Abstract
We present an imaging platform for stain-free quantitative imaging of biological cells using a simultaneous dual-wavelength holographic module. We use this module to experimentally solve the problem of 2π phase ambiguities that occurs in spatial locations where the optical thickness of the sample is larger than the wavelength. Thus, the process does not require using digital phase unwrapping that is computational heavy and impairs real-time processing. The proposed method is not limited to sequential acquisition of two quantitative phase maps in different times, but rather allows optical multiplexing of two off-axis holograms on the camera at once, enabling acquisition of fast dynamic processes. The module is simple and portable, making it attractive for clinical use. We demonstrate using the module for quantitative phase imaging of cancer and sperm cells.
Highlights
Digital holographic microscopy (DHM) can record the optical thickness profile of biological samples, such as cells in vitro
The optical path delay (OPD) profile can be used to calculate the cell dry mass, which is an indicator for the physical density of the cell, as well as other quantitative parameters of biological relevance [2]
The OPD profile can be recorded in a single camera exposure using off-axis holography, where two beams interfere on the camera at a small off-axis angle, one beam interacts with the sample and the other beam is the reference beam that does not contain sample modulation
Summary
Digital holographic microscopy (DHM) can record the optical thickness profile of biological samples, such as cells in vitro. We proposed an external, portable, and nearly common-path interferometric module, which is capable of forming a multiplexed off-axis hologram for two-wavelength phase unwrapping It externally creates the reference beam using spatial filtering, as implemented by two lenses and a confocally positioned pinhole [11]. In [13], we proposed flipping interferometry with doubled imaging area, an external portable module that combines simultaneous acquisition of two fields of view of the sample by spatial off-axis holographic multiplexing, without using a pinhole alignment It projects two holographic fields of view on the camera at once, where each field of view is encoded into interference fringes of different orientation. We use this module for simultaneous two-wavelength phase unwrapping and demonstrate dynamic quantitative phase imaging of microbeads, sperm cells and cancer cells
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