Digital Holographic Multimodal Cross-Sectional Fluorescence and Quantitative Phase Imaging System

Abstract

We present a multimodal imaging system based on simple off-axis digital holography, for simultaneous recording and retrieval of cross-sectional fluorescence and quantitative phase imaging of the biological specimen. Synergism in the imaging capabilities can be achieved by incorporating two off-axis digital holographic microscopes integrated to record different information at the same time. The cross-sectional fluorescence imaging is realized by a common-path configuration of the single-shot off-axis incoherent digital holographic system. The quantitative phase imaging, on the other hand, is achieved by another off-axis coherent digital holographic microscopy operating in transmission mode. The fundamental characteristics of the proposed multimodal system are confirmed by performing various experiments on fluorescent beads and fluorescent protein-labeled living cells of the moss Physcomitrella patens lying at different axial depth positions. Furthermore, the cross-sectional live fluorescence and phase imaging of the fluorescent beads are demonstrated by the proposed multimodal system. The experimental results presented here corroborate the feasibility of the proposed system and indicate its potential in the applications to analyze the functional and structural behavior of biological cells and tissues. Over the past decade, multimodal systems based on quantitative phase imaging in combination with fluorescence imaging have been developed considerably due to its several advantages 1-12. The phase imaging reveals the structural information by exploiting the optical path-length shifts through the biological specimen, while fluorescence imaging provides functional details of the specific molecules of interest in the biological specimen. Therefore, a hybrid multimodal imaging system comprising both these systems offers to precisely visualize and delineate structural and functional information in the biological specimen on a single platform at the same time. The quantitative phase imaging technique 13-16 is an emerging powerful technology of a new paradigm in general imaging and biomedical applications that provides quantitative information including the structure and dynamics of the transparent specimens, which is not easily obtained with the conventional optical imaging techniques. The technique employs the principle of optical interferometry to measure the optical field (i.e. amplitude and phase information). In recent years, quantitative phase imaging has received substantial interest and opened the door for quantifying dynamic structural information of biological cells with nanoscale sensitivity. Popescu et al. 17 reported the use of phase imaging for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells. The technique has also found applications for various investigations of cells and tissues including measurement of 3D profiling and tracking 15 , refractive index 18 , spectral dispersion 19 , optical path length 20 , dry mass localization 21 , and optim...

Coded aperture correlation holographic microscope for single-shot quantitative phase and amplitude imaging with extended field of view

“…Designing a digital holographic apparatus involves a well-known tradeoff between having a high space-bandwidth product at the expanse of a low temporal bandwidth, or vice-versa [27,28]. Usually, off-axis holography has higher temporal bandwidth while phase-shifting holography systems are higher in terms of spatial bandwidth.…”

Automated detection of apoptotic versus nonapoptotic cell death using label‐free computational microscopy

“…The biochemical assays have high accuracy but are typically resource and time intensive. Other commonly used methods include electrochemical assays, flow cytometry [28,29], microfluidic device [29], electron microscopy [15,28,30,31], atomic force microscopy [28,32], optical microscopy (fluorescence and phase contrast), single molecule spectroscopy, time-lapse phase contrast microscopy [33], and quantitative phase imaging [34,35]. Of these, optical techniques such as fluorescence microscopy and cytometry are most widely used due to their ability to accurately detect multiple different biomarkers simultaneously [36,37].…”

Single Shot Lensless Interferenceless Phase Imaging of Biochemical Samples Using Synchrotron near Infrared Beam

“…The development of multimodal imaging has proven useful for various applications such as cancer diagnosis and treatment [ 6 , 7 ]. Digital holography methods have been developed to achieve multimodal imaging consisting of quantitative phase imaging and fluorescence imaging [ 8 , 9 , 10 ]. The previously mentioned methods will be highly valuable if implemented at the IRM beamline as they can reveal additional information for connecting responses from different properties of the sample such as structural, functional, phase and compositional information.…”