Fluorescence-Encoded Time-Domain Coherent Raman Spectroscopy in the Visible Range.

Fluorescence-encoded vibrational spectroscopy has attracted increasing attention by virtue of its high sensitivity and high chemical specificity. We recently demonstrated fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS), which enables low-frequency vibrational spectroscopy of low-concentration fluorophores using near-infrared (800-900 nm) light excitation. However, the feasibility of this study was constrained by the scarcity of excitable molecules in the near-infrared range. Consequently, the broader applicability of FLETCHERS has not been investigated. Here we extend the capabilities of FLETCHERS into the visible range by employing a noncollinear optical parametric amplifier as a light source, significantly enhancing its versatility. Specifically, we use the method, which we refer to as visible FLETCHERS (vFLETCHERS), to individually acquire Raman spectra from five visible fluorophores that have absorption peaks in the 600-700 nm region. These results not only confirm the versatility of vFLETCHERS for a wide range of molecules but also allude to its widespread applicability in biological research through highly sensitive supermultiplexed imaging.

Highly Sensitive Low-Frequency Time-Domain Raman Spectroscopy via Fluorescence Encoding.

Fluorescence-encoded vibrational spectroscopy has become increasingly more popular by virtue of its high chemical specificity and sensitivity. However, current fluorescence-encoded vibrational spectroscopy methods lack sensitivity in the low-frequency region, which if addressed could further enhance their capabilities. Here, we present a method for highly sensitive low-frequency fluorescence-encoded vibrational spectroscopy, termed fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS). By first exciting molecules into vibrationally excited states and then promoting the vibrating molecules to electronic states at varying times, the molecular vibrations can be encoded onto the emitted time-domain fluorescence intensity. We demonstrate the sensitive low-frequency detection capability of FLETCHERS by measuring vibrational spectra in the lower fingerprint region of rhodamine 800 solutions as dilute as 250 nM, which is ∼1000 times more sensitive than conventional vibrational spectroscopy. These results, along with further improvement of the method, open up the prospect of performing single-molecule vibrational spectroscopy in the low-frequency region.