Article Figures & Data
Figures
- Figure 1.
Contrast declines with depth with two-photon excitation. A, Example three-photon images from 300, 600, 900, 1100, and 1400 µm below the pial surface of visual cortex. Emx1-IRES-Cre;CaMK2a-tTA;Ai94 mouse. B, Comparison of images acquired from a single Emx1-IRES-Cre;CaMK2a-tTA;Ai94 mouse (different fields of view) using two-photon and three-photon excitation, focused 200–600 µm below the pial surface of visual cortex.
- Figure 2.
Implementation of near-simultaneous two-photon and three-photon excitation. A, Schematic of the optical layout for near-simultaneous two-photon and three-photon excitation; 1300-nm beam (black) passed a Pockels cell (PC), prism compressor, a collimating telescope, combining dichroic mirror (CD), x-y galvanometer pair (G), scan lens (SL), tube lens (TL), FF735-DI02 primary dichroic mirror (PD), and objective lens; 910-nm beam (red) passed a PC, beam expansion to ∼1 cm in diameter, electrically-tunable lens (ETL), 0.3× beam expansion before being reflected by the combining dichroic mirror onto the galvanometer pair. B, Scanning strategy for near-simultaneous two-photon and three-photon excitation. Red: 920-nm excitation, no 1300-nm excitation. Black: no 920-nm excitation, 1300-nm excitation. Gray: both lasers blocked. Lines were sorted into two-photon and three-photon images. C, Example images from 350 µm below the pia, with traces from two somata.
- Figure 3.
Changes in two-photon image quality and apparent ΔF with depth. A–D, Plots of image brightness (A), contrast (B), corrected motion (C), and ROI count (D) for two-photon (red) and three-photon excitation (black), plot as a function of depth below the pial surface of cortex. Mean ± SEM of three experiments from two Slc17a7-Cre;Ai162 mice. E, ROI match (percentage of three-photon ROIs also segmented from two-photon images) as a function of depth. F, Pearson correlation coefficient between two-photon and three-photon fluorescence traces, plot as a function of depth. G, Two-photon and three-photon changes in fluorescence to grating stimuli for two neurons, 350 and 500 µm below the pia. Each panel shows change in fluorescence (in arbitrary fluorescence units) through time during presentation of the drifting grating (icon to left indicates orientation and direction) for 2 s (gray bar). Eight individual traces and the mean (thick line) per direction. Dashed line indicates zero fluorescence. Below, Resulting direction tuning curve. H, Plots comparing preferred direction of neurons measured with two-photon (y-axis) and three-photon (x-axis) excitation, for each depth. Colors indicate percentages of the total number of neurons at each depth (zero is white, 10% is black, see color bar). Directions progress at 30° intervals from the low left corner of each plot (icons). J, Percentage of neurons with matching direction preferences measured with two-photon and three-photon excitation, from 200 to 650 µm. Dashed line: 8.3%.
- Figure 4.
In-focus and out-of-focus fluorescence. A, Percentage of total fluorescence that originates from the focal plane, plot as a function of depth of the focal plane below the brain surface. Each point represents a single measurement (from a movie at one depth in one mouse). Lines are calculated from Equation 1 with scattering length constants of 200 µm (black) and 150 µm (gray). Dashed lines are relationships from the literature for scattering lengths constants of 200 µm: Equation 4 of Theer and Denk (2006) for two-photon excitation and Equation 7 of Xu and Webb (1996) for ballistic three-photon excitation. B, Plots showing the depth from which fluorescence originates with the focal plane at 200, 400, 600, and 800 µm below the brain surface. Fluorescence was calculated with Equation 1 and normalized to that in the focal plane. Note the difference in scale for two-photon and three-photon excitation. C, Breakdown of sources underlying the total fluorescence in panel B (Theer and Denk, 2006). Two-photon: 900-nm illumination, scattering length 200 µm (Eq. 1). Three-photon: 1300-nm illumination, scattering length 200 µm, equivalent formulation. Colors: fluorescence from ballistic incident photons (light green), from scattered photons (dark blue) and from a mixture of ballistic and scattered photons (cyan and deep green). Black: the sum of all fluorescence sources (reproduced in panel B).
Movies
- Movie 1.
Examples of simultaneous two-photon and three-photon image pairs at different depths. Examples of matched two-photon and three-photon movies 250, movies 450, and movies 650 μm below the pia. Two-photon and three-photon movie pairs were acquired pseudo-simultaneously. Each movie was acquired at a different illumination intensity and each was scaled differently for display purposes. Slc17a7-Cre;Ai162 mouse. Movies acquired at 8 Hz. Playback at 20 Hz.
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