SignificanceAbsorption microscopy is a promising technique that can detect single nonfluorescent molecules. However, fundamental limitations of existing optical absorption methods result in noisy detection signals for single molecules, which has hindered many anticipated applications. A promising method is to optically measure the photothermal heating of single molecules. In this paper, we present a photothermal microscopy technique where we detect the photothermal heating of single molecules mechanically with a temperature-sensitive nanomechanical drum. With our method, we achieve an unprecedented optical absorption sensitivity, enabling the detection of single molecules with large signal-to-noise ratios. This enables interesting applications such as the accurate localization of naturally occurring marker molecules or the identification of single molecules by measuring their absorption spectrum.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

It is the main function of T cells to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data has indicated that global clustering of the T cell receptor (TCR) occurs prior to T cell activation. Such pre-activation clustering has been interpreted as representing a potential regulatory mechanism that fine-tunes the T cell response. We found here that apparent TCR nanoclustering could be attributed to overcounting artifacts inherent to single-molecule-localization microscopy. Using complementary super-resolution approaches and statistical image analysis, we found no indication of global nanoclustering of the TCR on antigen-experienced CD4 + T cells under non-activating conditions. We also used extensive simulations of super-resolution images to provide quantitative limits for the degree of randomness of the TCR distribution. Together, our results suggest that the distribution of TCRs on the plasma membrane is optimized for fast recognition of antigen in the first phase of T cell activation.

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

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