共焦点光学系を用いたマイクロウェル内微量蛍光体の検出感度向上

概要

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In this study, we worked on the improvement of detection sensitivity for fluorescence dyes filled in the well of Microwell with Manageable Volume (MMV). For this purpose, we applied fluorescence lifetime imaging microscopy (FLIM) and time-resolved fluorescence (TRF) to method of fluorescence detection. In addition, we built up confocal optics system with a pinhole in front of the photomultiplier tube on the fluorescence microscopy in order to decrease noise from MMV. In the result, the confocal optics system increased the S/N ratio of photon count from dilute Alexa fluor 430. However, the pinhole limited available photon and mechanical vignetting of light path by lateral face of the well also restricted available photon. They caused increasing standard deviation of the result. These issues affected sensitivity of fluorescence detection and it is necessary to balance between noise reduction and amount of available photon for improvement of detection sensitivity by confocal optics system. We suggested suitable pinhole size and other parameters in order to keep the balance, and we succeeded to detect 1 pM Alexa fluor 430 filled into the well of MMV.