Multicolor super-resolution imaging using spectroscopic single-molecule localization microscopy with optimal spectral dispersion

Abstract

We developed a transmission diffraction grating-based spectroscopic single-molecule localization microscopy (sSMLM) to collect the spatial and spectral information of single-molecule blinking events concurrently. We characterized the spectral heterogeneities of multiple far-red emitting dyes in a high-throughput manner using sSMLM. We also investigated the influence of spectral dispersion on the single-molecule identification performance of fluorophores with large spectral overlapping. The carefully tuning of spectral dispersion in grating-based sSMLM permitted simultaneous three-color super-resolution imaging in fixed cells with a single objective lens at relatively low photon budget. Our sSMLM has a compact optical design and can be integrated with conventional localization microscopy to provide add-on spectroscopic analysis capability.

Symmetrically dispersed spectroscopic single-molecule localization microscopy

“…In addition to functional imaging based on spectral analysis 7 , sSMLM allows multicolour imaging with theoretically unlimited multiplexing capability. The multiplexing capability is predominantly determined by the spectral separation of selected dyes and the spectral precision under given experimental conditions 1,5,6 . We validated this capability of SDsSMLM using two types of nanospheres (200-nm diameter, F8806 and F8807, Invitrogen).…”

“…The ability of spectroscopic single-molecule localization microscopy (sSMLM) to capture the spectroscopic signatures of individual molecules along with their spatial distribution allows the observation of subcellular structures and dynamics at the nanoscale. As a result, sSMLM has shown great potential in understanding fundamental biomolecular processes in cell biology and material science [1][2][3][4][5][6][7] . It also enables the characterization of nanoparticle properties based on the emission spectrum at the single-particle level [8][9][10] .…”

“…Similar to other localizationbased super-resolution techniques, such as stochastic optical reconstruction microscopy (STORM) and point accumulation for imaging in nanoscale topography (PAINT), the localization precision of sSMLM is fundamentally limited by the number of collected photons per emitter 11 . However, sSMLM suffers from further photon budget constraints since the collected photons of each molecule need to be divided into two separate channels to simultaneously capture the spatial and spectral information [5][6][7][8][9][10] . Thus, the spatial localization precision of sSMLM also depends on the splitting ratio between the spatial and spectral channels and is typically limited to 15-30 nm in cell imaging 2,3,5,6 .…”

“…However, sSMLM suffers from further photon budget constraints since the collected photons of each molecule need to be divided into two separate channels to simultaneously capture the spatial and spectral information [5][6][7][8][9][10] . Thus, the spatial localization precision of sSMLM also depends on the splitting ratio between the spatial and spectral channels and is typically limited to 15-30 nm in cell imaging 2,3,5,6 . Although a dual-objective sSMLM design was previously demonstrated with improved spatial localization precision, it imposes a constraint on live-cell imaging and adds complexity to system alignment 1 .…”

Multi-modal Single-molecule Imaging with Continuously Controlled Spectral-resolution (CoCoS) Microscopy

“…Generally, these systems split the FOV to a diffraction limited localization channel and a spectral channel in which dispersive elements convert the spectra into spatial intensity distributions. Nevertheless, these methods divide the total number of emission photons between the channels resulting in reduced localization and spectral accuracy 16 and FOV sizes of 100-400 µm² 11,13,14,17,18 , 40 to 144 fold smaller compared to sequential imaging. A table summarizing the pros and cons of existing configurations is presented in supplementary Table S1.…”