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, Pavone and his colleagues 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. @“We found that the combined system, which we 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,” Pavone says. “This allows us to obtain sharp image slices that can be easily reconstructed into a macroscopic view of whole organs or entire organisms with a resolution of a few microns and faster acquisition time.” @The images obtained with CLSM, Pavone says, are 100 percent sharper than those acquired with conventional LSM. @The researchers validated the background rejection capabilities of CLSM by showing that it results in significantly sharper images than conventional LSM microscopy and than a variation of LSM that requires redundant slices and post-processing to remove scattered light. CLSM proved superior to the other two methods when used to view three samples from mouse brains: the hippocampus, the cerebellum and the whole brain. The 3-D reconstruction ability of CLSM also was put to the test when the researchers used it to map the micron-scale neuroanatomy of mouse Purkinje cells (large neurons found in the cerebellum) and trace an entire brain’s neuronal projections (the finger-like ends of a neuron by which neurochemical signals are received from other nerves).

Visit the explicative Web page

News by Baldanzi Elisabetta