Field Demonstration
system was developed to allow for simultaneous image capture from the two cameras (Figure 1(b)). Using the automated sample handing and image capture
described above, we examined ocean water collected from Newport Beach. Te results are described in detail in the fol- lowing section.
Results In the randomly collected sample from the ocean, we suc-
cessfully detected signs of both eukaryotes and prokaryotes. Eukaryotes. A variety of rapidly swimming eukaryotes,
3–50 µm in diameter, were apparent immediately in raw DHM and FLFM images. Many, but not all, of these were autofluorescent due to the presence of chlorophyll. For further analysis, we per- formed volumetric reconstructions on both DHM and FLFM raw images, followed by minimum z-projection for DHM and maxi- mum z-projection for FLFM. Te time-series of projected data was then minimum (DHM) and maximum (FLFM) projected to reveal their tracks. Figures 2(a) and 2(b) show a correlated FOV from both microscopes without a dye applied. It is interesting to note that some organisms were more visible in the FLFM, and
others could be seen only under the DHM. Tis is because of their contrast mechanisms: a fluorescent organism with low absorption at 405 nm appears only under FLFM, while a non-fluorescent one with high absorption at 405 nm appears only under DHM. Te addition of AO did not seem to reveal additional organ-
isms and caused an almost complete loss of organism motility (Figures 2(c) and 2(d)). Te motion shown in Figures 2(c) and 2(d) are due to the slight background flow and Brownian motion. Cells and subcellular features that appeared dark in ampli-
tude images usually showed high phase contrast and vice versa, but not always. For larger cells detected using ELVIS, we performed DHM reconstructions on the focal planes of the individual cells. Te reconstructions revealed subcellular structure in both ampli- tude and phase. Figures 2(e)–2(h) show a cell that appeared fairly transparent in the center under amplitude, but which had very high phase contrast. Tis pattern is typical of chlorophyll, which is highly dispersive and absorbs strongly at 405 nm. Figures 2(i)– 2(l) show a cell where the subcellular features are apparent in both amplitude and phase. Reconstruction and unwrapping were per- formed using our custom Fiji plug-ins [4] available from https://
github.com/sudgy.
Prokaryotes. Te detection of pro-
karyotic cells was performed mainly with the DHM because of its higher resolution. Marine bacteria could be identified on single-plane reconstruc- tions in amplitude as featureless par- ticles
(Figure 3(a)). Te volumetric
Figure 3: Prokaryotes. (a) Structureless particles seen at the edge of DHM resolution. (b) When the particles were motile, they could be readily identified as prokaryotic organisms. (c) A plot of the estimated index of refrac- tion difference between imaged particles and the surrounding water.
information yielded cell counts and motility patterns. Many marine bacteria show characteristic zig-zag swimming with velocities up to 40 µm/s [5]. Based on the high-resolution images from DHM, we were able to detect some particles that showed lifelike motility in the sample (Figure 3(b)). Estimates of the refractive index of particles could be calculated as in a previous study [6]. Te refractive index of the non-motile, micron-sized particles in the seawater differed from water only by ∼0.1, sug- gestive of cells rather than sand grains or other mineral particles (Figure 3(c)). Te total particle count in the seawa- ter was ∼103
eukaryotes and 105 pro-
karyotes/mL, consistent with the value obtained using a ground truth method of AO staining, paraformaldehyde fixation, and cell counting using high- resolution fluorescence microscopy.
Improvements to Performance: Lab Samples Following the field test at Newport
Figure 4: Improved FLFM system sensitivity by removal of fluorescence background. (a) Improved sensitivity proven by single bacteria detected under FLFM. The sample is 50 nM SYTO-9 stained E. coli imaged with FLFM at 60ms exposure. A single image was captured, volume reconstructed, and then maximum z-projected over 300 μm depth of field. (b) Simultaneously captured DHM image of the same FOV. DHM amplitude was volume reconstructed and minimum z projected. The inset shows a single cell under DHM.
16
Beach, we moved the instrument back to the Jet Propulsion Laboratory and analyzed the instrument performance. Te major limitations of the instru- ment identified in the field testing were
www.microscopy-today.com • 2020 July
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