Augmented line-scan focal modulation microscopy for multi-dimensional imaging of zebrafish heart in vivo

Abstract

Multi-dimensional fluorescence imaging of live animal models demands strong optical sectioning, high spatial resolution, fast image acquisition, and minimal photobleaching. While conventional laser scanning microscopes are capable of deep penetration and sub-cellular resolution, they are generally too slow and causing excessive photobleaching for volumetric or time-lapse imaging. We demonstrate the performance of an augmented line-scan focal modulation microscope (aLSFMM), a high-speed imaging platform that affords above video-rate imaging speed by the use of line scanning. Exceptional background rejection is accomplished by combining a confocal slit with focal modulation. The image quality is further improved by merging the information from simultaneously acquired focal modulation and confocal images. Such a hybrid imaging scheme makes it possible to use very low power excitation light in high-speed imaging, and therefore leads to reduced photobleaching that is desirable for three-dimensional (3D) and four-dimensional (4D) in vivo image acquisition.

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

“…This problem can be alleviated by introducing additional background rejection mechanisms beyond spatial filtering provided by confocal detection [41][42][43]. For example, focal modulation has been demonstrated to be very effective in suppressing scattering induced background in visible light confocal microscopy [44][45][46][47][48][49][50].…”

Multifunctional laser speckle imaging

“…We believe that the improved contrast and spatial resolution in LS-LSCI are achieved by the use of line illumination and confocal line detection, which are combined to enhance sensitivity to local flow. Similar to its fluorescence microscopy and optical coherence tomography counterparts, the line scan confocal laser speckle imaging method enjoys the intrinsical optical sectioning capability [29][30][31].…”

Quadrature demodulation in line‐scan focal modulation microscopy for imaging three‐dimensional zebrafish neural structure

“…In the field of signal processing, the most common method to demodulate a modulated carrier wave is to use a product detector . This method was also used in the previous LSFMM image demodulation . Basically, it multiplies the modulated image with a mask image with the same frequency f 0 to produce a copy of the original focal component and a modulated component at twice the carrier frequency 2 f 0 .…”

“…Thus, it is believed to be an excellent microscopic tool for basic biological research, translational research, as well as medical diagnosis. The prototyped line‐scan focal modulation microscope (LSFMM) has already proved its high‐performance in in vivo imaging of zebrafish model, such as zebrafish liver and zebrafish heart . Nowadays, zebrafish model has been more and more popular in neurobiological study due to its high optical transparency and small nervous system at larva stage .…”