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.