Correlated Microscope
Figure 4: Resolution and fluorescence sensitivity using beads. (a) 100 nm beads in a single plane amplitude reconstruction. (b) 100 nm beads in phase. (c) Amplitude in the xz plane of a single bead. (d) Note how the phase flips from dark to light across the focal plane; this is a manifestation of the Gouy phase anomaly. (e) Raw FLFM image of 3 μm fluorescent beads. (f) Maximum intensity projection of reconstructed LFM bead image.
carefully—many that are optimized for bright-field micros- copy have elements that affect polarization and thus the interference in the DHM. We have found that at least some high-quality objectives designed for metallurgy provide both good DHM and fluorescence performance. A second significant trade-off is in the lateral resolution the fluorescence microscope mode. In the implementa-
of
tion shown here the same model of camera was used for both microscopes for low cost and because our intended long-term application as an in situ planetary instrument drastically limits the rate and volume of data return compared to a laboratory environment [16]. Increasing the pixel count and enlarging the sensor size on the FLFM detector is a straightforward way to improve the lateral resolution without a loss of field of view. Te FLFM camera and detector could even be chosen to pro- vide resolution, depth of field, and field of view comparable to the DHM, at the expense of a substantially increased data vol- ume and likely a small increase in power consumption. One might ask then why the dual instrument is still valu-
able at the design point where both have comparable resolution and field. Why not just the FLFM? Te DHM mode offers label- free imaging of many otherwise transparent or translucent
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objects in the field, providing a survey capability that does not require any a priori knowledge of the characteristics of the objects in the field. Fluorescence microscopy typically requires some knowledge of the objects of interest—either excitation wavelengths of autofluorescent elements or chemical charac- teristics so that appropriate dyes can be selected. Te DHM mode can readily detect objects that have only a few percent index difference to the surrounding medium and provides dif- fraction-limited resolution of the volume. Another trade-off in the design of the instrument is the
selection of a color versus monochrome camera on the FLFM side. Te initial instrument described here was optimized for broad detection and discrimination of cells from mineral objects and so was designed around a small group of dyes. For fully correlated real-time observation of all dyes simulta- neously, a color camera filter set with isolated excitation and detection bands is valuable, but some applications could use a monochrome camera and selectable single-band filters.
Conclusion Te combined DHM-FLFM allows for fluorescence detec- tion in addition to volumetric amplitude and phase imaging.
www.microscopy-today.com • 2020 May
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