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Method for detecting and quantitating capture of organic molecules in hypervelocity impacts.

ABSTRACT: Enceladus is a prime candidate in the solar system for in-depth astrobiological studies searching for habitability and life because it has a liquid water ocean with significant organic content and ongoing cryovolcanic activity. The presence of ice plumes that jet up through fissures in the ice crust covering the sub-surface ocean, enables remote sampling and in-situ analysis via a fly-by mission. However, capture and transport of organic materials to organic analyzers presents distinctive challenges as it is unknown whether, and to what extent, organic molecules imbedded in ice particles can be captured and survive hypervelocity impacts. This manuscript provides a fluorescence microscopic method to parametrically determine the amount of an organic fluorescent tracer dye, Pacific Blue™ (PB) deposited on a metallic surface. This method can be used to measure the capture and survival outcomes of terrestrial hypervelocity impact experiments where an ice projectile labeled with Pacific Blue impacts a soft metal surface. This work is an important step in the advancement of instruments like the Enceladus Organic Analyzer for detecting biosignatures in an Enceladus plume fly-by mission. An apparatus consisting of a substrate humidification shroud coupled with an epifluorescence microscope with CCD detector is developed to measure the intensity of quantitatively deposited Pacific Blue droplets under controlled humidity. Calibration curves are produced that relate the integrated fluorescence intensity of humidified PB droplets on metal foils to the number of PB molecules deposited. To demonstrate the utility of this method, our calibrations are used to analyze and quantitate organic capture and survival (up to 11% capture efficiency) following ice particle impacts at a velocity of 1.7 km/s on an aluminum substrate.

SUBMITTER: Kazemi B

PROVIDER: S-EPMC8374173 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nuclear co-localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer-based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co-localization. We established a new ImageJ-based automated multichannel thresholding (MCT) method to quantitate nuclear co-localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL-positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross-sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as "typical" or high density "hotspot" TUNEL regions (n = 10/group). Images were analyzed and compared between the MCT method and the published techniques including both "standard" and "high" settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built-in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland-Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R2 = 0.8972, hotspot: R2 = 0.9000) with minor to non-significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R2 = 0.8036, standard/hotspot: R2 = 0.4309, high/typical: R2 = 0.7895, high/hotspot: R2 = 0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer-based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells.

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Method for detecting and quantitating capture of organic molecules in hypervelocity impacts.

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