Dr. Hemant Bhatta Profile

Dr. Hemant Bhatta

Research Associate at University of New South Wales

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Proceedings Article | 15 February 2006 Paper

Fluorescence and fluorescence-lifetime imaging microscopy (FLIM) to characterize yeast strains by autofluorescence

H. Bhatta, Ewa Goldys, J. Ma

Proceedings Volume 6088, 608801 (2006) https://doi.org/10.1117/12.645354

KEYWORDS: Luminescence, Yeast, Microscopy, Fluorescence lifetime imaging, Statistical analysis, Image analysis, Microscopes, Microorganisms, Laser scanners, Picosecond phenomena

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We characterised populations of wild type baking and brewing yeast cells using intrinsic fluorescence and fluorescence lifetime microscopy, in order to obtain quantitative identifiers of different strains. The cell autofluorescence was excited at 405 nm and observed within 440-540 nm range where strong cell to cell variability was observed. The images were analyzed using customised public domain software, which provided information on cell size, intensity and texture-related features. In light of significant diversity of the data, statistical methods were utilized to assess the validity of the proposed quantitative identifiers for strain differentiation. The Kolmogorov-Smirnov test was applied to confirm that empirical distribution functions for size, intensity and entropy for different strains were statistically different. These characteristics were followed with culture age of 24, 48 and 72 h, (the latter corresponding to a stationary growth phase) and size, and to some extent entropy, were found to be independent of age. The fluorescence intensity presented a distinctive evolution with age, different for each of the examined strains. The lifetime analysis revealed a short decay time component of 1.4 ns and a second, longer one with the average value of 3.5 ns and a broad distribution. High variability of lifetime values within cells was observed however a lifetime texture feature in the studied strains was statistically different.

Proceedings Article | 29 March 2005 Paper

Rapid identification of microorganisms by intrinsic fluorescence

Hemant Bhatta, Ewa Goldys, Robert Learmonth

Proceedings Volume 5699, (2005) https://doi.org/10.1117/12.588851

KEYWORDS: Yeast, Luminescence, Bacteria, Visible radiation, Microscopy, Spectroscopy, Microorganisms, Ultraviolet radiation, Proteins, Fluorescence spectroscopy

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Microbial contamination has serious consequences for the industries that use fermentation processes. Common contaminants such as faster growing lactic acid bacteria or wild yeast can rapidly outnumber inoculated culture yeast and produce undesirable end products. Our study focuses on a rapid method of identification of such contaminants based on autofluorescence spectroscopy of bacterial and yeast species. Lactic acid bacteria (Lac-tobacillus casei), and yeast (Saccharomyces cerevisiae) were cultured under controlled conditions and studied for variations in their autofluorescence. We observed spectral differences in the spectral range representative of tryptophan residues of proteins, with excitation at 290 nm and emission scanned in the 300 nm - 440 nm range. Excitation scans between 240 nm and 310 nm were also performed for the emission at 340 nm. Moreover, we observed clearly pronounced differences in the excitation and emission in the visible range, with 410 nm excitation. These results demonstrate that bacterial and yeast species can be differentiated using their intrinsic fluorescence both in UV and in the visible region. The comparative spectroscopic study of selected strains of Saccharomyces yeast showed clear differences between strains. Spectrally-resolved laser scanning microscopy was carried out to link the results obtained using ensembles of cells with spectral properties of individual cells. Strongly fluorescent subpopulation were observed for all yeast strains with excitation at 405 nm. The fluorescence spectra showed variations correlated with cell brightness. The presented results demonstrate that using autofluorescence, it is possible to differentiate between yeast and lactic acid bacteria and between different yeast species.

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