Chemical imaging innovations

1.1 Multiplex SRS microscopy: Spectral acquisition at microsecond scale

Our group invented a 32-channel tuned amplifier array, which enabled acquisition of an SRS spectrum within 5 microseconds to cover a window of 200 wavenumbers [Light S&A 2015]. This method enabled high throughput single cell analysis through multichannel SRS flow cytometry [Optica 2017]. Through multiplex modulation, we demonstrated spectroscopic SRS imaging in a spectrometer free manner [Science Advances 2015]. We further developed a resonant optical delay tuner for microsecond-scale hyperspectral SRS imaging with a pair of chirped fs pulses [Optica 2016].

1.2 Volumetric chemical imaging

Volumetric imaging allows global understanding of three-dimensional (3D) complex systems. Light-sheet fluorescence microscopy and optical projection tomography have been reported to image 3D volumes with high resolutions and at high speeds. Such methods, however, usually rely on fluorescent labels for chemical targeting, which could perturb the biological functionality in living systems. We demonstrated Bessel-beam-based stimulated Raman projection (SRP) microscopy and tomography for label-free volumetric chemical imaging [Nature Communications 2017]. More recently, by tissue clearance agent, we demonstrated 3D SRS chemical histology [Bio Opt Exp 2019]

1.3 Plasmon-enhanced CRS microscopy at single molecule sensitivity

Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of CRS microscopy is limited to ~10mM for endogenous biomolecules. Label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10−30 cm2 sr−1) remains unreachable. We demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity [Nature Communications 2019]. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical system.

1.4 Mid-infrared photothermal (MIP) microscopy: Breaking the diffraction limit by sensing the thermal effect

Infrared spectroscopy is highly sensitive in the fingerprint region, but the spatial resolution is limited by the long infrared wavelength. By using a visible beam to sense the thermal lensing effect induced by infrared absorption in fingerprint region, MIP imaging of living cells with  submicron resolution was first demonstrated [Science Advances 2016]. We also demonstrated epi-detected MIP [Anal Chem 2017] and DFG-based MIP (J Phys Chem C 2017]. Recently we pushed the speed limit of this method to 1000 frames per second via wide-field illumination [Science Advances 2019]. We also developed an iRaman platform which integrates IR photothermal imaging and Raman spectroscopy [Anal Chem 2019].

1.5 Bond-selective transient phase microscopy

Phase-contrast microscopy converts phase shift of light passing through a transparent specimen, e.g. a biological cell, into brightness variations in an image. This ability to observe structures without destructive fixation or staining has been widely utilized for applications in materials and life sciences. Despite these advantages, phase-contrast microscopy lacks the ability to reveal molecular information. To fulfill this gap, we developed a bond-selective transient phase (BSTP) imaging technique that excites molecular vibration by infrared light, resulting in a transient change of phase shift detected by a diffraction phase microscope. By developing a time-gated pump-probe camera system, we demonstrate BSTP imaging of live cells at 50 Hz frame rate with high spectral fidelity, sub-microsecond temporal resolution, and submicron spatial resolution. Our approach [Light S&A 2019] paves a new avenue for spectroscopic imaging investigation in biology and materials science.

1.6 Vibration-based photoacoustic tomography: listening to chemical bond vibration

To enable deep-tissue vibrational imaging, we invented a new modality based on optical excitation of harmonic vibration, subsequent relaxation of vibrational energy into heat, and acoustic detection of the generated ultrasonic waves [Phys Rev Lett  2011]. This method extended the vibrational imaging depth to centimeter scale, allowing us to see inside the body noninvasively. We further demonstrated two significant biomedical applications of this approach, one for in vivo imaging of lipid-laden plaque [Scientific Reports 2018; 2019] and the other for in situ detection of breast cancer margin [Medical Devices and Sensors 2018].