Applications of fluorescence microscopy for quantitative analysis of fluorescent molecules

Abstract

Fluorescent molecules are widely used not only in spectroscopy and biochemistry, but also in various fluorescence microscopes and single molecular detection techniques due to their high sensitivity and intuition. Fluorescent molecules have unique photophysical properties depending on the electronic structure. Therefore, an appropriate fluorophores may be selected for a specific purpose. In addition, fluorescence emission can be analyzed to obtain spatial and temporal information such as the distribution of target molecules and electron transition rates. In general, microscopes with optical filters and detectors are used to obtain photon signals for experiments. In this paper, applications for quantitative analysis of fluorescent molecules using a microscope will be mainly introduced. Also, quantum yield measurements, fluorescence correlation spectroscopy, and the magnetic field effect of emission in micelle will be briefly described. First, the magnetic field of the magnet was imaged using a pyrene-based magnetic field sensitive molecule. The magnetic field sensitive material forms an exciplex when excited, indicating that the intensity of fluorescence changes depending on the external magnetic field. Compared to the previous phenanthrene-based molecules, pyrene-based materials exhibit higher brightness (24.7 times, 355 nm), as well as are possible excitation at longer wavelengths (375 nm). Therefore, it is possible to build a setup that can image a magnetic field of a magnet just by installing a simple LED lamp in a conventional fluorescence microscope. With these advantages, a high S/N ratio can be obtained under milder conditions. In addition, it was confirmed that light stability over time was also improved because the experiment was possible at a longer wavelength with weak power. Second, differences in fluorescence mechanisms were found by analyzing the optical properties of upconversion nanoparticle (UCNP) through a microscope with PMT. UCNP is a material that is expected to be used for bioimaging and biosensors because it is known as a non-blinking and very stable light emitting body during upconversion light emission. I linked EMCCD and PMT to fluorescence microscopy, and found that UCNP exhibited stochastic photon emission (SPEM) phenomenon at several nanoseconds to microseconds time scale. In addition, I investigated that the off-time distribution was fitted to a single exponent. So it confirmed that there was one rate-determining step. Through this, it was expected that the green and red fluorescence of UCNP would result from different pathways. Finally, synthesis in which polystyrene microparticles are connected by functional polyelectrolyte chains was confirmed through a bright-field microscopy. In order to control the optical, physical, and chemical properties of polymer composites, it is important to efficiently connect between polymers. In this study, microparticle impregnation into the sodium polystyrene sulfonate (NaPSS) was synthesized using a single-pot strategy. Kaiser assay was used to confirm that the reaction was successful, and the result was analyzed by flowing microparticles into the flow cell and imaging them. It was confirmed that 65% microparticles were connected by analyzing them with MATLAB code.