Figure 1 Illustration of the LTRS setup used to acquire Raman spectra of individual spores. The trapping laser is reflected by a planar dichroic mirror and focused through an objective lens to form an optical potential well in the sample cavity. The captured spore Raman scattering excited by the captured laser is returned along the original path and focused into the spectrometer. To illuminate a sample, we used a lamp and acquired images using a CMOS camera. When the trapping laser is turned on, the trapped spore is imprisoned. When the trapping laser is turned off, the spore can move freely.
of the LTRS system is shown in Figure 1 . A diode laser of 532 nm wavelength is used for trapping and Raman excitation of the individual spores. The combination of two biconvex lens (L1 (f1=100mm) and L2(f2=150mm)) is used to expand the beam to overfill the inverted microscope objective (100×, 1.40 NA), which acts as a trapping and Raman excitation and collection objective. The spectrometer (HR Evolution, Horiba Jobin 7 Yvon, Japan) has an 1800 gr/mm grating blazed at 500 nm and a liquid nitrogen-cooled charge coupled device (CCD). The laser power was 2.5 mW and integration time was 30s. The characteristic peaks of CaDPA are located at 660, 825, and 1017 cm-1, the peak at 782 cm-1 is attributed to DNA, and the peak at 1004 cm-1 to phenylalanine [18, 19]. The content of each type of substance can be determined based on the relative intensities of the characteristic peaks [20].

Phase contrast micrography

To investigate CaDPA release from the core of the spores before and after sodium hypochlorite treatment, the state of spores was observed using phase contrast microscopy. Briefly, 2 μl of the budding suspension was added dropwise to a slide and dried in a vacuum desiccator for 10 min. Approximately 300 μl of sterile water was added to the sample chamber and a cover glass was added to form an airtight space. The sample chamber was fixed on an inverted microscope (Ti2; Nikon, Tokyo, Japan) with a 100× oil lens (Nikon). Images were obtained with a CMOS camera (2048×2048).

Cell viability analysis

Untreated and sodium hypochlorite-treated spore samples were diluted to a suitable concentration of spore suspension, coated on nutrient-rich LB agar medium, and placed in a constant temperature incubator at 37°C for 48 h, after which the colonies were counted and the survival of each group of spore samples was calculated.

AFM imaging of spores

Spore suspensions were added dropwise to Ploy-coated slides, allowed to stand in air, and dried for 30 min. Slides were fixed to sample holders and images were obtained in air. Images were acquired using a Bioscience AFM (NanoWizer4; Bruker, Wissembourg, France) operated in AC mode. The AFM probe used for imaging consisted of a silicon tip on a silicon nitride cantilever beam with a resonance frequency of –320 kHz and an elasticity coefficient of 42 N/m. All images were scanned at 512 pixels per line with a scanning frequency of 0.5–1.0 Hz.

Dynamic live-cell imaging

To analyze the sprouting and growth of untreated and sodium hypochlorite-treated spores, dynamic imaging of live cells dynamic imaging (N-storm; Nikon) was used to observe and record growth in real time. A small drop of budding solution was spread on a slide and dried naturally at 25°C for half an hour in order to distribute the spores evenly. A drop of melted 100% enriched LB agar medium was increased by approximately 400 μl to the top of the sample spores to form an agar pad with a thickness of about 3 mm. The agar on one side of the pad was removed to form some small holes for air flow, and was subsequently covered with a coverslip to form an airtight space above the agar. The slides were visualized under an inverted microscope (Ti2; Nikon) and the growth of the spores was recorded with a CCD camera (12bits, 2044 × 2048) at 30 s/frame for 6h at a constant temperature of 37°C.