Spatial Frequency Modulated Imaging (SPIFI) with amplitude or phase grating from a spatial light modulator

Distinguishing tumors from normal brain cells is important but challenging in glioma surgery due to the lack of clear interfaces between the two. The ability of label‐free third harmonic generation (THG) microscopy in combination with automated image analysis to quantitatively detect glioma infiltration in fresh, unprocessed tissue in real time is assessed. The THG images reveal increased cellularity in grades II–IV glioma samples from 23 patients, as confirmed by subsequent hematoxylin and eosin histology. An automated image quantification workflow is presented for quantitative assessment of the imaged cellularity as a reflection of the degree of glioma invasion. The cellularity is validated in three ways: 1) Quantitative comparison of THG imaging with fluorescence microscopy of nucleus‐stained samples demonstrates that THG reflects the true tissue cellularity. 2) Thresholding of THG cellularity differentiates normal brain from glioma infiltration, with 96.6% sensitivity and 95.5% specificity, in nearly perfect (93%) agreement with pathologists. 3) In one patient, a good correlation between THG cellularity and preoperative magnetic resonance and positron emission tomography imaging is demonstrated. In conclusion, quantitative real‐time THG microscopy accurately assesses glioma infiltration in ex vivo human brain samples, and therefore holds strong potential for improving the accuracy of surgical resection.

Multidimensional, multicontrast, spatial frequency modulation imaging with built-in femtosecond pulse characterization and dispersion compensation is presented. We present wavelength domain imaging which produces one-dimensional imaging with single element detection, and a multiplexed spatial and wavelength domain system that produces two-dimensional images and is compatible with single element detection imaging.

Multimodal multiphoton microscopy (MPM) can provide fast, label-free, non-invasive examination of cells, extracellular matrix, and lipids. Two-photon microscopy (2PM) can detect second harmonic generation (SHG) from fibrillar collagen and striated muscle myosin, whereas two-photon excitation fluorescence (2PEF) can detect intrinsic fluorophores such as NADH from cells. Meanwhile, three-photon microscopy (3PM) can detect third harmonic generation (THG) from lipids and tissue interfaces. We have developed a miniaturized multimodal multiphoton system which can perform label-free two-photon and three-photon imaging. An Er-doped fiber laser delivers fundamental pulses at 1580 nm and 80 fs for exciting THG. SHG and 2PEF are excited at 790 nm via the frequency doubling of 1580 nm pulses. For clinical applications, a compact probe is being developed with single-mode fiber for delivering the femtosecond excitation pulses and multi-mode fiber for collecting the MPM signals. A MEMS mirror performs lateral scanning at up to 4 frames/s. For objective lenses, a miniature aspherical lens (NA=0.64) is compared with a gradient index microobjective (NA=0.8). Shape memory alloy actuator used in smartphone cameras is evaluated for shifting the focal plane to acquire Z-stacks for 3D tissue imaging. High-resolution SHG, 2PEF, and THG images are acquired from biological tissues and show that multimodal MPM endomicroscopy has great potential for clinical applications as an alternative to histology.

Collagen is critical to the structure and function of skin tissues, with the collagen I/III ratios influencing fibrillogenesis, fiber organization, and skin mechanics. Abnormal collagen organization, such as in fibrosis or scar tissue, compromises both skin functionality and aesthetics. In this study, we employed label-free polarization resolved second harmonic generation (PSHG) microscopy to investigate collagen structure in artificial collagen matrices with various Col I/III ratios at the fibril scale (∼1 to 3μm) and in ex vivo human healthy and scarred skin at the fiber scale (∼10 to 20μm). Complementary third harmonic generation (THG) microscopy provided additional structural information. Our results indicate that an increasing Col I/III ratio is associated with longer fibril length, higher PSHG intensity, and a reduced effective α-helix pitch angle of fibrils. In pure Col I, the effective α-helix pitch angle is determined to be 47.72∘. These observations indicate alterations in fibril assembly. Furthermore, although the α-helix pitch angle of fibers in both healthy and scarred skin was approximately 46.7∘, healthy skin exhibited 24% greater variability in fiber orientation, suggesting a more randomized organization compared to scar tissue. THG imaging further revealed a higher cellular density in scar tissue, consistent with the inflammatory activity associated with wound healing. Immunohistochemical (IHC) staining using dermatansulphate and Col III-specific antibodies confirmed that the Col I/III ratio is higher in healthy skin (2.2) than in scarred skin (1.6). These findings underscore the potential of PSHG microscopy for label-free, quantitative assessment of collagen structure across multiple scales, with THG offering complementary cellular insights. This integrated approach represents a promising strategy for real-time, in vivo monitoring and automated quantification of collagen organization in clinical applications, including dermatology, burn treatment, and fibrosis monitoring.

The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy (MPM). Shifting to longer excitation wavelengths may help reduce skin scattering and aberration, potentially enabling larger imaging depths. However, previous demonstrations of skin MPM employ excitation wavelengths only up to the 1700[Formula: see text]nm window, leaving an open question as to whether longer excitation wavelengths are suitable for deep-skin MPM. Here, in order to explore the longer-wavelength territory, first, we demonstrate characterization of the broadband transmittance of excised mouse skin, revealing a high transmittance window at 2200[Formula: see text]nm. Then, we demonstrate third-harmonic generation (THG) imaging in mouse skin in vivo excited at this window. With 9[Formula: see text]mW optical power on the skin surface operating at 1[Formula: see text]MHz repetition rate, we can get THG signals of 250[Formula: see text][Formula: see text]m below the skin surface. Comparative THG imaging excited at the 1700[Formula: see text]nm window shows that as imaging depth increases, THG signals decay even faster than those excited at 2200[Formula: see text]nm. Our results thus uncover the 2200[Formula: see text]nm window as a new, promising excitation window potential for deep-skin MPM.

The lacuno-canalicular network (LCN) hosting the osteocytes in bone tissue represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. Using third-harmonic generation (THG) microscopy with sub-micrometer resolution, we investigate the impact of microgravity on the 3D LCN structure in mice following space flight. A specific analytical procedure to extract the LCN characteristics from THG images is described for ex vivo studies of bone sections. The analysis conducted in different anatomical quadrants of femoral cortical bone didn’t reveal any statistical differences between the control, habitat control and flight groups, suggesting that the LCN connectivity is not affected by one month space flight. However, significant variations are systematically observed within each sample. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.

In this paper we will describe the most recent advances in the development of Ti:Al2O3, unstable-resonator lasers. Results include demonstration of improved long-wavelength operation and efficient second, third and fourth harmonic generation. Second harmonic generation (SHG) results will be described for three nonlinear crystals: deuterated potassium di-hydrogen phosphate (D-KDP), beta barium borate (BBO) and lithium triborate (LBO). Third harmonic generation (THG) in BBO and D-KDP will be presented along with fourth harmonic generation (FHG) data obtained with BBO crystals.

The third harmonic generation (THG) and nonlinear transmission (NT) were used to probe the change of /spl chi//sup (3)/ in azo-doped polymethylmethacrylate (PMMA) thin films. The observed phenomena can be interpreted by a microscopic model based on angular hole burning (AHB) and angular redistribution (AR) mechanisms of photoisomerization effect. In this experiment, the pumping effect on the THG and NT at different polarization directions is investigated. The variations of THG here are mainly due to the change of third order susceptibility /spl chi//sup (3)/ (-/spl omega/; /spl omega/, /spl omega/, -/spl omega/). As we switch on the pumping beam, the THG in the co-polarized configuration experiences a fast decay associated with the AHB in few seconds and, thereafter, it reaches the stationary state after a slow decay corresponding to the AR lasting for several minutes. When we turn off the pumping beam, THG will recover its original level by thermal relaxation. However, there is a small G increase in the cross-polarized configuration. Since the nonlinear absorption is related to the image part of /spl chi//sup (3)/ (-/spl omega/; /spl omega/, /spl omega/, -/spl omega/), the decrease in trans molecule number density corresponds to the decrease in Im /spl chi//sup (3)/ (-/spl omega/; /spl omega/, /spl omega/, -/spl omega/). When nonlinear absorption decreases, the NT increases accordingly. The anisotropic molecular distribution, which appears during AHB and AR, will also result in the nonlinear dichroism in the sample. We present time evolutions of the NT in the sample measured in the vicinity of focus point of the probing beam for both co-polarized and cross-polarized configurations in NT measurements. A considerable increase of NT in the co-polarized configuration can be observed when we switch on the pumping beam. On the contrary, as we turn off the pumping beam, the NT decreases to its original level. However the NT remains nearly unchanged in the cross-polarized configuration. The considerable change of NT in the co-polarized configuration fits in with our expectation since, in the existence of pumping beam, the trans population density in the direction parallel to the pumping beam will decrease in both AHB and AR mechanisms. On the other hand, the thin film will restore its isotropic distribution by thermal relaxation after pump removal, so the NT returns its original level. Nevertheless, the nearly unchanged NT in cross-polarized configuration is beyond our expectation. It may be due to that the change is too small to be observed in the NT measurements. Furthermore, the expected fast growth associated with AHB disappears in the growing curve. This may be due to that we used another 1 kHz diode-pumped Nd:YAG laser as pumping source. The heat accumulated on thin film is inevitable, so thermal cis-trans relaxation speeds up and the raising population of trans molecules can reorient to the direction parallel to the pump polarization by fast angular diffusion. Accordingly, nonlinear absorption (NT) will not decrease (increase) as fast as expected in AHB.

Influence of temperature and hydrostatic pressure on the nonlinear optical properties in a GaAs/GaAlAs Kratzer-Fues confined quantum well

Spatial frequency modulated imaging (SPIFI) is a powerful imaging method that when used in conjunction with multiphoton contrast mechanisms has the potential to improve the spatial and temporal scales that can be explored within a single nonlinear optical microscope platform. Here we demonstrate, for the first time to our knowledge, that it is possible to fabricate inexpensive masks using femtosecond laser micromachining that can be readily deployed within the multiphoton microscope architecture to transform the system from a traditional point-scanning system to SPIFI and gain the inherent advantages that follow.

Strategic improvement of second and third harmonic generation in multifunctional Cu-Sn-S3 ternary semiconducting thin films

Third harmonic generation (THG) microscopy was used as a noninvasive technique for in vivo investigations of the osteocytic lacunar-canalicular network (LCN) in the mouse calvarium. The origin of THG contrast was investigated by simultaneous imaging with multiphoton excitation fluorescence. The THG signal in lacuna originates from the bone-interstitial fluid boundary and the interstitial fluid-osteocyte cell boundary. Canaliculi also emit intense THG allowing 3D investigations of the LCN network. Subsequently, THG microscopy was used to study the structural variations in the LCN in live wild type and transgenic mouse models. 2D and 3D analyses were performed to obtain the lacunar area, volume and surface area as well as the number of canaliculi per lacuna area and volume, differentiating the transgenic mice. THG microscopy of bone in live animals is a promising technique for noninvasive quantitative analysis of the LCN network.

Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in vivo, at the cellular or organismal level is challenging. We developed a label-free method for visualizing lipid depositions in vivo, based on third harmonic generation (THG) microscopy. THG imaging requires a single pulsed-laser light source, alleviating the technical challenges of implementing coherent anti-Stokes Raman scattering spectroscopy (CARS) to detect fat stores in living cells. We demonstrate that THG can be used to efficiently and reliably visualize lipid droplets in Caenorhabditis elegans. Thus, THG microscopy offers a versatile alternative to fluorescence and dye-based approaches for lipid biology research.

We experimentally achieved optical third harmonic generation (THG) from two-dimensional periodically arrayed gold-island films. The gold-island films were fabricated using micro-spherical lithography. By using a reflection-type optical system, these arrays can produce significant THG with the help of a spectrum-resolved femto-second laser. At the localized surface plasmon (LSP) excitation wavelength of the fundamental wave, THG is greatly enhanced due to the strong light localization effect. The radiation direction of THG satisfies the momentum matching condition.

We studied third harmonic generation (THG) of melanin solution with concentrations similar to melanocytes in human skin. In contrast to conventional observation of THG at interface, bulk THG was detected inside the solution due to the formation of melanin hydrocolloids. A linear relationship between melanin concentration and THG intensity was found, suggesting THG originated from high-order hyper-Rayleigh scattering. By fitting this linear relationship, third-order hyperpolarizability of melanin hydrocolloids was determined to be three orders larger than that of water. Our result will be useful for interpretation of THG signals in skin and other tissues containing colloidal particles.