研究目的
To develop a confocal laser scanning microscope for 3D cellular imaging in the NIR-II window and demonstrate its deep tissue penetration capabilities.
研究成果
The developed NIR-II confocal microscope achieves deep tissue imaging up to 800 μm in biological samples and 3.5 mm in artificial scattering media, with high spatial resolution. Future improvements could include better optical components, adaptive optics, and additional background rejection techniques to further enhance penetration depth and image quality.
研究不足
The system is based on a modified visible-light confocal setup, which may have suboptimal transmission for NIR-II light; photodetectors have high dark counts; organic dyes like IR-1061 have low quantum yield; sample aberration and photobleaching limit signal quality; background noise can overwhelm weak signals at great depths.
1:Experimental Design and Method Selection:
Adapted a Nikon C1 confocal setup for NIR-II wavelengths, using a pulsed laser diode and gated InGaAs/InP single-photon detection module to handle low light levels and reduce noise.
2:Sample Selection and Data Sources:
Used HeLa cells labeled with PbS QDs and chicken cartilage tissue labeled with IR-1061 dye, prepared with specific protocols for fixation and staining.
3:List of Experimental Equipment and Materials:
Includes laser diode, detection module, optical fibers, objective lens, and samples like Lipofundin solution.
4:Experimental Procedures and Operational Workflow:
Cells and tissues were labeled, mounted, and imaged with optimized parameters such as detection width and laser pulse settings; z-stack imaging was performed for 3D reconstruction.
5:Data Analysis Methods:
Images were acquired and processed using Nikon NIS-Elements software and ImageJ for 3D rendering; signal decay was analyzed with exponential fitting.
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Laser Diode
LU0808M250
Lumics GmbH Berlin
Excitation light source for fluorescence imaging
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Single-Photon Detection Module
id201
id Quantique
Detection of NIR-II fluorescence emissions at single-photon level
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Pulsed Laser Diode Driver
ALPLDC001
ALPHANOV
Driving the laser diode with nanosecond pulses and providing synchronization triggers
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Objective Lens
40X/0.8 NIR water dipping
Nikon
Focusing excitation light and collecting fluorescence from samples
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Confocal Microscope
Nikon C1
Nikon
Base system modified for NIR-II imaging, including scanning and control
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Optical Fiber
50μm core graded-index multimode
Coupling fluorescence emissions to the detection module for improved light collection
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Dichroic Mirror
1050nm long pass
Reflecting excitation light and transmitting fluorescence emissions
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Emission Filter
900nm long pass
Filtering out excitation light and passing NIR-II emissions
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Quantum Dots
PEG-NH2 PbS QDs
Creative Diagnostics
Fluorescent labeling agent for cells with NIR-II emission
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Organic Dye
IR-1061
Sigma
Fluorescent labeling agent for cell membranes in NIR-II range
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Lipofundin
Scattering medium to simulate biological tissue properties
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