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Super-resolution Fluorescence Microscopy

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...

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Updated: May 10, 2025

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

Design of Multiplexed, Live Cell Imaging Experiments Using Excitation Scan-Based Hyperspectral Imaging Microscopy.

Naga Annamdevula^1,2, Rebecca Tang-Holmes^1,2, Robert LeDoux²

¹Pharmacology, University of South Alabama, Mobile, AL 36688.

Proceedings of Spie--The International Society for Optical Engineering

|April 28, 2025

Summary

This summary is machine-generated.

This study provides protocols for selecting fluorescent labels for excitation scan-based hyperspectral imaging (HSI). Researchers can now better quantify multiple proteins and dyes simultaneously within cells using this advanced microscopy technique.

Keywords:

HSI Hyperspectral imaging multiplexed fluorescence imaging spectral microscopy

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Area of Science:

Cellular and Molecular Biology
Microscopy and Imaging Technologies
Biophotonics

Background:

Advances in microscopy and fluorescent probes have improved cellular tracking.
Real-time multiplexed imaging remains a challenge in live-cell studies.
Excitation scan-based hyperspectral imaging (HSI) offers potential for multiplexed imaging but lacks standardized protocols.

Purpose of the Study:

To address the lack of protocols for selecting multiple fluorescent labels for excitation scan-based HSI.
To outline considerations for choosing fluorescent labels and optimizing HSI parameters.
To demonstrate the capability of HSI for simultaneous quantification of various cellular components.

Main Methods:

Developed and utilized a custom-built excitation scan-based HSI microscope with tunable filters (360-550 nm).
Transfected HEK-293 cells with fluorescent proteins and loaded cells with specific dyes.
Measured excitation spectra of individual and combined fluorescent labels.

Main Results:

Successfully quantified the relative abundance and spatial distribution of multiple fluorophores, including NucBlue, AlexaFluor dyes, Cal dyes, and fluorescent proteins (GFP, Cerulean, Turquoise, Venus, tdTomato, mCherry).
Demonstrated the effectiveness of excitation scan-based HSI for distinguishing and measuring individual and combined labels.
Established a foundation for selecting optimal label combinations for multiplexed cellular imaging.

Conclusions:

Excitation scan-based HSI is a powerful technique for multiplexed cellular imaging.
Standardized protocols for label selection can enhance the adoption and application of HSI.
This work facilitates more comprehensive real-time analysis of protein dynamics and cellular activities.