(A) Power attenuation of 920 nm and 1320 nm excitation light in the mouse brain. The mouse brain vasculature was uniformly labeled with fluorescein dextran and imaged simultaneously by 920 nm 2PM …
A typical power attenuation with depth curve of 1320 nm and 920 nm excitation light in the mouse brain, plotted in Figure 1A.
The excitation pulse energy at the brain surface required to generate 0.1 signal photon per pulse at different depths in the mouse brain, measured in fluorescein-labeled vasculature and plotted in Figure 1B.
The excitation pulse energy at the brain surface required to generate 0.1 signal photon per pulse at different depths in the mouse brain, measured in the neurons of transgenic animals (CamKII-tTA/tetO-GCaMP6s) and plotted in Figure 1B.
Additional power attenuation with depth curves of 1320 nm and 920 nm excitation light in the mouse neocortex, plotted in Figure 1—figure supplement 1.
(A) Comparison of 3PM and 2PM images of fluorescein-labeled blood vessels at different depths. Scale bar 30 μm. (B) SBR measured simultaneously by 1320 nm 3PE and 920 nm 2PE on fluorescein-labeled …
The change of signal-to-background ratio with depth of 1320 nm 3PM and 920 nm 2PM in the mouse brain, measured in fluorescein-labeled vasculature and plotted in Figure 2B.
The change of signal-to-background ratio with depth of 1320 nm 3PM and 920 nm 2PM in the mouse brain, measured in the neurons of transgenic animals (CamKII-tTA/tetO-GCaMP6s) and plotted in Figure 2B.
Calcium traces recorded by 920nm 2PM on GCaMP6s-labeled neurons at different depths in transgenic animals (CamKII-tTA/tetO-GCaMP6s), based on which Figure 2—source data 5 is derived.
Calcium traces recorded by 1320nm 3PM simultaneously on the same GCaMP6s-labeled neurons as in Figure 2—source data 3 in transgenic animals (CamKII-tTA/tetO-GCaMP6s), based on which Figure 2—source data 5 is derived.
The ratio of calcium transient ΔF/F between simultaneously recorded by 1320 nm 3PM and 920 nm 2PM calcium traces, on the same GCaMP6s-labeled neurons as described in Figure 2—source datas 3 and 4.
This data is plotted in Figure 2D.
(A) Z-projection of the identified blood vessels by summing all the XY-images in a vertical image stack (0–400 μm deep, with 10 μm step size). Scale bar, 30 μm. (B) The fractional vascular area in …
The area fraction of vasculature measured in the mouse brain, plotted in Figure 2—figure supplement 1.
In the absence of the background fluorescence, is entirely determined by signal photon counts: when a neuron is sampled by 1000 excitation pulses in 1 s, it generates , which gives . …
(A) Monte Carlo simulation of light intensity of 1320 nm excitation light. The excitation light is focused at 1 mm below the brain surface in the cortex by an objective of 1.05 NA at ~75% filling of …
Quantification of the staining intensity of immunolabeld mouse brain slices post mortem after the exporsure to continuous 1320 nm 3PM scanning, plotted in Figure 3E.
(A) Power transmission versus immersion water thickness under the objective (Olympus XLPLN25XWMP2, 25X, NA=1.05, ~ 75% filling of the back aperture) for 5 spectra centered from 1280 nm to 1420 nm …
Power transmission through immersion water of different thicknesses under the objective lens, measured with different excitation spectra and plotted in Figure 3—figure supplement 1.
The imaging depth grows in the increment of the nominal EAL of the cortex, which is 150 μm for 920 nm and 300 μm for both 1320 nm and 1280 nm. The linear scanning FOV diameter is 300 μm, and other …
The simulation was performed for 1320 nm excitation wavelength at 1- and 1.2 mm imaging depth with various average powers after the objective lens. The NA and the back-aperture beam size remain the …
The scanning was performed with 230 μm FOV for 20 min at various average powers in the mouse brain. The window and scanning area are on the right side of each brain slice, and the location of …
Variable | Parameter | Wavelength | Value | Units |
---|---|---|---|---|
absorption coefficient | 920 | 0.039 | 1/mm | |
1280 | 0.078 | 1/mm | ||
1320 | 0.12 | 1/mm | ||
scattering coefficient | 920 | 6.7 | 1/mm | |
1280 | 3.2 | 1/mm | ||
1320 | 3.2 | 1/mm | ||
anisotropy coefficient | All | 0.9 | dimensionless | |
refractive index | All | 1.36 | dimensionless |
920 nm | 1320 nm | 1280 nm | |
---|---|---|---|
Contributing to heating | 20 | 63 | 49 |
Back scattered to window | 25 | 9 | 10 |
Back scattered to skull | 20 | 9 | 11 |
Escaped* | 35 | 19 | 30 |
*Photons escape the simulation volume by traveling too far (>6 mm) from the center of the cranial window or too deep (>6 mm) from the tissue surface.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus) | B6.Cg-Tg(CamK2a-tTA)1Mmay/J | The Jackson Laborartory Stock: 007004 | MGI:2179066 | |
Strain, strain background (Mus musculus) | B6;DBA-Tg(tetO-GCaMP6s)2Niell/J | The Jackson Laborartory Stock: 024742 | MGI:5553332 | |
Antibody | anti-HSP70/72 (mouse monoclonal) | Enzo Life Sciences, Cat# SPA-810PED | RRID:AB-2264369 | IHC(1:400) |
Antibody | anti-GFAP (mouse monoclonal) | Sigma-Aldrich, Cat# G3893 | RRID:AB_477010 | IHC(1:760) |
Antibody | anti-Iba1 (mouse monoclonal) | Sigma-Aldrich, Cat# SAB2702364 | RRID:AB_2820253 | IHC(1:1000) |
Antibody | Goat anti-mouse (polyclonal) | Thermo Fisher Scientific, Cat# A-11003 | RRID:AB_2534071 | IHC(1:500) |
Software, algorithm | Source code 1: matlab code for simulating the brain temperature distribution under continuous long-wavelength illumination by 3PM using Monte Carlo method and heat equation. | Mathworks, Matlab 2016b | RRID:SCR_001622 |
Variable | Parameter | Value | Units |
---|---|---|---|
Density | 1.04 × 10−3 | g/mm3 | |
Brain specific heat | 3.65 × 103 | mJ/g°C | |
Thermal conductivity | 0.527 | mW/mm °C | |
Blood density | 1.06 × 10−3 | g/mm3 | |
Blood specific heat | 3.6 × 103 | mJ/g°C | |
Blood perfusion rate | 8.5 × 10−3 | /s | |
Metabolic heat | 9.5 × 10−3 | mW/mm3 | |
Arterial temperature | 36.7 | °C |
Objective lens focal length (mm) | 1/e2beam diameter at the back aperture (mm) | Effective NA | Pulse duration (fs) | ||
---|---|---|---|---|---|
920 nm 2PE | 7.2 | 11 | 0.75 | 60 | 0.66 |
1320 nm 3PE | 7.2 | 11 | 0.75 | 60 | 0.51 |
Matlab code for simulating the brain temperature distribution under continuous long-wavelength illumination by 3PM using Monte Carlo method and heat equation, which was used to produce Figure 3B and C, Figure 3—figure supplements 3 and 4.