Lauren A. Ernst

Fluorophores for Confocal Microscopy: Photophysics and Photochemistry

Fluorescence is probably the most important optical readout mode in biological confocal microscopy, because it can be so much more sensitive and specific than absorbance or reflectance, and because it works so well with epi-illumination, which greatly simplifies scanner design. These advantages of fluorescence are critically dependent on the availability of suitable fluorophores that can either be tagged onto biological macro-molecules to show their location, or whose optical properties are sensitive to the local environment. Despite the pivotal importance of good fluorophores, little is known about how to rationally design good ones. Whereas the concept of confocal microscopy is only a few decades old and nearly all the optical, electronic and computer components to support it have been developed or redesigned in the last few years, the most popular fluorophores were developed more than a century ago (in the case of fluoresceins or rhodamines) or several billion years ago (in the case of phycobiliproteins). References

Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model.

This article describes an in vivo imaging method for visualizing and quantifying a specific cell population. Cells are labeled ex vivo with a perfluoropolyether nanoparticle tracer agent and then detected in vivo using 19F MRI following cell transfer. 19F MRI selectively visualizes only the labeled cells with no background, and a conventional 1H image taken in the same imaging session provides anatomical context. Using the nonobese diabetic mouse, an established model of type 1 diabetes, 19F MRI data were acquired showing the early homing behavior of diabetogenic T cells to the pancreas. A computational algorithm provided T cell counts in the pancreas. Approximately 2% of the transferred cells homed to the pancreas after 48 hr. The technique allows for both unambiguous detection of labeled cells and quantification directly from the in vivo images. The in vivo quantification and cell trafficking patterns were verified using 19F spectroscopy and fluorescence microscopy in excised pancreata. The labeling procedure did not affect T‐cell migration in vivo. This imaging platform is applicable to many cell types and disease models and can potentially be used for monitoring the trafficking of cellular therapeutics. Magn Reson Med 58:725–734, 2007.

Fluorescence Imaging of Tumors In Vivo

We review recent progress in tumor imaging in vivo using fluorescent tags, highlight the problems of fluorescence imaging in small animals, discuss recent advances in near-infrared fluorochromes and quantum dots, and point to some future possibilities. GFP-based fluorescence imaging is briefly discussed. The authors believe that improvements in near-infrared fluorochromes are required to enable practical imaging in tissues at centimeter depths.

Long Term Persistence and Spectral Blue Shifting of Quantum Dots in vivo

Quantum dots are a powerful fluorophore family with desirable attributes for fluorescence imaging. They have been used in several animal models with direct clinical relevance, including sentinel lymph node mapping, tracing vasculature and lymphatics, and targeting specific lesions for diagnosis and removal. (1-12) Despite significant interest for use in translational applications, little is known about the persistence and long-term fate of quantum dots in vivo. We have observed fluorescence of quantum dots injected into Balb/c and nude mice for up to two-years post injection using both whole-body and microscopic fluorescence techniques. Two-photon spectral microscopy was used to verify the existence of quantum dots within two-year tissues, but also revealed a range of significantly blue-shifted emission peaks with increased bandwidths. Systemically administered quantum dots persist and retain fluorescence for up to two-years in vivo, but with significantly blue-shifted emission.

Leishmania donovani: Surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Life in the Atacama: Searching for life with rovers (science overview)

[1] The Life in the Atacama project investigated the regional distribution of life and habitats in the Atacama Desert of Chile. We sought to create biogeologic maps through survey traverses across the desert using a rover carrying biologic and geologic instruments. Elements of our science approach were to: Perform ecological transects from the relatively wet coastal range to the arid core of the desert; use converging evidence from science instruments to reach conclusions about microbial abundance; and develop and test exploration strategies adapted to the search of scattered surface and shallow subsurface microbial oases. Understanding the ability of science teams to detect and characterize microbial life signatures remotely using a rover became central to the project. Traverses were accomplished using an autonomous rover in a method that is technologically relevant to Mars exploration. We present an overview of the results of the 2003, 2004, and 2005 field investigations. They include: The confirmed identification of microbial habitats in daylight by detecting fluorescence signals from chlorophyll and dye probes; the characterization of geology by imaging and spectral measurement; the mapping of life along transects; the characterization of environmental conditions; the development of mapping techniques including homogeneous biological scoring and predictive models of habitat location; the development of exploration strategies adapted to the search for life with an autonomous rover capable of up to 10 km of daily traverse; and the autonomous detection of life by the rover as it interprets observations on-the-fly and decides which targets to pursue with further analysis.

Robotic ecological mapping: Habitats and the search for life in the Atacama Desert

[1] As part of the three-year ‘Life in the Atacama’ (LITA) project, plant and microbial abundance were mapped within three sites in the Atacama Desert, Chile, using an automated robotic rover. On-board fluorescence imaging of six biological signatures (e.g., chlorophyll, DNA, proteins) was used to assess abundance, based on a percent positive sample rating system and standardized robotic ecological transects. The percent positive rating system scored each sample based on the measured signal strength (0 for no signal to 2 for strong signal) for each biological signature relative to the total rating possible. The 2005 field experiment results show that percent positive ratings varied significantly across Site D (coastal site with fog), with patchy zones of high abundance correlated with orbital and microscale habitat types (heaved surface crust and gravel bars); alluvial fan habitats generally had lower abundance. Non-random multi-scale biological patchiness also characterized interior desert Sites E and F, with relatively high abundance associated with (paleo)aqueous habitats such as playas. Localized variables, including topography, played an important, albeit complex, role in microbial spatial distribution. Site D biosignature trends correlated with culturable soil bacteria, with MPN ranging from 10-1000 CFU/g-soil, and chlorophyll ratings accurately mapped lichen/moss abundance (Site D) and higher plant (Site F) distributions. Climate also affected biological patchiness, with significant correlation shown between abundance and (rover) air relative humidity, while lichen patterns were linked to the presence of fog. Rover biological mapping results across sites parallel longitudinal W-E wet/dry/wet Atacama climate trends. Overall, the study highlights the success of targeting of aqueousassociated habitats identifiable from orbital geology and mineralogy. The LITA experience also suggests the terrestrial study of life and its distribution, particularly the fields of landscape ecology and ecohydrology, hold critical lessons for the search for life on other planets. Their applications to robotic sampling strategies on Mars should be further exploited.

Impermeant potential-sensitive oxonol dyes: I. Evidence for an “On-off” mechanism

SummaryThis series of papers addresses the mechanism by which certain impermeant oxonol dyes respond to membranepotential changes, denoted ΔEm. Hemispherical oxidized cholesterol bilayer membranes provided a controlled model membrane system for determining the dependence of the light absorption signal from the dye on parameters such as the wavelength and polarization of the light illuminating the membrane, the structure of the dye, and ΔEm. This paper is concerned with the determination and analysis of absorption spectral changes of the dye RGA461 during trains of step changes ofEm. The wavelength dependence of the absorption signal is consistent with an “on-off” mechanism in which dye molecules are driven by potential changes between an aqueous region just off the membrane and a relatively nonpolar binding site on the membrane. Polarization data indicate that dye molecules in the membrane site tend to orient with the long axis of the chromophore perpendicular to the surface of the membrane. Experiments with hyperpolarized human red blood cells confirmed that the impermeant oxonols undergo a potential-dependent partition between the membrane and the bathing medium.

Application of pulsed‐excitation fluorescence imager for daylight detection of sparse life in tests in the Atacama Desert

A daylight fluorescence imager was deployed on an autonomous rover, Zoe, to detect life on the surface and shallow subsurface in regions of the Atacama Desert in Chile during field tests between 2003 and 2005. In situ fluorescent measurements were acquired from naturally fluorescing biomolecules such as chlorophyll and from specific fluorescent probes sprayed on the samples, targeting each of the four biological macromolecule classes: DNA, protein, lipid, and carbohydrate. RGB context images were also acquired. Preparatory reagents were applied to enhance the dye probe penetration and fluorescence intensity of chlorophyll. Fluorescence imager data sets from 257 samples were returned to the Life in the Atacama science team. A variety of visible life forms, such as lichens, were detected, and several of the dye probes produced signals from nonphotosynthetic microorganisms.

A rapid and affordable screening platform for membrane protein trafficking

BackgroundMembrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed.ResultsThis high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane.ConclusionThe IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.