Fundamentally, LSFM techniques are made possible by decoupling the illumination and detection optical pathways, allowing for novel illumination strategies that optimize the photon collecting efficiency of the instrument, this general concept is illustrated by Figure 1 above. Light Sheet Fluorescence Microscopy ( LSFM) is a general name for a constantly growing family of planar illumination techniques that have revolutionized how optical imaging of biological specimens can be performed. (b) A typical episcopic illumination and detection geometry, where both excitation and detection are performed using a common objective and light path. Fluorescence is detected by the higher NA detection objective, oriented orthogonally to the illumination objective and the projected light sheet. (a) LSFM geometry with a low numerical aperture (NA) illumination objective projecting the light sheet into the sample. Figure 1 - Illumination and Detection GeometriesĬomparison of the illumination and detection geometries of LSFM compared to epifluorescence. Imaging such large and sensitive specimens thus necessitates an efficient approach to 3D imaging that minimizes exposure of the sample to light.
Light-induced photodamage and phototoxicity have long been problematic in the field of biological imaging, and can have quite a dramatic effect on health and function at all biological levels of organization. Such systems, including whole model organisms, tissue explants, and three-dimensional ( 3D) cell cultures, must furthermore be imaged in a manner minimizing perturbations in order to maintain their physiological integrity as experimental models.
Biological imaging has been moving consistently towards experimentation utilizing increasingly physiologically relevant systems for many years, and new technologies are making such experimental approaches increasingly feasible. The modern world of biological light microscopy is a demanding one, increasingly sophisticated research applications require instrumentation capable of keeping pace.
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