As a markerless imaging technique, multimodal nonlinear optical imaging (NLOI) has become a powerful tool for cancer evaluation. To avoid motion artifacts and optical damage associated with multimodal NLOI, one solution is to use a single ultrafast laser as an excitation source combined with multiple detection channels to collect signals from different modalities to observe different biomolecules. However, in this case, each mode cannot be optimized independently and a suitable excitation source is needed to excite all NLOI modes. Label-free spontaneous fluorescence multiplexed (SLAM) microscopy, with the excitation wavelength set at 1110 nm, enables the simultaneous collection of signals from four modes under a single excitation condition through different signal detection channels, acquiring two-photon fluorescence (2PAF) for FAD, three-photon fluorescence (3PAF) for NADH, two-octave frequency (SHG) for collagenous structures, and three-octave frequency (THG) for the refractive index mutation. frequency (THG) signals at refractive index mutations. Currently, most of the light sources used to drive SLAM microscopes need to couple ultrashort pulses into photonic crystal fibers or crystals to achieve wavelength conversion, which involves high cost, large footprint, complicated operation, and the inability of long-time stable operation.
To address the above problems and difficulties, the L07 group of Institute of Physics, Chinese Academy of Sciences/Beijing National Research Center for Condensed Matter Physics (NRCP), based on many years of research on ultrafast fiber lasers, proposed a Yb-doped fiber laser with dual management of pre-chirp and gain, and finally obtained a pulse with a wavelength of 1110 nm, an energy of more than 90 nJ, a pulse width of 34 fs, and peak power of close to 3 MW, by finely adjusting the input energy and pre-chirp. With a wavelength of >90nJ, a pulse width of 34fs, and a peak power of nearly 3MW, the light source is compact and stable while achieving excellent pulse quality to drive SLAM microscopes for medical imaging.
Figure 1 shows the schematic diagram of the ytterbium-doped fiber laser system with pre-chirp and gain dual management. It consists of a seed source, pre-amplification module, pre-chirp module, gain-managed amplification (GMA) module and compression module. The seed source provides a seed pulse with a center wavelength of 1040 nm, a pulse energy of 0.2 nJ, and a repetition frequency of 43 MHz. The seed pulse is pre-amplified by a 40 cm long Yb-doped fiber, and a pair of gratings is placed in front of the GMA module to introduce dispersion, and a negative or positive pre-chirp is added to the pre-amplified output pulse by adjusting the grating spacing. Further pre-chirped pulses are amplified in a 3.1 m long Yb-doped fiber for gain management. The second amplified pulse is compressed through another pair of transmission gratings. The effect of these parameters on the pulse compression quality is explored by finely tuning the input energy and pre-chirp, and the experimental results are shown in Figs. 2 and 3, which show that high compression quality pulses can be produced at a range of pump power, input energy, and appropriate negative chirp. When the pump power is 9 W, the input pulse energy is 0.6 nJ, and the pre-chirp is -36000 fs2, a pulse with a center wavelength of 1110 nm, a pulse width of 34 fs, an energy of 92.2 nJ, and a peak power of close to 3 MW is obtained, which is very suitable for driving SLAM microscopes for medical imaging.
Image Fig. 2. Effect of different input pulse energies on GMA pulse compression at a pump power of 9 W and a pre-chirp of -36000 fs2. (a) Compression pulse width and Strehl ratio at different input energies. (b) Output spectra at different input energies. (c) Red curve: measured autocorrelation trajectory of the compressed pulse, black curve: autocorrelation trajectory of the transformed limit pulse obtained by spectral calculation
Fig. 3. Effect of different pre-chirps on GMA pulse compression for an input pulse energy of 0.6 nJ and a pump power of 9 W. The results are summarized as follows (a) Compressed pulse pulse width and Strehl ratio for different pre-chirps. (b) Output spectra at different pre-chirps. (c) Red curve: measured autocorrelation trajectory of the compressed pulse, black curve: autocorrelation trajectory of the transformed limit pulse obtained by spectral calculation.
The team applied this ultrafast light source to study tumor pathology in different tissues, including intestinal adenocarcinoma, lung adenocarcinoma, and liver tissues, to simultaneously image cellular and extracellular components by SLAM technique. A SLAM image of intestinal adenocarcinoma tissue is shown in Figure 4, where green indicates SHG, magenta indicates THG, yellow indicates 2PEF, and blue indicates 3PEF.SLAM imaging can provide much richer cellular and tissue details than conventional H&E-stained images, which can help to understand the changes of biocomponents in both tumors and normal tissues, and to search for biomarkers for cancer diagnosis and prognosis.
Image Figure 4. (a) SHG/THG/2PEF/3PEF imaging of intestinal adenocarcinoma tissue. Different regions of interest are magnified in (c) - (e) (white dashed squares). (b) Corresponding H&E staining images. (c) 2PEF/3PEF imaging of normal intestinal mucosal tissue. (d) SHG/THG imaging of normal intestinal mucosal tissue. (e) SHG imaging of interstitial fibers and fat vacuoles, red arrow: intestinal gland, blue arrow: basement membrane, green arrow: mucus secreted by cup cells, white arrow: macrophage, yellow arrow: interstitial fibers, purple arrow: fat vacuoles. Scale bar: 200 μm
Overall, the research team achieved high-quality ultrafast pulse generation through the development of a pre-chirped and gain dual-managed Yb-doped fiber laser, which was successfully applied to SLAM imaging, a technique that can provide richer cellular and tissue details that can aid in oncopathology studies and cancer diagnosis. In addition, the ultrafast light source is compact and robust, making it ideal for use in a clinical setting for rapid and comprehensive evaluation of various physiological and pathological processes. The innovative results of this study are expected to advance the field of medical diagnostics and therapeutics by providing more accurate and comprehensive information for cancer diagnosis, efficacy assessment and individualized treatment. As the technology continues to advance and be optimized, SLAM imaging is expected to play a more important role in clinical practice in the future. The device and the core device associated with this advancement have been applied for national invention patents.
The results were published in a recent issue of Biomedical Optics Express, a journal of the Optical Society of America (10.1364/BOE.506915), and the first author of the paper is Yuting Xing, a doctoral student supervised by researcher Guoqing Chang.
This work was supported by the National Natural Science Foundation of China (Grant Nos. 92250307, 62227822, and 62175255) and the Important Instrument Development Program of the Chinese Academy of Sciences (Grant No. YJKYYQ20190034). Researcher Guoqing Chang and Dr. Yaobing Chen from Wuhan Tongji Hospital were the corresponding authors, and PhD students Runshi Chen, Lihao Zhang, Yang Liu, Xinzai Diao and Researcher Shu Zhang from Wuhan Tongji Hospital, Prof. Yishi Shi and Researcher Zhiyi Wei from University of Chinese Academy of Sciences were also involved in the design and discussion of this work.
