2.2.1. Optical set-up and the microscopic measurements
The microscopic imaging and measurements were performed on a commercial
inverted microscope (Nikon, Ti-E), which was equipped with a 60X
objective lens with a numerical aperture (NA) of 1.49. Depending on the
studied species, the microscope was operated under three different
modes: fluorescent, bright field, back-reflection. In the fluorescence
mode, a white-light diode (X-cite® 110LED, Excelitas
Technologies) coupled with a bandpass filter (450–490 nm) was used to
excite DHE-induced fluorophores. A digital CMOS camera (C11440,
Hamamastu) was used to collect the fluorescence signals, and a long-pass
filter (λ > 520 nm) to prevent the excitation light from
entering the camera. The back-reflection mode using the same light path
as that of the fluorescent mode was achieved by using a 520-560 nm
bandpass filter for the illumination light (to avoid fluorescent
excitation when the solution contained DHE molecules), and also removing
the long-pass filter. As such, only the back reflected green light
signals were collected. The two imaging modes were switched by
converting the filter cube manually. In the bright field mode, the light
source was a quartz tungsten halogen lamp (LV-LH50PC, Nikon) and used
without any optical filters. The three modes shared the same camera as
above.
For fluorescence analysis, the bacteria containing Ag particles were
prepared as a bacterial solution with a concentration of
2×108 mL-1 using PBS buffer, and DHE
was added into the latter with a final concentration of 100 μM. The
mixture was incubated at 30 ℃ for 1 hour and then washed using PBS
buffer to remove the excess DHE molecules. A 100 μL of the washed
mixture was added into a PDMS chamber with a sodium alginate-modified
glass substrate and let it stand still for 10 minutes. Consequently, the
bacterial cells were observed to adsorb strongly onto the glass wall
forming a monolayer. PBS was used to gently rinse the mixture in the
chamber for the removal of suspended bacteria. As such, the adhered
bacteria were studied using the fluorescence microscopy described above.
The sample preparation for the bright field and back reflection imaging
was all the same but the DHE molecules were not added in the experiments
described in Section 3.2.