Toward photoswitchable electronic pre-resonance stimulated Raman probes

Abstract

Reversibly photoswitchable probes allow for a wide variety of optical imaging applications. In particular, photoswitchable fluorescent probes have significantly facilitated the development of super-resolution microscopy. Recently, stimulated Raman scattering (SRS) imaging, a sensitive and chemical-specific optical microscopy, has proven to be a powerful live-cell imaging strategy. Driven by the advances of newly developed Raman probes, in particular the pre-resonance enhanced narrow-band vibrational probes, electronic pre-resonance SRS (epr-SRS) has achieved super-multiplex imaging with sensitivity down to 250 nM and multiplexity up to 24 colors. However, despite the high demand, photoswitchable Raman probes have yet to be developed. Here, we propose a general strategy for devising photoswitchable epr-SRS probes. Toward this goal, we exploit the molecular electronic and vibrational coupling, in which we switch the electronic states of the molecules to four different states to turn their ground-state epr-SRS signals on and off. First, we showed that inducing transitions to both the electronic excited state and triplet state can effectively diminish the SRS peaks. Second, we revealed that the epr-SRS signals can be effectively switched off in red-absorbing organic molecules through light-facilitated transitions to a reduced state. Third, we identified that photoswitchable proteins with near-infrared photoswitchable absorbance, whose states are modulable with their electronic resonances detunable toward and away from the pump photon energy, can function as the photoswitchable epr-SRS probes with desirable sensitivity (<1 μM) and low photofatigue (>40 cycles). These photophysical characterizations and proof-of-concept demonstrations should advance the development of novel photoswitchable Raman probes and open up the unexplored Raman imaging capabilities.

Bringing Vibrational Imaging to Chemical Biology with Molecular Probes

“…Furthermore, by harnessing the narrow line width of Raman peaks (50–100 times narrower than fluorescence peaks), researchers have developed highly sensitive Raman probe palettes. The matching dye palettes enable super-multiplexed (more than 20 channels) optical imaging for organelles or protein profiling with sensitivity down to 250 nM, bridging optical imaging’s subcellular spatial resolution with system biology’s high information throughput. − Moreover, chemically activatable and photochromic SRS probes have also been developed lately for intracellular sensing and multiplexed tracking, empowering functional SRS imaging. − …”

“…Such features are crucial to a variety of biological investigations including tracking protein dynamics, sensing subcellular environments and imaging ultrastructures beyond the diffraction limit. In 2021, three groups independently reported photophysical or photochemical approaches to achieve photoswitchable SRS imaging. − To name one example, the alkyne-tagged diarylethene showed impressive photoswitchable properties in the cell-silent spectral window (Figure k). The UV induced photoisomerization converts diarylethene from the open-ring state to the closed-ring state, while visible light induces the reverse conversion, accompanied by the switching of SRS peak intensities.…”

Super-resolution vibrational imaging based on photoswitchable Raman probe

“…Distinct from STED microscopy, reversible saturable optical fluorescence transitions (RESOLFT) microscopy based on photoswitchable fluorescence probes enables PSF modification under low power 25 . Recently, photoswitchable SRS spectroscopy and microscopy [26][27][28] have been demonstrated by exploiting photochromic molecules that can be photoswitched between two distinct isomers by applying ultraviolet (UV) or visible light, turning the Raman signal on or off. Inspired by RESOLFT microscopy, we propose the concept of RESORT microscopy, in which an additional CW laser light including a donut-shaped beam is introduced to regulate the SRS detection of a photoswitchable Raman probe (Fig.…”

Miniaturized Chemical Tags for Optical Imaging

“…To improve the detection sensitivity of photoactivatable Raman probes, Wei and co-workers have recently investigated the potential application of this methodology using epr-SRS. [105] Figure 6. Multiplexed Raman imaging and sensing in mammalian cells.…”