Field Demonstration
common dyes. By using a color camera, a single shot provides three images with three different color filters defined by the Bayer filter on the sensor. Terefore, a single shot can image the sample with a wavelength coverage from 500 nm to 750 nm, defined by the dichroic mirror (Torlabs, DMSP490R) and the camera’s spectral range. Tis advantage of distinguishing color adds extra specificity information without having to add extra spectral filters or having to take multiple images. To maximize specificity, we also built a stand-alone FLFM
system using a multi-band filter set (Chroma, 89402) (Figure 5(a)) and a white-light LED (Torlabs MNWHL4) so that each of the spectral bands was clearly separated from others. Figures 5(b) and 5(c) show a raw and reconstructed seawater sample stained with FM1-43, where a large cell shows green fluorescence on the membrane from the dye and red from the native chlorophyll. Although we did not have a priori knowledge of the existence of chlorophyll in the sample, the color (red) information provided us with a ground for deducing the existence of chloroplasts in the cell. Figure 5(d) shows a seawater sample stained with AO, illustrating the difference between the emission of AO bound to DNA and nonspecific binding/autofluorescence [7].
Discussion In our initial field trip with our combined microscope sys-
tem, we were able to successfully demonstrate several of the capabilities and potential of ELVIS for use in aquatic microbiol- ogy applications. Te multitude of data yielded simultaneously for each cell, including morphology, motion, and fluorescence, combined with the high throughput of the system provides high confidence for detection and enumeration of cells. Te system as originally built is especially well-suited for distin- guishing photosynthetic from non-photosynthetic microalgae while providing cell counts for both. Tis is an application of interest to many marine biologists, especially if it can be mea- sured in real time and at varying depths; a variety of submers- ible imaging spectrometers that gate on chlorophyll have been developed [8,9]. For marine biology applications focused on chlorophyll, the original system remains the simplest and is sufficient. However, for dye labeling with multiple dyes or to distin-
guish dye labeling from autofluorescence, fluorescence spectral information is needed. Tis may be obtained by using an RGB
camera with either a long-pass or multiple-bandpass filter. Careful selection of filters is required to separate chlorophyll autofluorescence from dye staining and to permit the same acquisition times to capture both simultaneously, since chlo- rophyll fluorescence may be much brighter than dye emission depending on the wavelength and intensity of excitation; this is a serious problem in plant biology [10]. Te properties of chlo- rophyll fluorescence are complex but have been well studied in the context of plants as well as microalgae [11–13]. Te system may eventually be interfaced with a liquid crys-
tal tunable filter (LCTF) or other multi-spectral imaging systems for full spectral information [14,15]. Separating the fluorescence excitation and emission from the DHM in a combined system poses some difficulty but can be achieved with narrow-band filters and either white-light or multiple-LED excitation.
Acknowledgements Portions of this work were performed under a contract
from the Jet Propulsion Laboratory, California Institute of Technology, and at the Jet Propulsion Laboratory, Califor- nia Institute of Technology, under contract with the National Aeronautics and Space Administration. Portions of this work were supported by NSF grant #1828793.
References [1] T Kim et al., Microscopy Today 28 (2020) 18–25. [2] DE Francisco et al., Trans Am Microsc Soc 92 (1973) 416–21. [3] I Fishov and CL Woldringh, Mol Microbiol 32 (1999) 1166–72. [4] D Cohoe et al., Frontiers in Physics 7 (2019) 1–15. [5] R Stocker, Proc Natl Acad Sci USA 108 (2011) 2635–36. [6] M Bedrossian et al., Astrobiology 17 (2017) 913–25. [7] JL Nadeau et al., Astrobiology 8 (2008) 859–75. [8] RJ Olson and HM Sosik, Limnol Oceanogr-Meth 5 (2007) 195–203.
[9] A Malkassian et al., Cytometry A 79 (2011) 263–75.
[10] Y Kodama, PLOS One 11 (2016) e0152484. [11] HM Kalaji et al., Photosynth Res 132 (2017) 13–66. [12] HM Kalaji et al., Photosynth Res 122 (2014) 121–58. [13] T Takahashi, Molecules 24 (2019) 4441–60. [14] T Zimmermann, Adv Biochem Eng Biotechnol 95 (2005) 245–65.
[15] Y Hiraoka et al., Cell Struct Funct 27 (2002) 367–74.
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