Prof. Aleksandra Radenovic, Laboratory of Nanoscale Biology
Prof. Edoardo Charbon, Advanced Quantum architecture Lab
In this project, the use of single photon avalanche diodes (SPAD) arrays for fluorescence microscopy was explored, with a focus on challenging imaging of single molecules. Firstly, SPAD cameras enable high-speed imaging of fast-moving emitters, which was applied to single proteins on lipid membranes, and to 2D crystals of hexagonal boron nitride. Single-molecule imaging was found to be possible, with the added value of the photon arrival sub-information at 10-100 us resolution, enriching the possibilities for the analysis of trajectories. Professors Aleksandra Radenovic and Edoardo Charbon teamed up with collaborators at Arizona State University to fully exploit the potential of the added data in single-molecule tracking.
In the second phase of their study, researchers explored the time-resolved capabilities of the SPAD cameras to perform lifetime measurements of fluorescent emitters on a wide-field basis, resulting in a new high-throughput method for such measurements, which are normally obtained by scanning one molecule at a time on a confocal microscope. This method could enhance techniques which are used in single-molecule localization microscopy, like STORM and PAINT, by adding lifetime information. This advancement may lead to improved imaging of multiple targets at once and better detection of environmental changes at the super-resolution level.
LBEN: Single-molecule localization microscopy was implemented on SPAD5122 camera, applied to imaging single fluorescently labeled proteins in lipid membranes and 2D hBN crystals. Scanning confocal microscopy using SPAD23 as the detector was also performed, showing limitations in measuring antibunching from emitters with short lifetime in the range of 1-2 nanoseconds due to crosstalk issues. High-speed binary tracking of single proteins and hBN surface emitters and their electrochemical characterization were performed. We also explored in-operando imaging of 2D nanoslits with SPAD512. We optimized gated imaging schemes for single molecule imaging, enabling high-throughput lifetime measurements.
In addition to exploring the proposed in-operando approach with artificial nanoslits, we also utilized the SPAD512 to monitor the memristive and gating behaviors of beta barrel pores biological pores such aerolysin, MspA and alpha-hemolysin. For painting bilayers that are optically addressable, we employed the Fluorescence Microscopy Kit from Nanion Technologies, Munich, DE, and 200 µm aperture MECA opto-inv chips from Ionera Technologies GmbH, Freiburg, and we measured in FLIM or FRET modality.
