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Related Experiment Video

Updated: Jun 16, 2026

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Published on: April 7, 2023

Depth of field multiplexing in microscopy.

Christian Maurer¹, Saranjam Khan, Stephanie Fassl

¹Innsbruck Medical University, Division for Biomedical Physics, Müllerstrasse 44, 6020 Innsbruck, Austria.

|February 23, 2010

Summary

This summary is machine-generated.

This study introduces depth of field multiplexing, a microscopy technique using a spatial light modulator (SLM) to image multiple focal planes simultaneously in one camera exposure. This enables real-time 3D motion recording in biological samples.

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Area of Science:

Microscopy and Imaging Technologies
Biophysics
Cell Biology

Background:

Standard microscopy techniques often require sequential imaging of different focal planes, limiting real-time observation of dynamic 3D processes.
Capturing rapid motion within a three-dimensional (3D) sample volume typically necessitates complex setups or compromises in resolution and speed.

Purpose of the Study:

To develop and demonstrate a novel microscopy method for simultaneous multi-focal plane imaging.
To enable high-speed, real-time recording of dynamic events within a 3D sample volume using a single camera exposure.

Main Methods:

Implementation of depth of field multiplexing using a high-resolution spatial light modulator (SLM) placed in the Fourier plane of a standard microscope.
Design of a phase mask on the SLM comprising superposed multi-focal off-axis Fresnel lenses to direct different focal planes to distinct camera regions.
Utilizing a single camera exposure to capture images from multiple focal planes simultaneously.

Main Results:

Successful demonstration of simultaneous imaging of distinct focal planes within a sample.
Achieved real-time recording of 3D motion, exemplified by observing cytoplasmic streaming in plant cells and the movement of protozoa.
The SLM-based phase mask effectively separated and focused different focal planes onto non-overlapping camera chip sections.

Conclusions:

Depth of field multiplexing offers a powerful approach for high-speed, 3D live-cell imaging.
This technique significantly enhances the capability of standard microscopes to capture dynamic biological processes in three dimensions.
The method provides a valuable tool for studying rapid cellular and subcellular movements in real-time.