Rapid diagnosis of biopsies, including core-needle biopsy and gastroscopic biopsy, is crucial for clinical decision makings. We applied stimulated Raman scattering (SRS) microscopy on fresh biopsy specimens without fixation, sectioning or staining. We further combined SRS with various deep neural networks for fast histological imaging and automated diagnosis. These include the integration of U-Net for femtosecond-SRS histology, as well as the use of convolutional neural network for histological classifications and gradings. Our results indicated that SRS histology integrated with deep learning algorithm provides potentials for delivering rapid diagnosis that could aid the surgical management of cancers.
Stimulated Raman scattering (SRS) microscopy has shown superior chemical resolution due to the much narrower vibrational spectral bandwidth than its fluorescence counterpart. However, breaking the diffraction-limited spatial resolution of SRS imaging is much more challenging because of the intrinsically weak scattering cross section and inert/stable nature of molecular bond vibrations. We report superresolution SRS (SR-SRS) nanoscopy based on reversible-switchable vibrational photochromic probes integrated with point spread function engineering strategy. By introducing a Gaussian-shaped ultraviolet excitation beam and a donut-shaped visible depletion beam in addition to the pump and Stokes beams, SR-SRS could reach sub-100 nm resolution on photoswitchable nanoparticles (NPs). Furthermore, NP-treated live cell imaging was demonstrated with resolution improvement by a factor of ∼4. Our proof-of-principle work provides the potential for SR vibrational imaging to assist research on complex biological systems.
Coherent anti-Stokes Raman scattering (CARS) can be used to excite vibrational bonds with chemical selectivity, high spatial and spectral resolution, and high sensitivity, which has many applications in biomedical research. The common way to realize CARS imaging is illuminating sample with two synchronized ultra-short pulses. Recent development of various fiber laser schemes based on nonlinear optical effect provides compact laser source for CARS imaging. However, the nonlinear conversion in optical fiber may inevitably introduce temporal or spectral noise to the newly generated pulses. In this paper, we have proposed a polarization-maintaining (PM) passive-synchronized picosecond fiber laser system that generates dual-color picosecond pulses for CARS. An Er-doped fiber laser and a Yb-doped fiber laser were passively synchronized by cross phase modulation based on master-slave injection scheme. In the experiment, the wavelength of one branch was fixed at 791 nm, which was generated by second harmonic generation of Er-doped fiber laser. The wavelength of the other branch can be continuously tuned from 1017-1047 nm, which was obtained by adding an active spectral broadening module and an optical bandpass filter after Yb-doped fiber laser. As a result, the laser source allows to probe vibrational bonds with frequencies difference between 2809 cm-1 and 3091 cm-1 . Finally, the achieved tunable synchronized pulses enabled us to microscopically image mouse ear samples. The compact optical fiber laser proposed with PM fiber design, stable synchronization and large wavelength tunability would become a promising laser source for CARS imaging in clinical use.
Rapid diagnosis on endoscopic biopsies is crucial for decision makings during gastrointestinal endoscopy. We applied stimulated Raman scattering (SRS) microscopy on fresh biopsy specimens without fixation, sectioning or staining. We further combined SRS with deep convolutional neural network for automated diagnosis of early gastric cancer. Our preliminary results indicated that SRS histology integrated with deep learning algorithm provides potential for delivering rapid diagnosis that could aid the surgical management of gastric cancer.
Stimulated Raman scattering (SRS) microscopy that is capable of both high speed imaging and rapid spectroscopy will be advantageous for detailed chemical analysis of heterogeneous biological specimens. We have developed a system based on spectral focusing SRS technology, with the integration of a rapid scanning optical delay line (RSODL), which allows continuous tuning of SRS spectra by scanning a galvo mirror. We demonstrated SRS spectral measurements of dimethyl sulfoxide solution at low concentrations, and multi-color imaging of rice pollens and HeLa cells with line-by-line delay tuning to reduce motion artifacts, as well as fast acquisition of SRS spectra at specific regions of interest.
Two-color Stimulated Raman scattering (SRS) microscopy has shown great potential in label-free digital histology with diagnostic results similar to H&E stain. However, achieving real-time two-color SRS imaging is challenging. We have precisely engineered the pulse profiles of the Stokes beams, and fully utilized the in-phase (X) and quadrature (Y) outputs of a phase sensitive lock-in amplifier to realize simultaneous two-color SRS imaging. We have demonstrated its robustness and advantages in rapid histology, as well as real-time in vivo imaging of live animals, both in transmission and epi modes. Moreover, we have also adapted this method to other pump-probe based microscopes, such as transient absorption (TA) microscopy.
A compact, alignment-free, and inexpensive fiber source for coherent Raman spectroscopy would benefit the field considerably. We present a fiber optical parametric oscillator offering the best performance from a fiber-source to date. Pumping the oscillator with amplified pulses from a 1 μm fiber laser, we achieve widely spaced, narrowband pulses suitable for coherent anti-Stokes Raman scattering microscopy. The nearly transform limited, 2 ps signal pulses are generated through the use of normal dispersion four wave mixing in photonic crystal fiber, and can be tuned from 779-808 nm, limited by the tuning range of the seed laser. The average signal power can reach 180 mW (pulse energies up to 4 nJ). The long-wavelength idler field is resonant in the oscillator, and the use of a narrow bandpass filter in the feedback loop is critical for stable operation, as seen in both simulation and experiment. Due to the self-consistent nature of the oscillator, this source provides lower relative intensity noise on its output pulses than parametric amplifiers based on the same frequency conversion process. We present high quality images of mouse tissues taken with this source that exhibit an outstanding signal to noise ratio at top imaging speeds.
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