SummaryThe plasma membrane is the site of intercellular communication and subsequent intracellular signal transduction. The specific visualization of the plasma membrane in living cells, however, is difficult using fluorescence-based techniques owing to the high background signals from intracellular organelles. In this study, we show that second harmonic generation (SHG) is a high-resolution plasma membrane-selective imaging technique that enables multifaceted investigations of the plasma membrane. In contrast to fluorescence imaging, SHG specifically visualizes the plasma membrane at locations that are not attached to artificial substrates and allows high-resolution imaging because of its subresolution nature. These properties were exploited to measure the distances from the plasma membrane to subcortical actin and tubulin fibers, revealing the precise cytoskeletal organization beneath the plasma membrane. Thus, SHG imaging enables the specific visualization of phenomena at the plasma membrane with unprecedented precision and versatility and should facilitate cell biology research focused on the plasma membrane.

Quantum-enhanced stimulated Raman scattering (QESRS) microscopy is expected to realize molecular vibrational imaging with sub-shot-noise sensitivity, so that weak signals buried in the laser shot noise can be uncovered. Nevertheless, the sensitivity of previous QESRS did not exceed that of state-of-the-art stimulated Raman scattering (SOA-SRS) microscopes mainly because of the low optical power (3 mW) of amplitude squeezed light [Nature 594, 201 (2021)10.1038/s41586-021-03528-w]. Here, we present QESRS based on quantum-enhanced balanced detection (QE-BD). This method allows us to operate QESRS in a high-power regime (>30 mW) that is comparable to SOA-SRS microscopes, at the expense of 3 dB sensitivity drawback due to balanced detection. We demonstrate QESRS imaging with 2.89 dB noise reduction compared with classical balanced detection scheme. The present demonstration confirms that QESRS with QE-BD can work in the high-power regime, and paves the way for breaking the sensitivity of SOA-SRS microscopes.

Coherent anti-Stokes Raman scattering (CARS) imaging is widely used for imaging molecular vibrations inside cells and tissues. Lipid bilayers are potential analytes for CARS imaging due to their abundant CH 2 vibrational bonds. However, identifying the plasma membrane is challenging since it possesses a thin structure and is closely apposed to lipid structures inside the cells. Since the plasma membrane provides the most prominent asymmetric location within cells, orientation sensitive sum-frequency generation (SFG) imaging is a promising technique for selective visualization of the plasma membrane labeled by a nonfluorescent and SFG-specific dye, Ap3, when using a CARS microscope system. In this study, we closely compare the characteristics of lipid bilayer imaging by dye-based SFG and CARS using giant vesicles (GVs) and N27 rat dopaminergic neural cells. As a result, we show that CARS imaging can be exploited for the visualization of whole lipid structures inside GVs and cells but is insufficient for identification of the plasma membrane, which instead can be achieved using dye-based SFG imaging. In addition, we demonstrate that these unique properties can be combined and applied to the live-cell tracking of intracellular lipid structures such as lipid droplets beneath the plasma membrane. Thus, multimodal multiphoton imaging through a combination of dye-based SFG and CARS can serve as a powerful chemical imaging tool to investigate lipid bilayers in GVs and living cells.

Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.