Field Demonstration
mainly in the FLFM side of the combined instrument: (a) high background fluorescence; and (b) lack of spectral capability to distinguish dye labeling from chlorophyll autofluorescence. Simple modifications aſter the field trip resulted in improved
performance. Te acrylonitrile butadiene styrene (ABS) (Ren- Shape SL 7820) 3D-printed objective lens holder was found to show significant autofluorescence at 470 nm, so it was replaced by a holder made of anodized aluminum. As a result, the background level of the FLFM was decreased down to 1–2 counts at maximum excitation intensity, when measured without a sample and using the same exposure and gain settings as the field test. With the previous objective lens holder, the background from the plastic autofluorescence saturated the 8-bit image (256 counts) at 20% of the maximum excitation intensity. Another source of background was from the residual fluorescent dyes in the background medium. We performed an optimization study for dye concentration using a bacterial test strain, Escherichia coli, which is approximately
1 µm wide by 2 µm long. By imaging the bacteria stained with various concentrations of the two dyes, we determined that 50 nM SYTO-9 and 10 nM FM1-43 provided the highest signal-to- background ratio (SBR), while maintaining high probability for each cell to be stained. Given the updates made to the system and the dye concentration determined, we were able to successfully image E. coli stained with 50 nM Syto-9 under FLFM. A single FOV of the stained sample was imaged, volume-reconstructed, and maximum-projected over the z-direction. Figure 4(a) shows the result, where the stained bacteria show up clearly over the low background. Figure 4(b) shows the DHM counterpart of the same image, taken simultaneously. Te image was volume recon- structed for amplitude and minimum z-projected. Tese results illustrate that although unresolved, single stained bacteria may be detected using the FLFM. Finally, an RGB camera (Basler, ace ac A4024-88gb) in our FLFM system was also used to distinguish chlorophyll from
Figure 5: Multispectral FLFM. Scale bar applies to all panels. (a) Multi-bandpass filter designed for simultaneous use of multiple dyes with white-light excitation. (b) Raw FLFM image of an FM1-43 stained seawater sample. The grid pattern results from the individual sub-apertures of the microlens array. (c) Reconstruction on one focal plane showing the distinction between dye staining the membranes (green) and chlorophyll (red). (d) Z-stack through a seawater sample. AO is red when bound to RNA, is green when bound to DNA, and shows general yellowish fluorescence when nonspecifically bound. Having RGB information permits specific binding to be readily identified. The cell nucleus is brighter than the rest of the cell, which is a uniform green; the yellow is nonspecific. The use of lower AO concentrations will reduce nonspecific staining and extranuclear staining in the cells.
2020 July •
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