Abstract
With the recent developments in optical imaging tools and techniques, scientists are now able to image deeper regions of the tissue with greater resolution and accuracy. However, light scattering while imaging deeper regions of a biological tissue remains a fundamental issue. Presence of lipids, proteins and nucleic acids in the tissue makes it inhomogeneous for a given wavelength of light. Two-photon fluorescence (TPF) microscopy supplemented with improved invasive optical tools allows functional imaging in awake behaving mammals in an unprecedented manner. Similarly, improved optical methods conjugated with previously existing scanning laser ophthalmoscopy (SLO) has paved diffraction-limited retinal imaging. With the evolving technology, scientists are now able to resolve biological structures and function at the sub-cellular level. Wavefront correcting methods like adaptive optics (AO) has been implemented in correcting tissue or optical-based distortions, shaping the excitation beam in 3D-holography to target multiple neurons. And more recently, AO-based SLO is implemented for eye imaging both in research and clinical settings. In this review, we discuss some of the recent improvements in TPF microscopy with the application of AO for wavefront corrections and its recent application in brain imaging as well as ophthalmoscopy.
Highlights
In confocal microscopy the 3D resolution is mainly achieved from the light originating from focus and not from the scattered light, a common problem associated mainly while imaging deep tissues [1,2,3]
Since its usage in correcting atmospheric aberrations in telescopes, adaptive optics (AO)-based optical imaging has paved its way into biological imaging
With AO, the diffraction-limited resolution could be achieved by correcting deep-tissue mediated aberrations
Summary
In confocal microscopy the 3D resolution is mainly achieved from the light originating from focus and not from the scattered light, a common problem associated mainly while imaging deep tissues [1,2,3]. Linear (one-photon) excitation microscopy is mainly suited for near-surface (100 μm with minimal scattering and better resolution. Deep tissue imaging at the single-cell level has provided adequate information on synaptic activity and function. The improvements in the optical approach have developed techniques that allow non-labeled imaging of tissues with high sensitivity, specificity, and spatial resolution under in vivo and in vitro conditions. Non-linear microscopy, in particular, TPF supplemented by the recent advances in optical labeling of deep tissues has become the method. It is best suited for gaining cellular level information from monolayer cultures
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