off the output beam after the 4th pass. (C) Photograph of table optics, including the compressor. HWP half wave plate mounted on rotation stage. PB polarizing beamsplitter. Table hole spacing is 1".
Single-prism compressor: construction and alignment
For the single prism compressor, construction is facilitated by first aligning the laser beam with the rail, reversing the final direction of travel (panel A, below). A pinhole mounted on a rail carriage facilitates this step. Insertion of the prism then indicates the location of the entry mirror for the compressor (panel B), adjustment of which produces a beam aligned with the rail after passing through the prism (panel C). Again, a pinhole mounted on a rail carriage is of assistance when adjusting the mirror. Addition of the remaining components is similar to that of a two-prism compressor: https://www.newport.com/medias/sys_master/images/images/hfb/hdd/9520061349918/DS-11065-Apps-Note-29-new-temp.pdf
Single-prism compressor: operation
Translation of RP1 away from the prism increases GVD compensation, introducing negative GVD. For example, Takasaki et al. (2019) used prism-to-RP1 separations of 44 and 92 cm to correct 4500 fs^2 and 11000 fs^2 GVD.
GVD compensation is optimized to minimize the pulse width. Pulse width can be measured by interferometric autocorrelation, with a commercial table-top free-space autocorrelator such as the APE PulseCheck. A table-top autocorrelator is usually positioned after the table optics and before the periscope, the last location at which the beam is static, collimated and of an appropriate diameter to enter the autocorrelator. In this configuration, tuning the compressor will correct for dispersion introduced by the table optics, but not the galvanometers, scan, tube or objectives lenses or the sample. Much of the total GVD is often in the table top components, making a table-top autocorrelator an excellent tool for course tuning of the compressor.
The remaining microscope components, particularly scan, tube and objective lenses can introduce modest, but undesirable GVD. Fine tuning of the compressor, to compensate GVD introduced by the remaining microscope components, is often desirable. A simple and effective solution method to fine tune the compressor prism - RP1 distance is to maximize the brightness of a fluorescent test sample, such as a fluorescein-filled cuvette.
In principle, one could dispense with the autocorrelator and simply tune the compressor to maximize sample brightness. Unfortunately this one-step approach can be confounded by local maxima in sample brightness as one tunes the compressor, raising the possibility that the final dispersion correction will be sub-optimal. Without a measure of pulse width to verify that the compressor configuration is in approximately the appropriate range, there's often considerable uncertainty surrounding the compressor and pulse width.
Measurement of pulse duration in the sample plane
Maximizing the brightness of a fluorescein-filled cuvette optimizes dispersion compensation, but doesn't provide an estimate of the pulse width in the sample plane. The pulse width in the sample can be estimated with a Michelson interferometer, a relatively simple device that can be constructed on the vibration isolation table from commercial parts and added to the beam path of a 3-photon microscope (Takasaki et al., 2019). The Michelson interferometer uses a half-silvered mirror to split the beam in two then adds a delay to one beam before collinear recombination of the two beams. The pulse duration can be estimated from the effects of the delay on fluorescence intensity. Again, a fluorescein-filled cuvette is a suitable sample at 1300 nm. In principle, a Michelson autocorrelator can be used to measure pulse duration when the microscope is focused into a biological preparation, enabling correction of pulse dispersion introduced by the tissue.
Third and higher-order dispersion
Third order dispersion has been observed from some laser sources (Farinella et al., 2020) and may be induced by passage through microscope optics and optical fibers (Klioutchnikov et al., 2020). Third and higher order dispersion may increase pulse duration, decreasing 3-photon excitation.