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Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

Published on: April 7, 2014

STED microscope with spiral phase contrast.

Marcel A Lauterbach¹, Marc Guillon, Asma Soltani

¹Wavefront Engineering Microscopy Group, Neurophysiology and New Microscopies Laboratory, CNRS UMR 8154, INSERM S603, University Paris Descartes, Sorbonne Paris Cité, Paris, France.

Scientific Reports

|June 22, 2013

Summary

This summary is machine-generated.

This study integrates Spiral Phase Contrast (SPC) microscopy with Stimulated Emission Depletion (STED) microscopy. This dual-mode imaging allows simultaneous super-resolution and label-free phase contrast visualization of biological specimens.

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Area of Science:

Biophysics
Microscopy
Cell Biology

Background:

Stimulated Emission Depletion (STED) microscopy provides super-resolution imaging of fluorescently labeled nanostructures.
Relating super-resolved structures to unlabeled features in biological samples remains a challenge.
Optical similarities between Spiral Phase Contrast (SPC) and STED microscopes suggest potential for integration.

Purpose of the Study:

To integrate a label-free phase contrast imaging channel into a STED microscope.
To enable dual-mode imaging (super-resolution and phase contrast) in fixed and live biological specimens.
To evaluate the performance of integrated SPC and STED microscopy.

Main Methods:

Implemented Spiral Phase Contrast (SPC) imaging into a STED microscope setup.
Utilized both widefield and scanning modes for SPC and STED imaging.
Imaged living neurons stained with Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP).

Main Results:

Successfully demonstrated dual imaging with STED and SPC channels, allowing overlay of contrast modes.
Achieved simultaneous super-resolution and label-free phase contrast imaging in living neurons.
Found that scanning confocal SPC provided superior optical contrast compared to widefield SPC.

Conclusions:

The integration of SPC into STED microscopy is straightforward and feasible.
This dual-mode approach overcomes limitations in correlating labeled and unlabeled cellular structures.
Scanning confocal SPC offers enhanced contrast, useful for contour detection and highlighting cellular features.