研究目的
To develop an optimized three-photon microscope for deep-tissue functional imaging in awake mice, overcoming limitations of two-photon microscopy, and to characterize visual responses across cortical layers and subplate neurons.
研究成果
The optimized three-photon microscope enables damage-free, deep-tissue functional imaging in awake mice with low laser power. It reveals distinct visual response properties across cortical layers and, for the first time, in subplate neurons, showing broader tuning in layer 5 and sharper tuning in layer 6, with subplate neurons being less responsive. This advances in vivo neuroscience studies and provides insights into cortical processing.
研究不足
The study is limited to mouse models and specific cortical regions (V1). The microscope design may not be directly applicable to other tissues or species. High repetition rates could increase heating risks, and pulse energy must be carefully controlled to avoid damage. The method requires specialized equipment and expertise.
1：Experimental Design and Method Selection:
Designed a custom three-photon microscope with optimized laser parameters (1300 nm wavelength, 40 fs pulse width, 800 kHz repetition rate) for deep imaging. Used Zemax software for optical modeling and included pre-chirp system and delay line to enhance performance.
2：Sample Selection and Data Sources:
Used adult mice (2-6 months old) of both sexes, including Thy1-GFP line M and wild-type C57BL6 mice. Injected AAV2/
3：Syn.GCaMP6s.WPRE.SV40 virus to label neurons in VList of Experimental Equipment and Materials:
Femtosecond laser (Spirit, Spectra Physics), NOPA (Spectra Physics), galvanometric mirrors (6215H, Cambridge Technologies), objective lens (XLPN25XWMP2, Olympus), PMTs (H7422A-40 and R7600U-200, Hamamatsu), scan and tube lenses, motorized stages (MMBP, Scienti?ca), and various optical components (e.g., waveplates, dichroic mirrors).
4：Experimental Procedures and Operational Workflow:
Performed craniotomy and virus injection in mice. Conducted in vivo imaging with visual stimuli (oriented gratings) at depths up to 1000 μm. Used laser ablation for optical property measurements and layer marking. Acquired images with ScanImage software.
5：Data Analysis Methods:
Analyzed images using custom scripts in ImageJ and MATLAB. Calculated orientation selectivity indices (gOSI, OSI, DSI) and performed statistical tests (t-tests, normality tests).
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Photomultiplier tubes
H7422A-40
Hamamatsu
Detect GCaMP6s, GFP, and retrobeads fluorescence signals.
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Photomultiplier tubes
R7600U-200
Hamamatsu
Detect THG signal.
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Half-wave plate
AHWP05M-1600
Thorlabs
Control laser power.
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Polarizing cube beam splitter
UFBS2080
Thorlabs
Control laser power with high extinction ratio.
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Dichroic mirror
FF705-Di01
Semrock
Separate emitted light in the collection path.
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Dichroic mirror
Di02-R488
Semrock
Separate emitted light in the collection path.
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Dichroic mirror
FF555-Di03
Semrock
Separate emitted light in the collection path.
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Laser blocking filter
FF01-670/SP
Semrock
Block laser light in emission path.
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Nonlinear imaging filter
FF01-433/24-25
Semrock
Filter for specific emission wavelengths.
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Nonlinear imaging filter
FF03-525/50
Semrock
Filter for specific emission wavelengths.
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Nonlinear imaging filter
FF01-630/92
Semrock
Filter for specific emission wavelengths.
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Objective lens
XLPN25XWMP2
Olympus
High transmission objective for deep imaging.
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Femtosecond laser
Spirit
Spectra Physics
Pump laser for generating ultrashort pulses at 1045 nm to pump the NOPA.
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Noncollinear optical parametric amplifier
NOPA
Spectra Physics
Generates excitation wavelength of 1300 nm for three-photon imaging.
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Galvanometric mirrors
6215H
Cambridge Technologies
Scan laser beams for imaging.
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Motorized stage
MMBP
Scienti?ca
Position mice and objective for imaging.
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