ELVIS: A Correlated Light-Field and Digital Holographic Microscope for Field and Laboratory Investigations
Taewoo Kim,3 Eugene Serabyn,1 J. Kent Wallace,1 Stephanie Rider,3
Pasadena, CA 91125 *
nadeau@pdx.edu
Abstract: This is the first of two articles on the Extant Life Vol- umetric Imaging System (ELVIS) describing a combined digital holographic microscope (DHM) and a fluorescence light-field micro- scope (FLFM). The instrument is modular and robust enough for field use. Each mode uses its own illumination source and cam- era, but both microscopes share a common objective lens and sample viewing chamber. This allows correlative volumetric imag- ing in amplitude, quantitative phase, and fluorescence modes. A detailed schematic and parts list is presented, as well as links to open-source software packages for data acquisition and analysis that permits interested researchers to duplicate the design. Instru- ment performance is quantified using test
targets and beads.
the second article on ELVIS, to be published in the next issue of Microscopy Today, analysis of data from field tests and images of microorganisms will be presented.
Keywords: holographic microscopy, light-field microscopy, volumet- ric imaging, lenslet arrays, reconstruction software
Introduction Recent technological developments, such as the FlowCy-
tobot [1], have revolutionized the in situ study of microorgan- isms such as diatoms, plankton, and microalgae at the 10 µm scale and above. However, smaller micron-scale organisms (bacteria and archaea) remain a neglected group of organisms for which limited environmental in situ imaging has been performed [2], largely because of the challenges placed on the imaging system. Prokaryotic life is limited by nutrient diffusion into the
cell, restricting most bacteria and archaea to sizes in the range of 0.4–2.0 µm [3]. Not only are cells small, but they lack dis- tinctive features such as nuclei and other membrane-bound organelles, making them difficult to distinguish microscopi- cally from debris (Figure 1). Imaging systems with sufficient resolution to identify objects at this size scale generally have very limited field of view and depth of field. Tis makes it difficult to observe a large number of interactive microorgan- isms and requires active focus and stage motion to track a single organism for even short times. Because of the need for instantaneous volume imaging, implementation of such fea- tures in a system for field use is daunting even if equipped with adaptive lenses. In this paper, we report a multi-modal microscope called
the Extant Life Volumetric Imaging System (ELVIS) designed for field use that integrates two modalities into a common instrument: digital holography microscopy
(DHM) 18 and doi:10.1017/S1551929520000899
fluorescence light-field microscopy (FLFM). ELVIS provides synchronous volumetric imaging of three types: fluorescence, intensity (bright-field), and quantitative phase. Both the DHM and FLFM components of the combined instrument are closely based on previously published designs [4,5]. DHM is an interferometric technique that results in
In
full electric field measurements of the sample volume. Off- axis holographic images may be reconstructed into intensity (bright-field) and quantitative phase images plane-by-plane through volumes ∼100 times deeper than those imaged using ordinary bright-field microscopy [6]. Amplitude and bright- field images are equivalent. However, quantitative phase imag- ing is an emerging technique in biology and materials science and has no direct counterpart in ordinary light microscopy. In quantitative phase imaging the phase shiſt of the light wave passing through the specimen is proportional to the product of the specimen’s thickness and the difference in the refractive index from the surrounding medium. Te phase shiſt infor- mation can be used to differentiate bacteria from minerals, to identify malignant cells in a tissue sample, to monitor the flux of water into and out of cells via ion channels, and to monitor neuronal function [7,8]. Fluorescence microscopy is a ubiquitous tool in biology
that allows for specific labeling of subcellular structures, enzy- matic processes, individual proteins or nucleic acid sequences, and much more. A combined intensity/quantitative phase/ fluorescence microscope would offer one-of-a-kind volumetric analysis. Te problem lies in adding fluorescence capability to a technique such as DHM that is focus-free. Because DHM works on the principle of interfering coherent light, it does not directly support volumetric fluorescence imaging, and when coupled with fluorescence using traditional microscopes, imaging has been limited to a single plane [9,10]. Tis means that either most of the sample is missed by the fluorescence analysis or that it must be filtered onto a flat substrate, which disturbs correspon- dence between the DHM and fluorescence images. In FLFM, the 3D light field of the sample is transformed
into a 2D image using a microlens array, thus enabling a 2D camera to image the 3D sample volume [11–13]. Computational reconstruction is then used to generate the 3D image of the sample from its 2D light-field image. FLFM suffers the trade- off of reduced lateral resolution compared to conventional microscopy carried out with similar numerical apertures.
www.microscopy-today.com • 2020 May
Maximilian Schadegg,2 Manuel Bedrossian,3
Kurt Liewer,1 Ave., Portland, OR 97201 Nathan Oborny,1 Christian Lindensmith,1
1Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Dr., Pasadena, CA 91109 2Department of Physics, Portland State University, 1719 SW 10th
3Andrew and Peggy Cherng Department of Medical Engineering, California Institute of Technology, 1200 E. California Blvd., and Jay Nadeau2 *
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84
