Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

Abstract

The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter, up to six subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and Förster resonance energy transfer fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.

Super-multiplex imaging of cellular dynamics and heterogeneity by integrated stimulated Raman and fluorescence microscopy

“…The efficient functionalization ( Hu et al., 2018 ; Tian et al., 2020 ; Fujioka et al., 2020 ) of the entire vibrational palette will be needed to realize the full multiplexing capability for targeted imaging in live cells. Besides, the development of excitation-based spectral fluorescence microscopy is promising to further increase the resolvable color number of fluorescence with low cross talk and at high speed ( Chen et al., 2021 ).…”

Combination of Structured Illumination Microscopy with Hyperspectral Imaging for Cell Analysis

“…Besides, those spectral imaging techniques providing better energy concentration in raw data are also worth considering in SIM. A special example for this is a recent spectral microscopy achieved by modulating the excitation wavelength, which is rarely seen in spectral imaging but theoretically compatible with SIM …”

“…A special example for this is a recent spectral microscopy achieved by modulating the excitation wavelength, which is rarely seen in spectral imaging but theoretically compatible with SIM. 43 One last thing to be mentioned is that although we demonstrated only planar hyperspectral SIM imaging in this work, the proposed hyperspectral SIM method can be directly applied to many other SIM configurations that use planar detector for data acquisition, including 3D SIM, 44 lattice lightsheet microscopy, 45 and instant SIM. 19 In summary, in this study, we developed a novel hyperspectral SIM method.…”

Single Molecules Are Your Quanta: A Bottom-Up Approach toward Multidimensional Super-resolution Microscopy

“…103,104,106,107 The latter strategy may be compared with the above-discussed polarization SMLM approach in which the illumination polarization direction is modulated in consecutive camera frames (Figure 2j,k). 50,51 Given the recent demonstration that frame-synchronized modulation of the illumination wavelength enables fast excitation spectral microscopy in the wide-field, 109 it is interesting to speculate whether similar tactics may extend SMLM to the new dimension of the fluorescence excitation spectrum.…”

“…Continued innovations in both illumination and detection designs have kept pushing the achievable spatiotemporal resolution, sample depth, and throughput of SMLM. In particular, methods have emerged that substantially enhance the localization precision of single molecules via the modulation of the illumination pattern (Figure a), remarkably, with patterns analogous to those used in STED microscopy , as well as those used in structured illumination microscopy. − Interestingly, the multiview referencing concept discussed above is utilized here to improve localization precision, whereby the brightness of the same single molecules is referred between altered illumination patterns in different views (Figure b) , or over time. ,,, The latter strategy may be compared with the above-discussed polarization SMLM approach in which the illumination polarization direction is modulated in consecutive camera frames (Figure j,k). , Given the recent demonstration that frame-synchronized modulation of the illumination wavelength enables fast excitation spectral microscopy in the wide-field, it is interesting to speculate whether similar tactics may extend SMLM to the new dimension of the fluorescence excitation spectrum.…”