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Total Internal Reflection Fluorescence Microscopy

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.

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Related Experiment Video

Updated: Jun 25, 2025

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Published on: January 18, 2017

High-throughput fluorescence quantification method based on inner filter effect and fluorescence imaging analysis.

Wanxiang Li¹, Yuchao Fu¹, Tianyuan Liu²

¹Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China.

Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy

|May 22, 2024

Summary

This summary is machine-generated.

This study introduces a new method for quantifying fluorescent substances using the inner filter effect (IFE) and fluorescence imaging. The developed high-throughput system accurately measures high concentrations of rhodamine B, improving fluorescence detection capabilities.

Keywords:

Fluorescence Fluorescence imaging High-throughput Inner filter effect (IFE)Quantification method

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Area of Science:

Analytical Chemistry
Spectroscopy
Biomedical Instrumentation

Background:

The inner filter effect (IFE) is increasingly utilized for fluorescent substance determination.
Previous research on the spatial micro-element method provides a foundation for understanding fluorescence distribution.

Purpose of the Study:

To derive a theory for fluorescence distribution along the excitation light path.
To propose a high-concentration and wide-range fluorescent substance quantification method using IFE and fluorescence imaging analysis.
To construct a high-throughput fluorescent substance quantification detection system.

Main Methods:

Derivation of fluorescence distribution theory based on the spatial micro-element method.
Development of a quantification method utilizing the relationship between fluorescence intensity summation and position.
Construction of a high-throughput system for simultaneous measurement of multiple samples.
Validation using rhodamine B solutions to assess concentration prediction and experimental influences.

Main Results:

Successful high-concentration (100-600 mg/L) and wide-range quantification of rhodamine B with high precision (R² = 0.9992, MRE = 2.34%).
Demonstration of simultaneous measurement of six samples.
Capability to measure fluorescent substances with different emission wavelengths by adjusting the filter wheel.

Conclusions:

The proposed method and system enable accurate, high-throughput quantification of fluorescent substances.
The system has potential applications in biomedicine, dye research, and enhancing fluorescence quantitative PCR instruments.
Future improvements aim to reduce sample volume and increase overall sample throughput.