Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle

Abstract

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

Temporal focusing‐based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging

“…(5) could be adopted for the HiLo technique, which would provide better reconstructed image contrast due to less background noise being involved in the original image. However, the HiLo reconstruction algorithm for two-dimensional modulated illumination patterns requires more complicated computation and reconstruction procedures as compared with those of the HiLo algorithm for one-dimensional modulated illumination patterns [34,35]. The algorithm is at present under consideration.…”

“…The low-frequency component, on the other hand, can be retrieved by processing the patterned illumination image according to the subsequent high-pass filtering, demodulation, and low-pass filtering [34,35]. Figure 6(a) shows the original TPEF image of the eosin-stained mouse cerebellar cortex via the TFMPEM.…”

“…With a single spatial frequency sinusoidal pattern, a well axially resolved widefield image can be analyzed with only a few images at different phase shifts [33]. HiLo microscopy combines the high spatial frequency component from a high-pass filtered image with the low spatial frequency component from the demodulated information of a patterned illumination image to improve the contrast of reconstructed widefield images [34,35]. In doing so, widefield images with rejection of the out-of-focus background can be obtained/produced [34].…”

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

“…Resonant scanners can help increase the frame rate to more than 30 Hz, but at the expense of reduced field of view and non-uniform illumination. Numerical postprocessing based background rejection methods, such as structured illumination microscopy [3], HiLo [4], and differential spinning disk microscopy [5], have been reported in the past decade. While these new techniques can provide high image speed, they suffer from limited dynamic range when imaging thick tissues, which is attributed to the fact that no physical device is present to prevent background photons from reaching the imaging sensor.…”

High speed, line-scanning, fiber bundle fluorescence confocal endomicroscopy for improved mosaicking

“…However, as HRME is non-sectioning, the range of applications is more limited [13]. Some degree of optical sectioning can be introduced using structured illumination [14,15], but this tends to produce noisier images from thick tissue, and as a multi-shot approach is subject to motion artefacts and a reduced net frame rate.…”