Pushing single photon array cameras to the limits : single molecule widefield lifetime imaging

Abstract

Microscopy has been an underpinning technology of the scientific community since it’s conception at the end of the 16th century. Significant inventions, which are still in use today, in the form of the fluorescence microscope and the development of fluorescent dyes, have confirmed and enhanced microscopy’s place as a staple in science today. However, these advancements would be dramatically reduced if the detector technology had not progressed in parallel. With the development of Single Photon Avalanche Diodes (SPAD) array detectors it is now possible to perform time resolved microscopy on-chip, without the need for any additional counting electronics. This allows for the possibility to perform widefield fluorescent lifetime imaging microscopy (FLIM) due to their fast frame rates and single photon sensitivity. The research presented here is based on the SPAD array technology, SPCImager. The aim is to push the SPCImager platform to its limits by performing widefield single molecule fluorescent lifetime imaging microscopy. The SPCImager is able to perform this due to a dual time-gated imaging modality that allows crude time tagging capabilities over the full array of 320 by 240 pixels. To provide SPCImager with the most optimal and stable conditions, an objective-based Total Internal Reflection Fluorescence (TIRF) microscope set up was designed and developed to allow operation in either CW or Pulsed laser modalities with data being captured on SPCImager and a commercial sCMOS/emCCD simultaneously. To gain a more complete understanding on the deployment of SPCImager’s dual time gates, a complete computational model was developed to simulate SPCImager’s response to a fluorescent event. By conducting a study into optimising imaging parameters, for a range of simulated events from 1µm carboxylate microspheres to quantum dots and single molecules, it was possible to determine optimal settings required to return accurate and reliable lifetimes. SPCImager was able to perform Widefield FLIM on a range of microspheres before demonstrating its potential and, identifying limitations, when applying this to single molecule widefield FLIM.