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.