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Confocal Fluorescence Microscopy

Confocal Fluorescence Microscopy

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

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A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Published on: May 30, 2016

Line-scanning hyperspectral imaging based on structured illumination optical sectioning.

Yu John Hsu¹, Chih-Chiang Chen², Chien-Hsiang Huang¹

¹Department of Optics and Photonics, National Central University, No.300, Zhongda Rd., Zhongli Dist., Taoyuan City 32001, Taiwan.

Biomedical Optics Express

|July 1, 2017

Summary

This summary is machine-generated.

Structured illumination enhances line-scanning hyperspectral imaging (LHSI) contrast by reducing out-of-focus signals. This technique improves axial imaging in biological tissues, enabling better spectral separation of fluorophores.

Keywords:

(110.4234) Multispectral and hyperspectral imaging (170.2520) Fluorescence microscopy (180.5810) Scanning microscopy

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Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Published on: August 22, 2019

Area of Science:

Optics and Photonics
Biomedical Imaging
Spectroscopy

Background:

Line-scanning hyperspectral imaging (LHSI) offers high acquisition rates but limited sectioning capability compared to point-scanning methods.
Enhancing axial imaging contrast in LHSI is crucial for detailed analysis of biological samples.
Off-focus and scattered fluorescence signals can degrade image quality in conventional LHSI.

Purpose of the Study:

To improve the axial imaging contrast of line-scanning hyperspectral imaging (LHSI).
To investigate the effectiveness of integrating structured illumination into line excitation for fluorescence signal isolation.
To demonstrate the capability of the enhanced LHSI system for spectral unmixing of complex samples.

Main Methods:

Integration of structured illumination into the line excitation pathway of LHSI.
Application of the enhanced LHSI technique to unsectioned leaf and sectioned mouse skin tissues.
Spectral data acquisition with a resolution of 1.15 nm.
Linear unmixing algorithms applied to resolve overlapping fluorophore spectra.

Main Results:

Significant enhancement in imaging contrast was achieved: 8-fold in unsectioned leaf and 4.5-fold in sectioned mouse skin.
Structured illumination effectively removed off-focus and scattered on-focus fluorescence signals.
Fluorophores with severely overlapped spectra were successfully separated without cross-talk using linear unmixing.

Conclusions:

Structured illumination is a highly effective method for enhancing axial imaging contrast in LHSI.
The developed technique improves the performance of LHSI for imaging biological tissues.
High spectral resolution and linear unmixing enable precise spectral discrimination of fluorophores in complex biological environments.