Combination of Raman tweezers and quantitative differential interference contrast microscopy for measurement of dynamics and heterogeneity during the germination of individual bacterial spores

Raman tweezers and quantitative differential interference contrast (DIC) microscopy are combined to monitor the dynamic germination of individual bacterial spores of Bacillus species, as well as the heterogeneity in this process. The DIC bias phase is set properly such that the brightness of DIC images of individual spores is proportional to the dipicolinic acid (DPA) level of the spores, and an algorithm is developed to retrieve the phase image of an individual spore from its DIC image. We find that during germination, the rapid drop in both the intensity of the original DIC image and the intensity of the reconstructed phase image precisely corresponds to the release of all DPA from that spore. The summed pixel intensity of the DIC image of individual spores adhered on a microscope coverslip is not sensitive to the drift of the slide in both horizontal and vertical directions, which facilitates observation of the germination of thousands of individual spores for long periods of time. A motorized stage and synchronized image acquisition system is further developed to effectively expand the field of view of the DIC imaging. This quantitative DIC technique is used to track the germination of hundreds or thousands of individual spores simultaneously

Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering▿

Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water. Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90°C, the level of the 1:1 complex of Ca2+ and dipicolinic acid (CaDPA; ∼25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (Tlag), and then CaDPA was released within 1 to 2 min. (ii) The Tlag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all three species were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T1 when ∼80% of spore CaDPA was released, then increased rapidly until T2 when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T1. (vi) Carotenoids in B. megaterium spores' inner membranes exhibited two changes during heat treatment. First, the carotenoid's two Raman bands at 1,155 and 1,516 cm−1 decreased rapidly to a low value and to zero, respectively, well before Tlag, and then the residual 1,155-cm−1 band disappeared, in parallel with the rapid CaDPA release beginning at Tlag

Multiple-trap laser tweezers Raman spectroscopy for simultaneous monitoring of the biological dynamics of multiple individual cells

We report the development of a multiple-trap laser tweezers Raman spectroscopy (LTRS) array for simultaneously acquiring Raman spectra of individual cells in physiological environments. This LTRS-array technique was also combined with phase contrast and fluorescence microscopy, allowing measurement of Raman spectra, refractility, and fluorescence images of individual cells with a temporal resolution of~5 s. As a demonstration, we used this technique to monitor multiple Bacillus cereus spores germinating in a nutrient medium for up to 90 min and observed the kinetics of dipicolinic acid release and uptake of nucleic acid-binding stain molecules during spore germination

Direct Analysis of Water Content and Movement in Single Dormant Bacterial Spores Using Confocal Raman Microspectroscopy and Raman Imaging

Heavy water (D₂O) has a distinct molecular vibration spectrum, and this has been used to analyze the water content, distribution, and movement in single dormant Bacillus cereus spores using confocal Raman microspectroscopy and Raman imaging. These methods have been used to measure the kinetics of D₂O release from spores suspended in H₂O, the spatial distribution of D₂O in spores, and the kinetics of D₂O release from spores during dehydration in air at room temperature. The results obtained were as follows. (1) The Raman spectrum of single D₂O-loaded dormant spores suggests that D₂O in spores is in a relatively weak hydrogen-bonded mode, compared to the strong hydrogen-bonded mode in pure D₂O. (2) The D₂O content of individual spores in a population was somewhat heterogeneous. (3) The spatial distribution of D₂O in single dormant spores is uneven, and is less dense in the central core region. Raman images of different molecular components indicate that the water distribution is somewhat different from those of proteins and Ca-dipicolinic acid. (4) Exchange of spore D₂O with external H₂O took place in less than 1 s. (5) However, release of spore D₂O during air dehydration at room temperature was slow and heterogeneous and took 2–3 h for complete D₂O release