We present a theoretical formulation to quantify the imaging properties of volume holographic microscopy (VHM).
Volume holograms are formed by exposure of a photosensitive recording material to the interference of two mutually
coherent optical fields. Recently, it has been shown that a volume holographic pupil has spatial and spectral sectioning
capability for fluorescent samples. Here, we analyze the point spread function (PSF) to assess the imaging behavior of
the VHM with a point source and detector. The coherent PSF of the VHM is derived, and the results are compared with
those from conventional microscopy, and confocal microscopy with point and slit apertures. According to our analysis,
the PSF of the VHM can be controlled in the lateral direction by adjusting the parameters of the VH. Compared with confocal microscopes, the performance of the VHM is comparable or even potentially better, and the VHM is also able to achieve real-time and three-dimensional (3D) imaging due to its multiplexing ability.
Phase contrast imaging is a specific technique in optical microscopy that is able to capture the minute structures of
unlabeled biological sample from contrast generated in the variations of the object's refractive index. It is especially
suitable for living cells and organisms that are hardly visible under conventional light microscopy as they barely alter the
intensity and only introduce phase shifts in transmitted light. Optical phase imaging provides sensitivity to measure
optical path length (OPL) differences down to nanometers, which has great potential in biomedical applications from
examining both topological and three-dimensional biophysical properties of cells and organisms.
Conventional DIC microscopy with partially coherent light source is a very powerful technique for phase imaging, and is
able to yield higher lateral resolution compared to other interferometric phase imaging methods. However, it is
inherently qualitative and the information obtained is a phase-gradient image rather than a true linear mapping of OPL
differences. This hinders its further application as it is difficult to infer the results directly. Work has been done
previously to obtain the quantitative phase information out of DIC. However, some of these methods not only involves
costly hardware modification but also complicated computation. Here we investigate another approach that combines the
correlation of light intensity and phase with polarization-modulated differential interference contrast (DIC) microscopy.
The required hardware modification is simple, and numerically solving the relationship of light propagation in a series of
through-focus DIC images allows phase information to be restored from phase gradients in two-dimensional planes.
Quantitative phase information in three-dimensional space can then be reconstructed from 3D rendering of the calculated
phase images. Initial results of application in biological cells are also demonstrated.
Phase imaging is an invaluable tool for observation of biological sample, especially for living cells, where staining might
not be appropriate, or for materials that do not absorb stain. Imaging of phase distributions with high spatial resolution
can be used to derive the actual thickness and refractive index variations in the specimen. The detection of very small
phase variations enables the detailed structure in the specimen to be revealed. As a result, the development and
utilization of various phase imaging modalities have been important aspects of microscopy research. Differential
Interference Contrast (DIC) and Quantitative Phase Microcopy (QPM) are based on partially coherent light, thus
enabling high-resolution imaging. However, the low coherence requirement prevents the acquisition of quantitative
phase data directly. On the other hand, Digital Holography Microscopy (DHM) is able to yield quantitative phase
information but is compromised on resolution and cannot give full three dimensional (3D) reconstructions. In this paper,
we present the 3D theoretical formalism of the above mentioned phase imaging methods with the focus on DHM. A
comparative analysis here through visualization of 3D optical transfer functions gives an insight into the behaviors of
these phase imaging methods.
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