ELVIS: A Correlated Light-Field and Digital Holographic Microscope for Field and Laboratory Investigations – Field Demonstration
Taewoo Kim,1 Eugene Serabyn,2
Pasadena, CA 91125 2
Kurt Liewer,2 Stephanie Rider,1 Manuel Bedrossian,1 Nathan Oborny,2 Christian Lindensmith,2
J. Kent Wallace,2 and Jay Nadeau3
3Department of Physics, Portland State University, 1719 SW 10th Ave., Portland, OR 97201 *
nadeau@pdx.edu
Abstract: Following the previous article, here we describe the first field demonstration of the ELVIS system, performed at Newport Beach, CA. We examined ocean water to detect microorganisms using the combined holographic and light-field fluorescence microscope and successfully detected both eukaryotes and prokaryotes. The shared field of view provided simultaneous bright-field (amplitude), phase, and fluorescence information from both chlorophyll autofluorescence and acridine orange staining. The entire process was performed in a nearly autonomous manner using a specifically designed sample pro- cessing unit (SPU) and custom acquisition software. We also discuss improvements to the system made after the field test that will make it more broadly useful to other types of fluorophores and samples.
Keywords: holographic microscopy, light-field microscopy, volumet- ric imaging, eukaryotes, prokaryotes
Introduction Te Extant Life Volumetric Imaging System (ELVIS) is a
combined digital holographic microscope (DHM) and fluores- cence light-field microscope (FLFM) that uses the same objectives and sample chamber to provide overlapping volumes of view for instantaneous 3D measurement in amplitude, quantitative phase, and fluorescence. Te construction and operation of the system is described in the May 2020 issue of Microscopy Today [1].
Materials and Methods A first field test of the instrument was performed at the
Kerckhoff Marine Laboratory, Newport Beach, CA, in July 2019. Te instrument was transported to an indoor laboratory near the beach and set up within an hour. In transporting the system, there was no special care taken other than placing the microscope into a foam-filled carrying case to keep it free from vibrations and shock. Once the microscope was set up at the lab, we performed initial tests using microbeads and U.S. Air Force (USAF) targets, as described in our previous article, to confirm that the transport caused no misalignment [1]. Te first field sample was obtained directly from the nearby
ocean. A sterile container was immersed in seawater ∼10m off- shore and ∼1 m deep to collect approximately 4 L of sample to be shared among the present instrument and others being tested in parallel. Subsequent sample handling for the combined instru- ment was performed in a nearly autonomous manner using a custom sample processing unit (SPU). Te SPU was designed to route a 100 μL sample to a microfluidic sample chamber mounted in the field of view (FOV) of the combined instrument. Once within the FOV, the cells within the sample were imaged
14 doi:10.1017/S1551929520001133
by ELVIS. For each 100 μL sample, we imaged 5 fields of view for 30 seconds each. Following this, the sample was routed to a mixing chamber where one of three possible cellular dyes was applied. Tese dyes were chosen to target cellular structures likely to be common to all life, including cellular membranes and nucleic acids. In our field test, we selected two dyes. Acri- dine orange (AO) for nucleic acids (both DNA and RNA) [2] (Sigma-Aldrich 01640) and FM1-43 for cellular membranes [3] (TermoFisher T-3163). In applying the dyes, care was taken to keep both the sample and the stock dyes away from light to min- imize photobleaching. Te dyes chosen were water-soluble and sold as powders for compatibility with the fluidic system, which was printed from WaterShed XC 11122 (Protolabs, Maple Plain, MN). It is important to note that many commonly used dyes for bacterial enumeration, such as SYTO9/Propidium iodide (“live/ dead”) (Boulos, 1999 #443), are oſten sold in dimethylsulfoxide (DMSO) solution. Lyophilized, water-soluble forms are available and should be used whenever material compatibility of the flu- idic channels is a potential concern. Once the first dye (AO) had been applied, the sample was
allowed to react for 20 minutes before being routed back to the sample chamber. Once there, the sample was again imaged by the combined system. Following the imaging, the sample was re-routed to a mixing chamber, and the second dye (FM1-43) was applied. Finally, in addition to control of sample delivery and stain-
ing of the sample, the SPU also allowed the system to be rinsed with water and disinfected with a 70% ethanol solution. Te addition of these functions allowed the system to perform in a largely autonomous mode as the system cleaned itself between runs, waited for a sample to operate, and performed staining operations automatically (Figure 1(a)). Image acquisition was also performed in an automated
manner. Based on the soſtware development kit (SDK) pro- vided by Allied Vision, we developed custom acquisition soſt- ware to control each camera’s exposure, gain, and capture separately. Te acquisition soſtware, called DHMx, includes features for holographic image processing and is available as an open-source package on GitHub (
https://github.com/dhm- org/dhm_suite). Two copies of the soſtware were run simulta- neously to control the DHM and FLFM cameras. By adding an external trigger system built using Arduino, we were able to synchronize the two cameras within a 0.2 ms delay. Finally, a script controlling the DHMx soſtware as well as the trigger
www.microscopy-today.com • 2020 July *
1Andrew and Peggy Cherng Department of Medical Engineering, California Institute of Technology, 2500 E. California Blvd., Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Dr., Pasadena, CA 91109
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
