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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

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Published on: December 11, 2021

Calibrating differential interference contrast microscopy with dual-focus fluorescence correlation spectroscopy.

Claus B Müller¹, Kerstin Weiss, Walter Richtering

¹Institute of Physical Chemistry II, RWTH-Aachen University, Landoltweg 2, 52056 Aachen, Germany. mueller@pc.rwth-aachen.de

|June 11, 2008

Summary

We developed a new method to precisely measure the shear distance of Nomarski prisms. This calibration technique is crucial for accurate imaging in microscopy and spectroscopy applications.

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Area of Science:

Optical microscopy
Spectroscopy
Nanotechnology

Background:

Nomarski Differential Interference Contrast (DIC) prisms are essential components in DIC microscopy and dual-focus fluorescence correlation spectroscopy (FCS).
Accurate knowledge of the shear distance introduced by the Nomarski prism is critical for quantitative data analysis in these techniques.
In DIC microscopy, shear distance impacts spatial resolution; in dual-focus FCS, it defines the length scale for diffusion coefficient measurements.

Purpose of the Study:

To present a novel and highly accurate calibration technique for determining the shear distance of Nomarski DIC prisms.
To provide a method that enhances quantitative data evaluation in DIC microscopy and dual-focus FCS.

Main Methods:

The calibration technique utilizes a combination of fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS).
This integrated approach allows for precise measurement of the shear distance induced by the Nomarski prism.

Main Results:

The presented method achieves nanometer accuracy in determining the shear distance.
The technique is demonstrated to be straightforward to implement.

Conclusions:

The novel calibration method offers a reliable way to quantify Nomarski prism shear distance.
This advancement is vital for improving the precision of measurements in DIC microscopy and dual-focus FCS, particularly for diffusion studies.