Abstract
Quantitative phase imaging of cells provides important morphological information about them, leading to their characterization, comparison, and identification. The interference principle when applied to microscopy provides high-contrast quantitative phase images of otherwise transparent objects along with their thickness information. The two-beam off-axis geometry of interference microscopes, in which the light beam interacting with the object interferes with a separate reference beam, is preferred since it leads to single shot quantitative phase imaging methodologies. But these techniques lead to bulky setups, with lower temporal stability not suitable for the measurement of nanometer-level cell thickness fluctuations. Self-referencing interference microscopes manipulate a portion of the light beam interacting with the cells to act as the reference, leading to compact, temporally stable geometries ideal for the measurement of cell dynamics. Here we present an overview of our efforts in the development of self-referencing digital holographic microscopes and their use in quantitative phase imaging of cells.
Highlights
The most commonly employed technique to image living cells is to use visible electromagnetic radiation as a probe and a lens to magnify the object.[1]
Inherently, living cells have a low absorption coefficient for visible light and they have small thickness and even smaller thickness variation, leading to a low-contrast output, when the intensity of the probe beam is used to image them in the case of bright-field microscopes
In contrast to the common path geometry where a separate reference beam is generated, self-referencing interferometers use a portion of the object beam, which is unmodulated by cell information as the reference beam
Summary
The most commonly employed technique to image living cells is to use visible electromagnetic radiation as a probe and a lens to magnify the object.[1]. Common path techniques in which the object and a separate reference beam travel along the same path improve the time stability, but such methods require specialized optical components for beam filtering and may not be compact in nature.[13,14,15,18,26,27,28,29,44,45,47] In contrast to the common path geometry where a separate reference beam is generated, self-referencing interferometers use a portion of the object beam, which is unmodulated by cell information (without spatial filtering) as the reference beam This reference beam is made to interfere with the object beam to create interferograms. These microscopes require only few optical elements compared to other two-beam techniques and are easy to implement and compact.[48,49,50,51,52,53,54,55,56,57,58,59,60,61,62] Here we provide an overview of our efforts in the design and development of compact self-referencing microscopes for quantitative phase contrast imaging of cells by enumerating the advantages and disadvantages of such microscopes over the other two-beam geometries including common-path geometries
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