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Two-photon microscopy in brain tissue: parameters influencing the imaging depth.
M Oheim1, E Beaurepaire, E Chaigneau
1Laboratoire de Neurophysiologie et Nouvelles Microscopies, INSERM EPI 00-02, Ecole Supérieure de Physique et Chimie Industrielles, 10 Rue Vauquelin, 75005, Paris, France. martin.oheim@espci.fr
Journal of Neuroscience Methods
|September 28, 2001
Summary
Light scattering in brain tissue limits two-photon microscopy depth. Optimizing optical collection efficiency can significantly increase imaging depth for functional brain imaging.
Area of Science:
- Neuroscience
- Biomedical Optics
- Microscopy
Background:
- Light scattering in biological tissues, particularly brain tissue, impedes deep imaging with two-photon microscopy.
- This scattering affects both the excitation of fluorescence and the subsequent collection of emitted photons, limiting functional brain imaging in vivo.
Purpose of the Study:
- To investigate the impact of light scattering on fluorescence excitation and collection in two-photon microscopy.
- To identify key tissue and instrument parameters that dictate imaging depth in the brain.
Main Methods:
- Measured scattering length in juvenile and adult cortical brain slices at 800 nm.
- Analyzed fluorescence collection efficiency in a simulated in vivo detection geometry.
- Compared different objective lenses (magnification and numerical aperture) for their effect on fluorescence collection.
Main Results:
- Scattering length in juvenile cortical tissue was found to be twice that of adult tissue.
- Collected fluorescence fraction decreases with depth, proportional to the square of angular acceptance.
- Matching microscope angular acceptance to the objective lens increased collection efficiency by ~3x at depths >500 µm.
- A 20x, 0.95 NA objective increased fluorescence collection ~10x compared to standard 60x-63x objectives, without compromising resolution.
Conclusions:
- Optimizing optical collection parameters, such as angular acceptance and objective choice, can significantly enhance imaging depth in scattering brain tissue.
- These improvements enable deeper functional brain imaging, potentially allowing measurements of neuronal or vascular activity over 100 µm deeper than with conventional methods.