Browsing by Author "Bhardwaj, C."

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Differentiation of microbial species and strains in coculture biofilms by multivariate analysis of laser desorption postionization mass spectra
(2013-09) Bhardwaj, C.; Cui, Y.; Hofstetter, T.; Liu, S. Y.; Bernstein, Hans C.; Carlson, Ross P.; Ahmed, M.; Hanley, L.
7.87 to 10.5 eV vacuum ultraviolet (VUV) photon energies were used in laser desorption postionization mass spectrometry (LDPI-MS) to analyze biofilms comprised of binary cultures of interacting microorganisms. The effect of photon energy was examined using both tunable synchrotron and laser sources of VUV radiation. Principal components analysis (PCA) was applied to the MS data to differentiate species in Escherichia coliâ€“Saccharomyces cerevisiae coculture biofilms. PCA of LDPI-MS also differentiated individual E. coli strains in a biofilm comprised of two interacting gene deletion strains, even though these strains differed from the wild type K-12 strain by no more than four gene deletions each out of approximately 2000 genes. PCA treatment of 7.87 eV LDPI-MS data separated the E. coli strains into three distinct groups, two â€œpureâ€ groups, and a mixed region. Furthermore, the â€œpureâ€ regions of the E. coli cocultures showed greater variance by PCA at 7.87 eV photon energies compared to 10.5 eV radiation. This is consistent with the expectation that the 7.87 eV photoionization selects a subset of low ionization energy analytes while 10.5 eV is more inclusive, detecting a wider range of analytes. These two VUV photon energies therefore give different spreads via PCA and their respective use in LDPI-MS constitute an additional experimental parameter to differentiate strains and species.
Laser desorption VUV postionization MS imaging of a cocultured biofilm
(2013-09) Bhardwaj, C.; Moore, J. F.; Cui, Y.; Gasper, G. L.; Berstein, H. C.; Carlson, Ross P.; Hanley, L.
Laser desorption postionization mass spectrometry (LDPI-MS) imaging is demonstrated with a 10.5 eV photon energy source for analysis and imaging of small endogenous molecules within intact biofilms. Biofilm consortia comprised of a synthetic Escherichia coli K12 coculture engineered for syntrophic metabolite exchange are grown on membranes and then used to test LDPI-MS analysis and imaging. Both E. coli strains displayed many similar peaks in LDPI-MS up to m/z 650, although some observed differences in peak intensities were consistent with the appearance of byproducts preferentially expressed by one strain. The relatively low mass resolution and accuracy of this specific LDPI-MS instrument prevented definitive assignment of species to peaks, but strategies are discussed to overcome this shortcoming. The results are also discussed in terms of desorption and ionization issues related to the use of 10.5 eV single-photon ionization, with control experiments providing additional mechanistic information. Finally, 10.5 eV LDPI-MS was able to collect ion images from intact, electrically insulating biofilms at âˆ¼100 Î¼m spatial resolution. Spatial resolution of âˆ¼20 Î¼m was possible, although a relatively long acquisition time resulted from the 10 Hz repetition rate of the single-photon ionization source.
Molecular imaging and depth profiling of biomaterials interfaces by femtosecond laser desorption postionization mass spectrometry
(2013-10) Cui, Y.; Bhardwaj, C.; Milasinovic, S.; Carlson, Ross P.; Gordon, R. J.; Hanley, L.
Mass spectrometry (MS) imaging is increasingly being applied to probe the interfaces of biomaterials with invasive microbial biofilms, human tissue, or other biological materials. Laser desorption vacuum ultraviolet postionization with 75 fs, 800 nm laser pulses (fs-LDPI-MS) was used to collect MS images of a yeastâ€“Escherichia coli co-culture biofilm. The method was also used to depth profile a three-dimensionally structured, multispecies biofilm. Finally, fs-LDPI-MS analyses of yeast biofilms grown under different conditions were compared with LDPI-MS using ultraviolet, nanosecond pulse length laser desorption as well as with fs laser desorption ionization without postionization. Preliminary implications for the use of fs-LDPI-MS for the analysis of biomaterials interfaces are discussed and contrasted with established methods in MS imaging.