Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy

Abstract

As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with SMLM was next utilized to achieve f-SRM. By encoding functional information into the spectral responses of environment-sensing fluorescent probes, f-SRM transcends the structural information provided by typical SRM techniques and reveals the spatiotemporal distribution of physicochemical parameters with single-molecule sensitivity and nanoscale spatial resolution. As one example, by employing the solvatochromic dye Nile Red to sense local chemical polarity, we revealed nanoscale heterogeneity in the membranes of live mammalian cells. This enabled us to unveil substantial polarity differences between the plasma membrane and the membranes of nanoscale intracellular organelles, a result we determined to be due to differences in local cholesterol levels. With the addition of cholesterol or cholera toxin, we further observed the formation of low-polarity, raftlike nanodomains in the plasma membrane. In another study, we generalized SR-SMLM to fluorogenic single-molecule reactions. As a wide-field technique, SR-SMLM readily captures the emission spectra of indiv...

Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

“…Whereas fascinating results were obtained here with free FPs, we expect SMdM with FP-tagged proteins to be powerful in probing specific protein-protein interactions. The further integration of SMdM with other emerging super-resolution methods, e.g., spectrally resolved SMLM (Yan et al, 2018), represents additional exciting possibilities.…”

Toward Precision Measurement and Manipulation of Single‐Molecule Reactions by a Confined Space

“…Since a huge amount of other intriguing approaches, e.g., electron microscopy has been developed for SMR research as well, it is unlikely to include all of them here due to the limit of space, and therefore the selection of papers mainly reflects the authors' own research interest. It is also highly recommended that the readers can refer to some other comprehensive reviews for additional information on each specific single‐molecule technique that is covered in this review, e.g., fluorescence, Raman, scanning probe, electric conductance, force, etc.…”

Information-rich localization microscopy through machine learning

“…Originally developed toward the specific goal of superior spatial resolution, single-21 molecule localization (super-resolution) microscopy (SMLM), including STORM/(F)PALM [1][2][3] 22 and PAINT 4 , has evolved in recent years into a generic tool for sampling diverse biologically 23 relevant information at the nanoscale 5 . For example, when combined with environment-sensitive 24 dyes, intracellular heterogeneity in the local chemical environment can be mapped by 25 concurrently obtaining the spatial positions and emission spectra of millions of single fluorescent 26 molecules 6 .…”

“…For example, when combined with environment-sensitive 24 dyes, intracellular heterogeneity in the local chemical environment can be mapped by 25 concurrently obtaining the spatial positions and emission spectra of millions of single fluorescent 26 molecules 6 . Consequently, the full potential of SMLM is expected to be unleashed through the 27 proper multidimensional analysis of the emission wavelength 5 , brightness, dipole orientation 7 , as 28 well as the axial (3D) position 8 of single molecules. 29 To date, to extract information beyond the in-plane location, e.g., the emission 30 wavelength and the axial position, of single emitters, would often oblige the explicit encoding of 31 such high-dimensionality information into the diffraction pattern of single molecules (point 32 spread functions; PSFs) through optical aberrations and alterations 5,8 , including astigmatism 9 , 33 interference 10 , wavelength-dependent splitting 11 , dispersion 12 , and wave-front modification 13 .…”

“…Consequently, the full potential of SMLM is expected to be unleashed through the 27 proper multidimensional analysis of the emission wavelength 5 , brightness, dipole orientation 7 , as 28 well as the axial (3D) position 8 of single molecules. 29 To date, to extract information beyond the in-plane location, e.g., the emission 30 wavelength and the axial position, of single emitters, would often oblige the explicit encoding of 31 such high-dimensionality information into the diffraction pattern of single molecules (point 32 spread functions; PSFs) through optical aberrations and alterations 5,8 , including astigmatism 9 , 33 interference 10 , wavelength-dependent splitting 11 , dispersion 12 , and wave-front modification 13 . 34 The resultant, engineered PSF shape and intensity then help establish best-fit models between 35 experimental observables and fluorophore characteristics.…”