Confocal Light Sheet Microscopy Improves Imaging of Neural Pathways

Confocal Light Sheet Microscopy Improves Imaging of Neural Pathways

This shows micron-scale neuroanatomy of a whole thy1-GFP-M brain. (a) Isosurface perspective (P) and transverse (T), coronal (C) and sagittal (S) contours of an entire PND-15 mouse brain. The volumes highlighted by the blue and red boxes are magnified in panel (b) and (c) respectively. (b) Volume rendering of a portion of hippocampus. (c) Volume rendering of a portion of superior colliculus. (d) Soma segmentation and process tracing of selected fluorescent neurons present in the red box (c). For clarity, each neuron was drawn with a different color. Scale bars, 200 µm. Credit: Optics Express

Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies.

A popular method of studying neural pathways in brains of laboratory animals has been to image them with light-sheet based microscopy (LSM), an optical technique that uses tightly focused thin sheets of light emanating from a laser to illuminate only narrow crossections of a sample. LSM works well for small tissue samples, but when imaging something even as large as a mouse brain, the additional tissue scatters too much light to maintain a high quality image. Now a team of Italian researchers have overcome much of the image degradation due to scattering by introducing confocal microscopy into the mix and calling the new technique confocal light sheet microscopy (CLSM).

2fjf888f83 Confocal Light Sheet Microscopy Improves Imaging of Neural Pathways

This shows Purkinje cells from a mouse cerebellum imaged (a) with light-sheet microscopy and (b) with the significantly higher contrast provided by confocal light-sheet microscopy. The scale bar at the bottom is 100 micrometers across. Credit: Optics Express/European Laboratory for Non-Linear Spectroscopy, University of Florence, Italy

Confocal microscopy provides higher resolution imaging over traditional (wide-field) microscopy by using a tiny slit to limit the volume that is being looked at. Coupling it with LSM allows scientists to penetrate an whole mouse brain with a sheet of light while only looking at the slice being imaged.

From the study abstract:

The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies.

Optical Society of America press release: Novel microscopy method offers sharper view of brain’s neural network

Source : http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-20-18-20582

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