Guiwen Wang

Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers

This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled microscope sample holder containing a germinant solution plus a nucleic acid stain; (ii) capturing a single spore with optical tweezers; (iii) simultaneously measuring phase-contrast images, Raman spectra and fluorescence images of the optically captured spore at 2- to 10-s intervals; and (iv) analyzing the acquired data for the loss of spore refractility, changes in spore-specific molecules (in particular, dipicolinic acid) and uptake of the nucleic acid stain. This information leads to precise correlations between various germination events, and takes 1–2 h to complete. The method can also be adapted to use multi-trap Raman spectroscopy or phase-contrast microscopy of spores adhered on a cover slip to simultaneously obtain germination parameters for multiple individual spores.

NIR Raman spectroscopic investigation of single mitochondria trapped by optical tweezers

Raman spectroscopy is a vibration spectroscopic technique that has been widely used to probe biochemical changes of biological sample such as tumor tissue, blood cells, bacteria and yeast. Here, we applied near-infrared Raman spectroscopy to analyze the chemical composition changes of intact or swollen mitochondria induced by calcium ions. We used a confocal Laser Tweezers Raman Spectroscopy (LTRS) system that combined optical trapping and near infrared Raman spectroscopy to confine a single mitochondrion and consequently measure its Raman spectra following the addition of calcium ion solution. We analyzed Raman spectra of mitochondria isolated from rat liver, heart muscle and kidney, respectively. The major Raman peaks at 1654, 1602, 1446, 1301 and 1226 cm(-1) were observed from individual intact mitochondria. We examined the differences in near infrared spectra between intact and Ca(2+) damaged mitochondria. We found that after the exposure of the intact mitochondria to the 100 muM Ca(2+) solution the band of 1602 cm(-1) decreased very rapidly in the first period and then disappeared after 30minutes, while the intensities of the phospholipids and protein bands changed slowly in the first period and then suddenly disappeared, corresponding to the Ca(2+) induced swelling process. These results demonstrate the potential of LTRS technique as a valuable tool for the study of bioactivity and molecular composition of mitochondria.

Kinetics of Germination of Wet-Heat-Treated Individual Spores of Bacillus Species, Monitored by Raman Spectroscopy and Differential Interference Contrast Microscopy

ABSTRACT Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores of Bacillus cereus and Bacillus megaterium, as well as of several isogenic Bacillus subtilis strains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca2+ and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time, T lag, at which spores began release of the great majority of their CaDPA during the germination of B. subtilis spores with different nutrient germinants and also increased the variability of T lag values. (iii) The time period, ΔT release, between T lag and the time, T release, at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values of I release, the intensity of a spores DIC image at T release, than did untreated spores and had much longer time periods, ΔT lys, for the reduction in I release intensities to the basal value due to hydrolysis of the spores peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases in T lag and ΔT release were also observed when wet-heat-treated B. subtilis spores were germinated with the nonnutrient dodecylamine, while the change in I release was less significant. (vi) The effects of wet-heat treatment on nutrient germination of B. cereus and B. megaterium spores were generally similar to those on B. subtilis spores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Aims:  To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens.

Effects of wet heat treatment on the germination of individual spores of Clostridium perfringens

To analyse the effect of wet heat treatment on nutrient and non‐nutrient germination of individual spores of Clostridium perfringens.

Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

Aim:  To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores.

Monitoring and rapid quantification of total carotenoids in Rhodotorula glutinis cells using laser tweezers Raman spectroscopy.

Rhodotorula glutinis is known to accumulate large amounts of carotenoids under certain culture conditions, which have very important industrial applications. So far, the molecular mechanism of regulating carotenogenesis is still not well understood. To better understand the carotenogenesis process, it requires methods that can detect carotenogenesis rapidly and reliably in single live cells. In this paper, a method based on laser tweezers Raman spectroscopy (LTRS) was developed to directly detect carotenoids, as well as other important biological molecules in single live R. glutinis cells. The data showed that the accumulation of carotenoids and lipids occurred mainly in the late exponential and stationary phases when the cell growth was inhibited by nutrient limitation. Meanwhile, the carotenoid concentration changed together with the concentration of nucleic acids, which increased in the first phase and decreased in the last phase of the culture. These data demonstrate that LTRS is a rapid, convenient, and reliable method to study the carotenogenesis process in vivo.

Probing the Kinetic Anabolism of Poly-Beta-Hydroxybutyrate in Cupriavidus necator H16 Using Single-Cell Raman Spectroscopy

Poly-beta-hydroxybutyrate (PHB) can be formed in large amounts in Cupriavidus necator and is important for the industrial production of biodegradable plastics. In this investigation, laser tweezers Raman spectroscopy (LTRS) was used to characterize dynamic changes in PHB content—as well as in the contents of other common biomolecule—in C. necator during batch growth at both the population and single-cell levels. PHB accumulation began in the early stages of bacterial growth, and the maximum PHB production rate occurred in the early and middle exponential phases. The active biosynthesis of DNA, RNA, and proteins occurred in the lag and early exponential phases, whereas the levels of these molecules decreased continuously during the remaining fermentation process until the minimum values were reached. The PHB content inside single cells was relatively homogenous in the middle stage of fermentation; during the late growth stage, the variation in PHB levels between cells increased. In addition, bacterial cells in various growth phases could be clearly discriminated when principle component analysis was performed on the spectral data. These results suggest that LTRS is a valuable single-cell analysis tool that can provide more comprehensive information about the physiological state of a growing microbial population.

Dual-trap Raman tweezers for probing dynamics and heterogeneity of interacting microbial cells

We report on development of dual-trap Raman tweezers for monitoring cellular dynamics and heterogeneity of interacting living cells suspended in a liquid medium. Dual-beam optical tweezers were combined with Raman spectroscopy, which allows capturing two cells that are in direct contact or closely separated by a few micrometers and simultaneously acquiring their Raman spectra with an imaging CCD spectrograph. As a demonstration, we recorded time-lapse Raman spectra of budding yeast cells held in dual traps for over 40 min to monitor the dynamic growth in a nutrient medium. We also monitored two germinating Bacillus spores after the initiation with L-alanine and observed their heterogeneity in the release of CaDPA under identical microenvironment.

Application and Progress of Raman Tweezers in Single Cells

Abstract Raman tweezers is a new optical technique that combines laser tweezers with Raman spectroscopy, which has the capability of studying single biological cells or organelles in nearly natured solution. Owing to its characteristics of noncontact, noninvasion, fast identification, and real-time detection, it has found application in improving the Signal Noise Ratio of Raman spectroscopy, optimizing the detection and sorting single cells or organelles, practicing the real-time detection of biochemistry dynamics process so as to make the activity and mechanisms of biomolecules understood deeply. The source, principle of Raman tweezers, and its optical application in biology are depicted and reviewed particularly in this article.