We discuss our recent efforts to achieve highly multiplexed fast fluorescence imaging and quantitative biosensing through camera frame-synchronized scanning of the excitation wavelength in the wide field. For live-cell microscopy, we thus attain low (~1%) crosstalks and ~10 ms temporal resolutions for up to six fluorophores via linear unmixing, and further develop novel, quantitative imaging schemes for both bi-state and FRET fluorescent biosensors. These capabilities are further integrated to multiplex absolute pH imaging with three additional target proteins in the mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging without fluorescence dispersion.
Semicrystalline polymers are an ubiquitous class of materials incorporated into everyday life, and their functional properties are contingent on their structure over a hierarchical range of length scales. By correlating micro- and nano- structural crystalline and amorphous components of lithium triflate doped PEO thin films with fluorescent single particle trajectories, we find that crystalline fibers anisotropically constrain probe transport without altering the intrinsic diffusivity of probes. Our findings suggest controlled and periodic arrangement of crystalline fibers is a promising design principle for mass transport in semicrystalline polymers that enables the requisite mechanical stability for device applications.
While single-molecule localization microscopy (SMLM) offers superior super-resolution for biology, typical SMLM system using highly-inclined off-axis illumination limits an imaging depth to only a few microns from a coverslip surface. Alternative SMLM system using light-sheet illumination has extended the accessible depth for whole cells or small embryos, but may be less practical as it requires specialized or dedicated sample devices. Furthermore, for typical tissue samples (laterally a few millimeters or wider), the lateral lightsheet illumination is no longer applicable. Here, we demonstrate oblique light-sheet SMLM (obSTORM) that provides a facile and practical platform with a full compatibility with tissue samples. By using a single-objective, inclined lightsheet and directly detecting single molecules along the oblique plane, obSTORM opens new doors for tissue-level super-resolution imaging.
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