Because of the coatings needed to solubilize and passivate quantum dots for biological applications, their use in fluorescent resonance energy transfer (FRET) has been limited. However, hydrophobic particles without polymer coatings may be embedded into lipid membranes, as demonstrated here with biomimetic vesicles. FRET is seen to a lipid-soluble dye (DiD) and a water-soluble dye (Cy3.5) in which the vesicles are suspended. The degree of energy transfer to each dye suggests that most of the QDs are located deep within the lipid, as confirmed by electron microscopy of whole mounts and thin sections of vesicles. Energy transfer is also seen to a voltage-sensitive, lipid-soluble dye (di-4-ANEPPS) only when the potassium ionophore valinomycin is present in the membrane. The effect is dependent upon potassium ion concentration rather than absolute membrane potential.

Traditional fluorophores often impose inconvenient limitations because of their narrow excitation spectra, broad emission bands, and significant photobleaching. Quantum dots (QDs) have grown in popularity because of their high emission quantum yields, broad absorbance spectra, and narrow, tunable emission spectra. Here, coated CdSe/ZnS QDs with emission maxima at 496 nm (T2−496), ∼520 nm (QD520), and ∼560 nm (QD560 and Qdot565) were characterized while freely diffusing in solution using confocal fluorescence correlation spectroscopy (FCS) and were compared with well-known fluorophores such as Alexa 488 to reveal critical photophysical properties. Comparisons are made between dots synthesized by similar methods (QD520 and QD560 nm) differing in their emission spectra and outer coating for biofunctionalization. The same photophysical principles also describe the T2−496 and Qdot565 dots, which were synthesized by different, proprietary methods. All of the tested QDs had larger hydrodynamic radii and slower diffusion coefficients than Alexa 488 and underwent numerous transitions between bright and dark states, especially at high illumination intensities, as described here by a new FCS fitting function. The QDs with the fastest transitions between the bright and dark states had the lowest average occupancies in dark states and correspondingly higher maximum brightness per particle. Although these QDs were in some cases brighter than Alexa at low excitation intensities, the QDs saturated at lower intensities than did Alexa and had generally somewhat lower maximum brightness per particle, except for the Qdot565s. Thus, it appears that intermittency (at least in part) limits maximum brightness in QDs, despite the potential for high fluorescence emission rates that is expected from their large extinction coefficients. These results suggest possibilities for significant improvement of QDs for biological applications by adjustments of manufacturing techniques and environmental conditions.

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There is no reductionist definition of life, so the way organisms look, behave, and move is the most definitive way to identify extraterrestrial life. Life elsewhere in the Solar System is likely to be microbial, but no microscope capable of imaging prokaryotic life has ever flown on a lander mission to a habitable planet. Nonetheless, high-resolution microscopes have been developed that are appropriate for planetary exploration. Traditional light microscopy, interferometric microscopy, light-field microscopy, scanning probe microscopy, and electron microscopy are all possible techniques for the detection of extant micro-organisms on Mars and the moons of Jupiter and Saturn. This article begins with a general discussion of the challenges involved in searching for prokaryotic life, then reviews instruments that have flown, that have been selected for flight but not flown or not flown yet, and developing techniques of great promise for life detection that have not yet been selected for flight.

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Multiwavelength digital holographic microscopy (DHM) has been used to improve phase reconstructions of digital holograms by reducing 2π phase ambiguities. However, most samples used as test images have been solid or adhered to a surface, making it easy to determine focal planes and correct for chromatic aberration. In this study we apply 3-wavelength off-axis DHM to swimming protozoa containing distinct spectral features such as chlorophyll and carotenoids. We reconstruct the holograms into amplitude and phase images using the angular spectrum method. Methods for noise subtraction, chromatic aberration correction, and image registration are presented for both amplitude and phase. Approaches to phase unwrapping are evaluated and compared to expected results from simulated holograms. The algorithms used are implemented in plug-ins using the open source Fiji platform and are available for use, significantly expanding the open-source software available for DHM.