Super-resolution vibrational imaging based on photoswitchable Raman probe

Abstract

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.

Super-resolution Raman probe imaging

“…The SRS signal intensity at 1503 cm -1 exhibited a more than 25-fold change during this photoswitching process, underscoring the effective control of the Raman signal with the CW light. We calculated the relative Raman intensity versus EdU (RIE) value for the closed form of DAE620 to be 9.78, which surpasses the RIE values of CMTE (RIE: 1.43) 26 . The high SRS signal intensity of DAE620 is attributed to the epr effect due to the longer absorption wavelength.…”

“…Recently, photoswitchable SRS spectroscopy and microscopy [29][30][31] have been achieved by leveraging photochromic molecules that can transition between two distinct isomers when exposed to ultraviolet (UV) or visible light, thereby enabling the control of Raman signal emission. Inspired by the principles of RESOLFT microscopy, we introduced the concept of RESORT microscopy 26 , wherein an additional continuous-wave (CW) laser beam, configured in a donutshaped pattern, is employed to modulate the SRS detection of a photoswitchable Raman probe (see Fig. 1).…”

“…We thank Yuusaku Wachi, Minoru Kawatani, Hiroyoshi Fujioka, Spencer John Spratt, Takaha Mizuguchi, Kenichi Oguchi, Hikaru Akaboshi, Fumiaki Obata, Ryo Tachibana, Shun Yasunaga, Yoshio Mita, Yoshihiro Misawa, Ryosuke Kojima, Yasuteru Urano for their contribution to our original work 26…”

“…Herein, we describe our recent demonstration of reversible saturable optical Raman transitions (RESORT) microscopy 26 , a novel optical far-field super-resolution Raman microscopy technique that enables vibrational imaging beyond the optical diffraction limit using low-intensity photoswitching lasers and a simplified system configuration. We selected stimulated Raman scattering (SRS) as the foundational Raman imaging technology due to its capacity to offer high vibrational contrast for intracellular structures without non-resonant background interference [9][10][11][12] .…”

Stain-Free Histopathology with Stimulated Raman Scattering Microscopy

“…Taking advantage of chemical engineering, photoswitchable SRS microscopy has been developed based on photoreaction or photoisomerization of vibrational probe molecules. 65−67 Further integrated with the reversible saturable/switchable optical fluorescence transition (RESOLFT) strategy, Shou et al 68 and Ao et al 69 reported super-resolution SRS nanoscopy with sub-100 nm optical resolution. In this scheme, a Gaussianshaped ultraviolet (UV) activation beam and a donut-shaped visible depletion beam were implemented to confine the final SRS point spread function (PSF) (Figure 2e).…”