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

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Published on: April 7, 2014

Numerical refractive index correction for the stitching procedure in tomographic quantitative phase imaging.

Piotr Stępień¹, Michał Ziemczonok¹, Małgorzata Kujawińska¹

¹Warsaw University of Technology, Institute of Micromechanics and Photonics, ul. Sw. A. Boboli 8, Warsaw, 02-525, Poland.

Biomedical Optics Express

|February 3, 2023

Summary

Quantitative phase imaging (QPI) struggles with refractive index (RI) baselines for large objects. This study introduces a method using overlapping regions to establish RI offsets, improving volume stitching and accuracy.

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Area of Science:

Biomedical Imaging
Optical Physics

Background:

Tomographic quantitative phase imaging (QPI) requires a stable refractive index (RI) baseline for accurate measurements.
Large, dense biological samples often exceed the field of view, necessitating stitching of multiple volumes.
This stitching process can introduce RI baseline errors when the mounting medium is not accessible for all measurements.

Purpose of the Study:

To address the challenge of establishing a consistent RI baseline in tomographic QPI for large, multi-volume samples.
To propose a novel method for calculating RI offsets using overlapping regions between reconstructed volumes.
To validate the enhanced 2D baseline correction (2D-BC) method for improved tomographic QPI accuracy.

Main Methods:

Developing a method to calculate RI offsets based on overlapping regions of adjacent reconstructed volumes.
Demonstrating the analogy between the proposed 3D RI offset calculation and established 2D baseline correction (2D-BC) techniques.
Validating the enhanced 2D-BC procedure on both synthetic datasets and a real biological sample (liver organoid).

Main Results:

Successfully established a method for determining RI offsets in QPI volumes lacking a direct mounting medium baseline.
Showcased that RI offset determination can be simplified to a 2D-BC analogous procedure.
Validated the enhanced 2D-BC approach, demonstrating its effectiveness in improving the accuracy of stitched QPI volumes.

Conclusions:

The proposed method effectively resolves the RI baseline problem in tomographic QPI for extended samples.
The reduction of the 3D offset problem to a 2D-BC analogous procedure simplifies and enhances data processing.
This work provides a validated solution for accurate volumetric reconstruction in QPI of complex biological structures.