Figures and data in Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain

Version of Record: February 18, 2020
Accepted Manuscript: January 30, 2020

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Tianyu Wang

Chunyan Wu

Dimitre G Ouzounov

Wenchao Gu

Fei Xia

Minsu Kim

Xusan Yang

Melissa R Warden

Chris Xu

(2020)
Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain

eLife 9:e53205.

https://doi.org/10.7554/eLife.53205
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Figures
Tables
Additional files

3 figures, 5 tables and 2 additional files

Figures

Figure 1 with 1 supplement

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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
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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
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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
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Figure 1—figure supplement 1

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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
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Figure 2 with 2 supplements

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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
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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
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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
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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
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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
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Figure 2—figure supplement 1

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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
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Figure 2—figure supplement 2

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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 $F_{0} = 1000 p u l s e s \times 0.1 p h o t o n / p u l s e = 100 p h o t o n s / s$ , which gives $d^{'} \sim 3$ . …

Figure 3 with 5 supplements

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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
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Figure 3—figure supplement 1

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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
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Figure 3—figure supplement 2

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Spatial dimension, coordinate system, and boundary conditions for Monte Carlo and heat conduction simulation.

Figure 3—figure supplement 3

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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

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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

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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.

Variable	Parameter	Wavelength	Value	Units
$μ_{a}$	absorption coefficient	920	0.039	1/mm
		1280	0.078	1/mm
		1320	0.12	1/mm
$μ_{s}$	scattering coefficient	920	6.7	1/mm
		1280	3.2	1/mm
		1320	3.2	1/mm
$g$	anisotropy coefficient	All	0.9	dimensionless
$n$	refractive index	All	1.36	dimensionless

Table 2

Percentage of Excitation Photons for Various Final Destinations.

	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.

Key resources table

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

Table 3

Thermal and mechanical properties of gray matter (Stujenske et al., 2015).

Variable	Parameter	Value	Units
$ρ$	Density	1.04 × 10⁻³	g/mm³
$c$	Brain specific heat	3.65 × 10³	mJ/g°C
$k$	Thermal conductivity	0.527	mW/mm °C
$ρ_{b}$	Blood density	1.06 × 10⁻³	g/mm³
$c_{b}$	Blood specific heat	3.6 × 10³	mJ/g°C
$w_{b}$	Blood perfusion rate	8.5 × 10⁻³	/s
$q$	Metabolic heat	9.5 × 10⁻³	mW/mm³
$T_{A}$	Arterial temperature	36.7	°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 NA	Pulse duration $τ$ (fs)	$g_{p}^{(n)}$
920 nm 2PE	7.2	11	0.75	60	0.66
1320 nm 3PE	7.2	11	0.75	60	0.51

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

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.

Figure 1—source data 1

Figure 1—source data 2

Figure 1—source data 3

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

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.

Figure 2—source data 1

Figure 2—source data 2

Figure 2—source data 3

Figure 2—source data 4

Figure 2—source data 5

Measurement of the staining density in uniformly labeled vasculature.

Figure 2—figure supplement 1—source data 1

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.

Brain heating and thermal damage induced by continuous scanning by 1320 nm 3PM.

Figure 3—source data 1

Light attenuation by immersion water for various excitation wavelengths.

Figure 3—figure supplement 1—source data 1

Spatial dimension, coordinate system, and boundary conditions for Monte Carlo and heat conduction simulation.

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 maximum brain temperature decreases with the scanning field-of-view.

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

Tables

Optical parameters of gray matter.

Percentage of Excitation Photons for Various Final Destinations.

Thermal and mechanical properties of gray matter (Stujenske et al., 2015).

Two- and three-photon excitation parameters.

Additional files

Source code 1

Transparent reporting form

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Cite this article

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.

Figure 1—source data 1

Figure 1—source data 2

Figure 1—source data 3

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

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.

Figure 2—source data 1

Figure 2—source data 2

Figure 2—source data 3

Figure 2—source data 4

Figure 2—source data 5

Measurement of the staining density in uniformly labeled vasculature.

Figure 2—figure supplement 1—source data 1

The pulse energy required at the brain surface to generate the same d′<math id="inf20"><mstyle displaystyle="true" scriptlevel="0"><mrow><msup><mi>d</mi><mo>′</mo></msup></mrow></mstyle></math> per pulse sampling the neuron for 2PE and 3PE of GCaMP6s at different imaging depths.

Brain heating and thermal damage induced by continuous scanning by 1320 nm 3PM.

Figure 3—source data 1

Light attenuation by immersion water for various excitation wavelengths.

Figure 3—figure supplement 1—source data 1

Spatial dimension, coordinate system, and boundary conditions for Monte Carlo and heat conduction simulation.

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 maximum brain temperature decreases with the scanning field-of-view.

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

Optical parameters of gray matter.

Percentage of Excitation Photons for Various Final Destinations.

Thermal and mechanical properties of gray matter (Stujenske et al., 2015).

Two- and three-photon excitation parameters.

Source code 1

Transparent reporting form

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.