Lingbo Kong

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.

Characterization of Bacterial Spore Germination Using Integrated Phase Contrast Microscopy, Raman Spectroscopy, and Optical Tweezers

We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus spores in both nutrient (l-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of approximately 2 s. Phase contrast microscopy assesses changes in refractility of individual spores during germination, while Raman spectroscopy gives information on changes in spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant spore is proportional to the level of CaDPA in that spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in spore refractility and complete CaDPA release was observed not only for spores germinating in the well-controlled environment of an optical trap but also for spores germinating when adhered on a microscope coverslip. Using this latter method, we also simultaneously characterized the distribution of the time-to-complete-CaDPA release (T(release)) of hundreds of individual B. cereus spores germinating with both saturating and subsaturating concentrations of l-alanine and with CaDPA.

Rapid confocal Raman imaging using a synchro multifoci-scan scheme for dynamic monitoring of single living cells

We developed a rapid multifoci-scan confocal Raman microscopy system for label-free molecular imaging of single living cells. A pair of galvo-mirrors were used to raster scan a single laser to generate multifoci excitations and another galvo-mirror synchronously projected Raman scattering from each foci onto a multichannel spectrograph such that multiple spectra were collected simultaneously. The image acquisition time is ∼40 times faster than in conventional point-scan Raman microscopy with diffraction-limited resolution retained. We demonstrated that this system can be used to monitor the germination dynamics of single bacterial spores with about 1.0 min resolution and 2.5 mW power at each focal point.

Multifocus confocal Raman microspectroscopy for rapid single-particle analysis

We have developed a multifocus confocal Raman microspectroscopy system that allows simultaneous analyses of ≈ 80 individual biological or airborne microparticles based on a precise image-guided technique. Multiple individual particles adhered in random positions on a coverslip were illuminated by a multifocus excitation pattern formed by rapidly steering a single laser beam with a pair of galvo-mirrors, and their Raman scatterings were synchronously projected with another galvo-mirror to different rows of a CCD chip for parallel spectroscopic analyses. We show that this technique can be used to rapidly identify single airborne particles or bacteria collected on a slide and to monitor germination dynamics of multiple bacterial spores in real-time.

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.

Analysis of the Raman spectra of Ca2+-dipicolinic acid alone and in the bacterial spore core in both aqueous and dehydrated environments

The core of dormant bacterial spores suspended in water contains a large depot of dipicolinic acid (DPA) chelated with divalent cations, predominantly Ca(2+) (CaDPA), and surrounded by water molecules. Since the intensities of the vibration bands of CaDPA molecules depend significantly on the water content in the CaDPAs environment, the Raman spectra of CaDPA in spores may allow the determination of the spore cores hydration state. We have measured Raman spectra of single spores of three Bacillus species in different hydration states including the spores suspended in water, air-dried and vacuum-dried. As a comparison, we also measured the Raman spectra of CaDPA and DPA in different forms including in aqueous solution, and as amorphous powder and crystalline form. We also monitored changes in Raman spectra of an individual spore during dehydration under vacuum. The results indicated that (1) the state of CaDPA in the core of a spore suspended in water is close to an amorphous solid or a glassy state, but still mixed with water molecules; (2) the ratio of intensities of Raman bands at 1575 and 1017 cm(-1) (I(1575)/I(1017)) is sensitive to the water content in the CaDPAs environment; (3) variations in I(1575)/I(1017) are small (∼4%) in a population of dormant Bacillus spores suspended in water; and (4) the I(1575)/I(1017) ratio increases significantly during dehydration under vacuum. Consequently, measurement of the I(1575)/I(1017) ratio of CaDPA in spores may allow a qualitative estimation of the degree of hydration of the bacterial spores core.

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 90min 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.

Observation of the dynamic germination of single bacterial spores using rapid Raman imaging

Abstract. The dynamics of bacterial spore germination were successfully observed using a fast Raman imaging system, in combination with real-time phase contrast microscopy. By using a multifocus scan scheme, the spontaneous Raman-scattering imaging acquisition speed was increased to ∼30  s per frame while maintaining diffraction-limited resolution, which allowed monitoring of the dynamics of spore germination on a time scale of tens of seconds to a few minutes. This allowed simultaneous gathering of rich spatial distribution information on different cellular components including time-lapse molecular images of Ca-dipicolinic acid, protein, and nucleic acid during germination of single bacterial spores for the periods of 30 to 60 min.

Isolation and characterization of Bacillus subtilis spores that are superdormant for germination with dodecylamine or Ca2+‐dipicolinic acid

To isolate and characterize spores superdormant (SD) for germination with either Ca2+‐dipicolinic acid (CaDPA) or dodecylamine.