Identification of distinctive features in human intracranial tumors by label‐free nonlinear multimodal microscopy

Nonlinear multimodal microscopy offers a series of label‐free techniques with potential for intraoperative identification of tumor borders in situ using novel endoscopic devices. Here, we combined coherent anti‐Stokes Raman scattering, two‐photon excited fluorescence (TPEF) and second harmonic generation imaging to analyze biopsies of different human brain tumors, with the aim to understand whether the morphological information carried by single field of view images, similar to what delivered by present endoscopic systems, is sufficient for tumor recognition. We imaged 40 human biopsies of high and low grade glioma, meningioma, as well as brain metastases of melanoma, breast, lung and renal carcinoma, in comparison with normal brain parenchyma. Furthermore, five biopsies of schwannoma were analyzed and compared with nonpathological nerve tissue. Besides the high cellularity, the typical features of tumor, which were identified and quantified, are intracellular and extracellular lipid droplets, aberrant vessels, extracellular matrix collagen and diffuse TPEF. Each tumor type displayed a particular morphochemistry characterized by specific patterns of the above‐mentioned features. Nonlinear multimodal microscopy performed on fresh unprocessed biopsies confirmed that the technique has the ability to visualize tumor structures and discern normal from neoplastic tissue likewise in conditions close to in situ.      

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near‐infrared spectrum can improve the present situation. Here, the capability of saturated two‐photon saturated excitation (TP‐SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering‐enlarged point spread function (PSF), we find that TP‐SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single‐photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering‐enlarged PSF, TP‐SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.