Abstract
A single-molecule total internal reflection fluorescence microscope (TIRFM) is necessary to study the crucial interaction between PCNA and CAF-1 in replication-coupled nucleosome assembly to contribute to epigenetic inheritance. The microscope measures fluorescence following total internal reflection (TIR) to accomplish single molecule imaging (SMI). TIR provides a high signal-to-noise ratio (SNR) by generating a hundred-nanometer-thick evanescent field which excites fluorophores within this zone. SMI provides an observation of the interactions between a single molecule sparsely surface-tethered and other interacting partners labeled with fluorophores with a high time resolution. We built a single-molecule TIRFM from a laser system, two TIR-generating systems (through objective and prism), an inverted microscope, an EMCCD camera, and a computer with analysis programs. A customized prism-lens-holder directed the laser to enter a fused silica prism horizontally so that TIR always occurred with an 74° angle at the sample plane (refractive index (RI) is 1.3 ~ 1.4). Initial attempts to measure the penetration depth with polystyrene microspheres in aqueous solution were ineffective because of the high RI mismatch and the resolution limit. In parallel, initial efforts to image PCNA – CAF-1 interactions with Alexa Fluor 488 at 5 mW observed flickering signals as outliers, and no detection of binding. SMI was established with a compatible 488 nm-50 mW laser for Alexa Fluor 488, but not for Cy3 that would require another appropriate laser. During the development of our TIRFM, the problems that we encountered and addressed are discussed, promising progress in the PCNA – CAF-1 study as well as other binding studies.