Figure 3. Flow cytometric detection of the bacterial populations in terms of side scattering and forward scattering without (a) and with (b) Ag+ incubation. (c) Side scattering intensity distribution of the bacteria with and without Ag+ incubation. (d) Schematics of back-illumination microscopy. (e) Bright-field image and (f) back-illumination image of bacteria in a same region of interest.
We further seek to distinguish the individual ‘strongly reducing’ bacteria visibly. Bright field microscopy is first applied to the bacteria mixture after Ag+ incubation and washing with PBS buffers. Figure 3e shows that in the view over 100 bacteria is seen which yet exhibit very different contrasts. While most bacteria appear to be greyer than the background, a small portion shows somewhat even darker which is not observed in the control group (see Figure S2). Although bright field microscopy allows the observations of all the bacteria in the optical field, the difference in the contrasts between different bacteria is sometimes not very distinct (e.g. the upper left inset in Figure 3e). To improve the situation, we have developed an alternative imaging mode utilising the back reflection of a light beam that is able to presumably better distinguish between different types of bacterial cells. As shown in Figure 3d, the technique basically uses the same light path as that in fluorescent microscopy where a light beam illuminates the bacteria and reflect back to the camera, but in this back-reflection mode the light is not restricted to the narrow range of wavelength for fluorophore excitation. Consequently, Figure 3f insets show a more significant contrast between these bacteria – much greater optical intensity for the darker bacteria seen using the bright field mode (Figure 3e insets), while far lower intensity for the rest population. Moreover, the former type of bacteria is observed to be apparently fewer than the latter in the back reflection imaging mode, which is consistent with the results of the flow cytometric measurements. We thus assume the imaging mode is more sensitive to the internal silver nanoparticles. This imaging mode is therefore considered to be a well-suited approach to discern the bacteria of a strong capacity of silver metal metabolism from those of an average ability.