Light sheet based microscopy LSM

 

Light-sheet based microscopy (LSM), also known as single plane illumination microscopy (SPIM), is a state-of-the-art microscope imaging method in which a biological sample is illuminated with a thin sheet of light—provided by a laser beam narrowed to just a few microns, or millionths of a meter, across—coming from the side rather than from above or below as with traditional light sources. Fluorescence bouncing off the illuminated sample radiates upward through a lens, gets focused and is captured by a digital camera.
Because the light sheet illuminates the part of the sample directly in the same plane, only a single section of the target is imaged at a time. Raising and lowering the illumination plane, as well as rotating the sample, rapidly produces a series of two-dimensional sectional views known as “slices” that can yield a 3-D map of a whole organism or any of its organs/systems when the individual 2-D visual pieces are brought together.
One region that scientists have tried to survey in mice with LSM is the neural pathway, the billion-fold network of neurons that underlie the functioning of the brain. While the LSM method yields high-resolution views of tissue excised from mouse brains and fixed in position, whole brain samples scatter the emitted light and create background fluorescence that reduces contrast and blurs the perceived image. Obviously, this aberration makes it impossible to resolve and reconstruct the entire neuronal network with a high contrast.
To correct the problem, scientists combined the advantages of light sheet illumination with confocal microscopy, an imaging method that uses a filter to remove photons that stray from the single plane of the thin sheet.
They found that the combined system, which they call confocal light sheet microscopy or CLSM, filtered the scattered photons that were emitted and recovered the normally lost image contrast in real time without the need for multiple acquisitions or any post-processing of the acquired data.

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