3 figures, 5 tables and 2 additional files

Figures

Figure 1 with 1 supplement
Comparison of the power attenuation of 1320 nm and 920 nm excitation light and their respective 3-photon and 2-photon excitation efficiency in the mouse brain.

(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 …

Figure 1—source data 1

A typical power attenuation with depth curve of 1320 nm and 920 nm excitation light in the mouse brain, plotted in Figure 1A.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig1-data1-v2.csv
Figure 1—source data 2

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig1-data2-v2.csv
Figure 1—source data 3

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig1-data3-v2.csv
Figure 1—figure supplement 1
Additional power attenuation curves of 920 nm and 1320 nm excitation light in the mouse neocortex (n = 3, >12 weeks old mice, all males).
Figure 1—figure supplement 1—source data 1

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 2 supplements
Comparison of signal-to-background ratio for 1320 nm 3PM and 920 nm 2PM in the non-sparsely labeled mouse brain and its effect on calcium imaging sensitivity.

(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 …

Figure 2—source data 1

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig2-data1-v2.csv
Figure 2—source data 2

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig2-data2-v2.csv
Figure 2—source data 3

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig2-data3-v2.csv
Figure 2—source data 4

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig2-data4-v2.csv
Figure 2—source data 5

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig2-data5-v2.csv
Figure 2—figure supplement 1
Measurement of the staining density in uniformly labeled vasculature.

(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 …

Figure 2—figure supplement 1—source data 1

The area fraction of vasculature measured in the mouse brain, plotted in Figure 2—figure supplement 1.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig2-figsupp1-data1-v2.csv
Figure 2—figure supplement 2
The pulse energy required at the brain surface to generate the same d per pulse sampling the neuron for 2PE and 3PE of GCaMP6s at different imaging depths.

In the absence of the background fluorescence, d is entirely determined by signal photon counts: when a neuron is sampled by 1000 excitation pulses in 1 s, it generates F0=1000 pulses × 0.1 photon/pulse = 100 photons/s, which gives d3. …

Figure 3 with 5 supplements
Brain heating and thermal damage induced by continuous scanning by 1320 nm 3PM.

(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 …

Figure 3—source data 1

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig3-data1-v2.zip
Figure 3—figure supplement 1
Light attenuation by immersion water for various excitation wavelengths.

(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 …

Figure 3—figure supplement 1—source data 1

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.

https://cdn.elifesciences.org/articles/53205/elife-53205-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Spatial dimension, coordinate system, and boundary conditions for Monte Carlo and heat conduction simulation.
Figure 3—figure supplement 3
The maximum brain temperature as a function of the average power at the brain surface, shown for different imaging depths at 920 nm, 1320 nm, and 1280 nm.

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 …

Figure 3—figure supplement 4
The maximum brain temperature decreases with the scanning field-of-view.

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 …

Figure 3—figure supplement 5
Immunostaining reveals brain tissue damage by 1320 nm 3PM with 150 mW imaging power after the objective lens at 1.2 mm imaging depth.

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 …

Tables

Table 1
Optical parameters of gray matter.
VariableParameterWavelengthValueUnits
μaabsorption coefficient9200.0391/mm
12800.0781/mm
13200.121/mm
μsscattering coefficient9206.71/mm
12803.21/mm
13203.21/mm
ganisotropy coefficientAll0.9dimensionless
nrefractive indexAll1.36dimensionless
Table 2
Percentage of Excitation Photons for Various Final Destinations.
920 nm1320 nm1280 nm
Contributing to heating206349
Back scattered to window25910
Back scattered to skull20911
Escaped*351930
  1. *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.

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)B6.Cg-Tg(CamK2a-tTA)1Mmay/JThe Jackson Laborartory
Stock: 007004
MGI:2179066
Strain, strain background (Mus musculus)B6;DBA-Tg(tetO-GCaMP6s)2Niell/JThe Jackson Laborartory
Stock: 024742
MGI:5553332
Antibodyanti-HSP70/72
(mouse monoclonal)
Enzo Life Sciences,
Cat# SPA-810PED
RRID:AB-2264369IHC(1:400)
Antibodyanti-GFAP (mouse monoclonal)Sigma-Aldrich,
Cat# G3893
RRID:AB_477010IHC(1:760)
Antibodyanti-Iba1 (mouse monoclonal)Sigma-Aldrich,
Cat# SAB2702364
RRID:AB_2820253IHC(1:1000)
AntibodyGoat anti-mouse
(polyclonal)
Thermo Fisher Scientific,
Cat# A-11003
RRID:AB_2534071IHC(1:500)
Software, algorithmSource 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
Table 3
Thermal and mechanical properties of gray matter (Stujenske et al., 2015).
VariableParameterValueUnits
ρDensity1.04 × 10−3g/mm3
cBrain specific heat3.65 × 103mJ/g°C
kThermal conductivity0.527mW/mm °C
ρbBlood density1.06 × 10−3g/mm3
cbBlood specific heat3.6 × 103mJ/g°C
wbBlood perfusion rate8.5 × 10−3/s
qMetabolic heat9.5 × 10−3mW/mm3
TAArterial temperature36.7°C
Appendix 1—table 1
Two- and three-photon excitation parameters.
Objective lens focal length (mm)1/e2beam diameter at the back aperture (mm)Effective NAPulse duration τ (fs)gp(n)
920 nm 2PE7.2110.75600.66
1320 nm 3PE7.2110.75600.51

Additional files

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, which was used to produce Figure 3B and C, Figure 3—figure supplements 3 and 4.

https://cdn.elifesciences.org/articles/53205/elife-53205-code1-v2.zip
Transparent reporting form
https://cdn.elifesciences.org/articles/53205/elife-53205-transrepform-v2.docx

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