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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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Updated: Jun 20, 2026

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

Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator.

Reto Fiolka¹, Markus Beck, Andreas Stemmer

¹Department of Mechanical and Process Engineering, ETH Zurich, Zurich, Switzerland.

|July 17, 2008

Summary

This summary is machine-generated.

Standing wave illumination in total internal reflection fluorescence microscopy enhances lateral resolution over twofold. This versatile setup achieves 91 nm resolution by controlling illumination fields with a spatial light modulator and diffraction grating.

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Area of Science:

Microscopy
Optics
Biophysics

Background:

Wide-field fluorescence microscopy offers valuable insights into biological systems.
Achieving higher lateral resolution is crucial for detailed cellular imaging.
Total internal reflection fluorescence microscopy (TIRFM) provides enhanced signal-to-noise ratio.

Purpose of the Study:

To develop a versatile setup for standing wave illumination in TIRFM.
To improve lateral resolution in fluorescence microscopy.
To enable fine control over illumination penetration depth.

Main Methods:

Utilizing a phase-only spatial light modulator (SLM) to create an adjustable diffraction grating.
Implementing standing wave illumination within a TIRFM setup.
Selecting specific diffraction orders and employing sheared gratings for penetration depth tuning.

Main Results:

Achieved a twofold increase in lateral resolution.
Demonstrated fine step control over the illumination penetration depth.
Attained a lateral resolution of 91 nm for green emission.

Conclusions:

The developed setup offers a versatile and effective method for enhancing lateral resolution in TIRFM.
The use of SLM-controlled diffraction gratings provides precise control over illumination parameters.
This technique significantly advances the capabilities of fluorescence microscopy for high-resolution imaging.