Advanced optical microscopy methods for in vivo imaging of sub-cellular structures in thick biological tissues

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 di®raction 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 ltering 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.

Method of lateral image reconstruction in structured illumination microscopy with super resolution

“…To solve this problem, by combining adaptive optics with SIM to compensate the aberrations, the image resolution beyond the di®raction limit can be obtained through thick tissue. [28][29][30] …”

“…30 And the SIM system should be set up stably to avoid the sample movement or beamjitter in optical path.…”

Dual-slit confocal light sheet microscopy for in vivo whole-brain imaging of zebrafish

“…Consequently, more out-of-focus fluorophores are excited, giving rise to an increase in background noise. Second, before the fluorescence is collected by the wide-field detection system, it has been scattered by the turbid medium [28]. The heavily scattered fluorescent photons received by the detector lose the position information of corresponding fluorophores, and increase image noise.…”

Line-scan focal modulation microscopy

“…[14][15][16] It employs a spatiotemporal phase modulator in the excitation light path to modulate the phase of the excitation beam spatially and temporally (at a fixed frequency). The spatially and temporally phase modulated beam, after being condensed by an objective, is subjected to intensity modulation that is essentially confined to the focal plane.…”