Total internal reflection fluorescence microscopy for visualizing the bottom layer of an evaporating sessile droplet.*

Total internal reflection fluorescence (TIRF) microscopy, unlike epifluorescence (EPI) microscopy which captures fluorescence from the entire depth of a sample, selectively visualizes only a single layer of it. TIRF achieves this by the generation of a thin evanescent field near the sample-substrate interface by total internal reflection of laser light. Conventional TIRF systems are designed with a substrate placed above the sample, making the total internal reflection occur at the upper interface of the sample. However, this design of TIRF is unsuitable for imaging sessile droplet samples, where the substrate is placed beneath the droplet. To overcome this limitation, we developed a TIRF system specifically optimized for sessile droplet imaging. Our design includes a prism, slide glass, air slit, and optical trap to create the TIRF effect. We calculated the proper light path through the prism-based TIRF system and used an optical trap to eliminate uncertainties. We then conducted a comparative analysis between TIRF and EPI microscopy by imaging the bottom layer of sessile droplets with fluorescent particles during the evaporation. Our research demonstrated that TIRF provides distinct visualization of particle sedimentation sequence and resultant drying pattern at the bottom of the sessile droplet.