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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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Simultaneous two-wavelength transmission quantitative phase microscopy with a color camera.

Matthew T Rinehart¹, Natan T Shaked, Nathan J Jenness

¹Department of Biomedical Engineering, Fitzpatrick Institute for Photonics, Duke University, Durham, North Carolina 27708, USA. matt.rinehart@duke.edu

|August 4, 2010

Summary

We developed a quantitative phase microscopy technique using a color camera and a two-wavelength algorithm. This method accurately measures optical path delays in microstructures, overcoming common phase ambiguities.

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Area of Science:

Optics
Microscopy
Biophysics

Background:

Quantitative phase microscopy (QPM) is crucial for label-free imaging of transparent specimens.
Traditional QPM methods often face limitations in phase ambiguity and measurement range.
Simultaneous multi-wavelength acquisition can address these limitations.

Purpose of the Study:

To present a novel QPM method enabling extended optical path delay measurements.
To demonstrate the technique's capability in resolving phase ambiguities in microstructures.
To quantify phase noise introduced by spectral crosstalk in color cameras.

Main Methods:

Utilizing a Bayer mosaic color camera for simultaneous off-axis interferogram acquisition at two wavelengths.
Implementing a two-wavelength algorithm for processing wrapped phase information.
Experimental validation using optically clear microstructures.

Main Results:

Successfully acquired phase profiles of microstructures without 2pi ambiguities.
Demonstrated extended range of optical path delay measurements in a single acquisition.
Quantified the phase noise contribution from spectral channel crosstalk.

Conclusions:

The presented two-wavelength QPM method effectively overcomes phase ambiguities and extends measurement range.
This technique offers a robust solution for precise optical path delay measurements of transparent samples.
Understanding spectral crosstalk is important for optimizing color camera-based phase imaging systems.