Fiber lasers have transformed microscopy with their robustness and portability. Fiber laser-based stimulated Raman scattering (SRS) offers label-free chemical imaging but faces challenges like noise and high light collection requirements. To overcome these, we introduce stimulated Raman photothermal (SRP) microscopy with fiber lasers. SRP detects thermal effects induced by SRS, reducing sensitivity to laser noise and eliminating balance detection needs. Our setup allows light collection through a low NA air condenser, simplifying operation for non-experts. SRP demonstrates superior sensitivity, particularly in media with high thermos-optic coefficients, outperforming fiber laser-based SRS. We showcase SRP's efficacy in imaging biological samples, promising enhanced sensitivity, resolution, and user-friendliness. This innovation signifies a significant step forward in biological microscopy, offering potential for widespread adoption and groundbreaking discoveries in vibrational imaging.
Deep-tissue chemical imaging is essential for many biomedical applications. Here, we present a shortwave infrared photothermal (SWIP) microscope for millimeter-deep vibrational imaging with micron lateral resolution. By pumping the overtone transition of carbon-hydrogen bonds and probing the subsequent photothermal lens with shortwave infrared light, SWIP can obtain chemical contrast from one-micron polymer particles located at 800-μm depth in a highly scattering phantom. We demonstrated that SWIP can resolve intracellular lipids across an intact tumor spheroid and the layered structure in thick liver, skin, brain, and breast tissues. SWIP microscopy fills a gap in vibrational imaging with sub-cellular resolution and millimeter-level penetration, which heralds broad potential for life science and clinical applications.
Deep-tissue chemical imaging is essential for many biomedical applications. Here, we present a shortwave infrared photothermal (SWIP) microscope for millimeter-deep vibrational imaging with micron-resolution and nanoparticle detection sensitivity. By using short-wave infrared light for overtone excitation of CH bonds and photothermal probing, our modality can reach millimeter deep into scattering phantom and tissue. With a fast digitization method, we showed that in our setting the photothermal signal amplitude is 100 times larger than photoacoustic thus enabling higher sensitivity. SWIP can resolve intracellular lipids in tumor spheroid and layered structure in the liver, skin, and brain tissue.
Stimulated Raman photothermal (SRP) is a newly developed chemical imaging scheme that probes the SRS induced thermal effect with a third laser beam. SRP shows high sensitivity, where a >500-fold boost of modulation depth comparing to SRS has been achieved. To further improve the sensitivity of SRP, we introduce spSRP with a ~1 MHz repetition rate, high peak power OPA laser serving as the SRP excitation source. The high peak power increases the energy deposition of SRS process, enables SRP measurement with a single pair of pump and Stokes laser pulses. spSRP is a firm step towards vibrational spectroscopic imaging with ultrahigh sensitivity.
The detection sensitivity of SRS microscopy is fundamentally limited by shot noise. To address this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrational excited states. The thereafter relaxation heats up the surrounding and induces a change in refractive index, as suggested by simulation and fluorescence thermometer measurement. By introducing a CW laser beam to probe the refractive index change, we demonstrate stimulated Raman photothermal (SRPT) imaging of particles, cells, and tissues with high signal to noise ratio at the fingerprint region. SRPT microscopy opens a new way to vibrational spectroscopic imaging with ultrahigh sensitivity.
Spectroscopic stimulated Raman scattering (SRS) imaging has become a useful tool finding a broad range of applications. Due to the limited spectral coverage of current SRS systems, most SRS applications utilized a narrow Raman band (<300 cm-1). Multi-window SRS imaging covering C-H, C-D and fingerprint regions offers richer chemical information. In this work, we present a multi-window SRS imaging system with a rapid widely tunable fiber laser. We implemented auto-balanced detection to enhance the signal-to-noise ratio of stimulated Raman loss by 23 times. We demonstrated SRS metabolic imaging of fungi, cancer cells, and Caenorhabditis elegans across the C-H, silent and fingerprint Raman windows. Our results showcase the potential of the multi-window SRS system for a broad range of applications.
The World Health Organization (WHO) published a catalogue of 12 families of antibiotic-resistant bacteria which pose an alarming threat to human health in 2017. These bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), could cause a wide range of infections from minor subcutaneous infection to toxic shock syndrome, and bacteremia. As the body’s second line of host defense, phagocytosis could eliminate the majority of the invasive bacteria. However, the survival of microbial pathogens within the macrophage cells which act as ‘Trojan horses’ largely provides a reservoir relatively related protected from antibiotics, thus causing recurrent infections from the dissemination of intracellular bacteria. Moreover, the pace of antibiotic development can’t keep with the resistance acquisition from bacteria. Therefore, there is an unmet need for alternative approaches to eradicate multi-drug resistant intracellular bacteria. Here, we develop an effective photonic approach to assist macrophage cell (RAW 264.7) to efficiently eradicate intracellular MRSA, P. aeruginosa along with Salmonella enterica. This approach selectively targets intracellular bacteria without damaging macrophage cells through photoinactivation of a microbial detoxifying enzyme existing in most of the bacteria. Moreover, we utilize advanced nonlinear optical imaging methods to record the in situ photoinactivation process and to visualize the real-time phagocytosis difference with or without photoinactivation of this enzyme. Our findings and approach reported here could provide an effective method to eliminate multi-drug resistant intracellular bacteria, and also treat the clinical bacterial infection in the future.
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