The Hsp90 chaperone is a central node of protein homeostasis activating a large number of diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies define distinct conformational states of the mechanistic core implying structural changes that have not yet been observed in solution. Here, we engineered one-nanometer fluorescence probes based on photo-induced electron transfer into yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement are mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilizes the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.

Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.

No abstract

The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1−8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

Cyanine dyes are exceptionally useful probes for ar ange of fluorescence-based applications,b ut their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending ar ing system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime.H ere,w er eport an optimizeds ynthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins.Wedemonstrate that ab ifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in ar ange of contexts.T hese include their use in the plasmonic hotspot of aD NA-nanoantenna, in single-molecule Fçrster-resonance energy transfer (FRET) applications,f ar-red fluorescence-lifetime imaging microscopy(FLIM), and single-molecule localization microscopy( SMLM). These efforts define contexts in whiche liminating cyanine isomerization provides meaningful benefits to imaging performance.