Correlated Microscope
resulting low sensitivity so that even labeled bacteria could not be identified under the FLFM; and (b) monochromatic fluorescence that did not permit distin- guishing dye labeling from chlorophyll autofluorescence, or the use of 2 differ- ent dyes. Simple modifications resulted in improved performance. Te 3D-printed objective lens holder was found to show significant autofluorescence, so it was replaced by a holder made of anodized aluminum. Te use of an RGB camera was also used on the FLFM side to dis- tinguish chlorophyll from common dyes (Figure 5) without the need to insert or change filters. Soſtware. Acquisition and real-time
DHM reconstruction were performed using a custom package, DHMx, written by our group. DHMx runs under Linux and is open-source (
https://github.com/ dhm-org/dhm_suite). FLFM reconstruc- tion was performed using another group’s open-source package [14]. Full amplitude and phase reconstructions were made using custom Fiji plug-ins that we have published previously [15].
Discussion Te combined DHM/FLFM system
provides multimodal volumetric imag- ing, but trade-offs were made to permit the combination, which can be further optimized for particular applications. Te first major trade-off in the
Figure 3: DHM/FLFM test target (TT) images. Only a portion of the field of view is shown. The total field of view was 478×478 μm. (a) Amplitude reconstruction of USAF TT showing resolution better than group 9, element 2 (0.87 μm line widths). (b) The reconstructed best-focus position versus the actual position, allowing an estimate of axial resolution of 4.4 μm (see text). (c) Phase reconstruction of USAF phase TT with known widths. (d) Phase delay in nm versus the actual thickness in nm (data points shown as open circles) with best linear fit, permitting estimate of phase resolution (see text). (e) Raw FLFM image of USAF TT. (f) Reconstructed FLFM image showing resolution of group 6, element 5 (4.92 μm line widths).
reconstructions of the beads are shown in Figures 4a and 4b. Both images were de-noised by translating the stage while imaging and using the median image to subtract amplitude background; a reference hologram containing no beads was used to remove noise in phase. Tese procedures are described in detail elsewhere [15]. Amplitude and phase x-z slices are shown in Figures 4c and 4d. Fluorescence sensitivity was ascertained using fluorescent SiO2 beads (Polysciences, Inc. 24330-15). Figure 4e shows a raw
image of 3 µm beads, and Figure 4f shows a maximum inten- sity projection through the sample. Early
issues with the instrument that were identified
and ameliorated were (a) high background fluorescence, with 2020 May •
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design of the instrument was the choice to use a shared set of objectives for both modes of the microscope. In early development we considered both this shared-objective design and a second design that used two fully independent microscopes observing the same volume at crossed angles. Te shared objectives architecture
ensures straightforward
co-registration of the fields of view. Te choice of objectives then sets both the capability and to some extent the size of the instrument. We have been working with simple aspheric and achromat objectives in order to make an instrument that is more rugged for field use and to avoid optical elements that might adversely affect the ability to obtain high fringe con- trast in the DHM. Simple aspheric objectives are sufficient for the DHM, but their chromatic aberration can move the focus for many desired wavelengths far enough out of focus as to be effectively useless for imaging broad bands of light. Simple achromatic doublet objectives help mitigate this, and com- pound-apochromat or super-apochromat objectives could improve this further. Compound objectives must be selected
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