Abstract
Optical microscopy has become an indispensable tool for visualizing sub-cellular structures and biological processes. However, scattering in biological tissues is a major obstacle that prevents high-resolution images from being obtained from deep regions of tissue. We review common techniques, such as multiphoton microscopy (MPM) and optical coherence microscopy (OCM), for diffraction limited imaging beyond an imaging depth of 0.5 mm. Novel implementations have been emerging in recent years giving higher imaging speed, deeper penetration, and better image quality. Focal modulation microscopy (FMM) is a novel method that combines confocal spatial filtering with focal modulation to reject out-of-focus background. FMM has demonstrated an imaging depth comparable to those of MPM and OCM, near-real-time image acquisition, and the capability for multiple contrast mechanisms.
Highlights
Optical imaging into biological tissue is limited by absorption, and more importantly, by scattering.[1]
Another advantage of darkeld optical coherence microscopy (OCM) is that the illumination power can be increased to improve the signal level of high spatial frequency content, which is not possible with classical OCM where the illumination power is limited by the signal level of the low spatial frequencies that use most of the available dynamic range of the detection system
multiphoton microscopy (MPM) and OCM are well established as deep tissue imaging methods
Summary
Optical imaging into biological tissue is limited by absorption, and more importantly, by scattering.[1]. Therst, which is the subject of the present review, is to reject the scattered light, by some gating method In this case, resolution can be in the wavelength range, while the penetration can be a few millimeters. The scattered light is used to reconstruct This is an Open Access article published by World Scientic Publishing Company. Methods including di®use optical tomography (DOT), and in vivo °uorescence imaging In such cases, penetration can be as large as several centimeters, but resolution is severely limited to the millimeter regime, or worse. Absorption of water exhibits several windows of comparatively low absorption, at about 480, 805, 1060 and 1250 nm, and can vary by several orders of magnitude in between It increases as we progress from the visible to the infrared.
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