Since two-photon microscopy (TPM) can obtain high-resolution images at cellular and subcellular level and moxifloxacin has multiphoton fluorescence characteristic, our study aimed to explore the feasibility and diagnostic value of moxifloxacin-assisted TPM in different human colorectal diseases, including low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN) and cancer tissues. Excitation power for TPM imaging with and without moxifloxacin was (2.74 ± 0.16) mW and (0.28 ± 0.02) mW, respectively (p < 0.05). Whether labeled with moxifloxacin or not, images of normal, LGIN, HGIN and cancer tissues all reached the strongest signal at 30 μm from the mucosa. Normalized fluorescence intensity of TPM images with moxifloxacin was approximately 10 times stronger than that without moxifloxacin. Fluorescence signal was differed significantly in normal, LGIN, HGIN and cancer tissues with or without moxifloxacin (p < 0.05). Besides, moxifloxacin-assisted TPM could present variant tissue features with different colorectal diseases, such as the crypt opening, glandular structure, adjacent glandular space and fluorescence distribution.

The progressive miniaturization of photonic components presents the opportunity to obtain unprecedented microscopic images of colonic polyps in real time during endoscopy. This information has the potential to act as "optical biopsy" to aid clinical decision-making, including the possibility of adopting new paradigms such as a "resect and discard" approach for low-risk lesions. The technologies discussed in this review include confocal laser endomicroscopy, optical coherence tomography, multiphoton microscopy, Raman spectroscopy, and hyperspectral imaging. These are in different stages of development and clinical readiness, but all show the potential to produce reliable in vivo discrimination of different tissue types. A structured literature search of the imaging techniques for colorectal polyps has been conducted. The significant developments in endoscopic imaging were identified for each modality, and the status of current development was discussed. Of the advanced imaging techniques discussed, confocal laser endomicroscopy is in clinical use and, under optimal conditions with an experienced operator, can provide accurate histological assessment of tissue. The remaining techniques show potential for incorporation into endoscopic equipment and practice, although further component development is needed, followed by robust prospective validation of accuracy. Optical coherence tomography illustrates tissue "texture" well and gives good assessment of mucosal thickness and layers. Multiphoton microscopy produces high-resolution images at a subcellular resolution. Raman spectroscopy and hyperspectral imaging are less developed endoscopically but provide a tissue "fingerprint" which can distinguish between tissue types. Molecular imaging may become a powerful adjunct to other techniques, with its ability to precisely label specific molecules within tissue and thereby enhance imaging.

Multiphoton microscopy (MPM) facilitates three-dimensional, high-resolution functional imaging of unlabeled tissues in vivo and ex vivo. This systematic review discusses the diagnostic value, advantages and challenges in the practical use of MPM in surgical oncology.

A Medline search was conducted in April 2019. Fifty-three original research papers investigating MPM compared to standard histology in human patients with solid tumors were identified. A qualitative synopsis and meta-analysis of 14 blinded studies was performed. Risk of bias and applicability were evaluated. MPM can image fresh, frozen or fixed tissues up to a depth 1000 μm in the z-plane. Best results including functional imaging and virtual histochemistry are obtained by in vivo imaging or scanning fresh tissue immediately after excision. Two-photon excited fluorescence by natural fluorophores of the cytoplasm and second harmonic generation signals by fluorophores of the extracellular matrix can be scanned simultaneously, providing high resolution optical histochemistry comparable to standard histology. Functional parameters like fluorescence lifetime imaging or optical redox ratio provide additional objective information. A major concern is inability to visualize the nucleus. However, in a subpopulation analysis of 440 specimens, MPM yielded a sensitivity of 94%, specificity of 96% and accuracy of 95% for the detection of malignant tissue.

MPM is a promising emerging technique in surgical oncology. Ex vivo imaging has high sensitivity, specificity and accuracy for the detection of tumor cells. For broad clinical application in vivo, technical challenges need to be resolved.

Endoscopic imaging is a rapidly progressing field and benefits from miniaturization of advanced imaging technologies, which may allow accurate real-time characterization of lesions. The concept of the "optical biopsy" to predict polyp histology has gained prominence in recent years and may become clinically applicable with the advent of new imaging technology. This review aims to discuss current evidence and examine the emerging technologies as applied to the optical diagnosis of colorectal polyps.

A structured literature search and review has been carried out of the evidence for diagnostic accuracy of image-enhanced endoscopy and emerging endoscopic imaging technologies. The image-enhanced endoscopy techniques are reviewed, including their basic scientific principles and current evidence for effectiveness. These include the established image-enhancement technologies such as narrow-band imaging, i-scan, and Fuji intelligent chromoendoscopy. More recent technologies including optical enhancement, blue laser imaging, and linked color imaging are discussed. Adjunctive imaging techniques in current clinical use are discussed, such as autofluorescence imaging and endocytoscopy. The emerging advanced imaging techniques are reviewed, including confocal laser endomicroscopy, optical coherence tomography, and Raman spectroscopy.

Large studies of the established image-enhancement techniques show some role for the optical diagnosis of polyp histology, although results have been mixed, and at present only the technique of narrow-band imaging is appropriate for the diagnosis of low-risk polyps when used by an expert operator. Other image-enhancement techniques will require further study to validate their accuracy but show potential to support the use of a "resect-and-discard" approach to low-risk polyps. New technologies show exciting potential for real-time diagnosis, but further clinical studies in humans have yet to be performed.

Previous studies using nonlinear microscopy have demonstrated that osteoarthritis (OA) is characterized by the gradual replacement of Type II collagen with Type I collagen. The objective of this study was to develop a prototype nonlinear laser scanning microendoscope capable of resolving the structural differences of collagen in various orthopaedically relevant cartilaginous surfaces. The current prototype developed a miniaturized femtosecond laser scanning instrument, mounted on an articulated positioning system, capable of both conventional arthroscopy and second-harmonic laser-scanning microscopy. Its optical system includes a multi-resolution optical system using a gradient index objective lens and a customized multi-purpose fiber optic sheath to maximize the collection of backscattered photons or provide joint capsule illumination. The stability and suitability of the prototype arthroscope to approach and image cartilage were evaluated through preliminary testing on fresh, minimally processed, and partially intact porcine knee joints. Image quality was sufficient to distinguish between hyaline cartilage and fibrocartilage through unique Type I and Type II collagen-specific characteristics. Imaging the meniscus revealed that the system was able to visualize differences in the collagen arrangement between the superficial and lamellar layers. Such detailed in vivo imaging of the cartilage surfaces could obviate the need to perform biopsies for ex vivo histological analysis in the future, and provide an alternative to conventional external imaging to characterize and diagnose progressive and degenerative cartilage diseases such as OA. Moreover, this system is readily customizable and may provide a suitable and modular platform for developing additional tools utilizing femtosecond lasers for tissue cutting within the familiar confines of two or three portal arthroscopy techniques.

High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

Preoperative radiochemotherapy improves outcomes in patients with locally advanced rectal carcinoma, and has been used increasingly in patient management. However, there is a strong clinical need to assess tumor response to neoadjuvant treatment, and a non-invasive technique that allows the precise identification of morphologic changes in tumors would be of considerable clinical interest.

In this study, we used multiphoton microscopy (MPM) to detect morphologic alterations in rectal adenocarcinomas in patients treated with preoperative radiochemotherapy.

MPM was able to identify histopathologic alterations in rectal cancer following preoperative radiochemotherapy, and allowed the qualitative assessment of treatment efficacy and feasibility in relation to dose or strategy.

These findings may provide the groundwork for evaluating tumor response to neoadjuvant treatment, thus allowing the tailoring of effective treatment doses and strategies.

Nonlinear optical imaging (NLOI) has emerged to be a promising tool for bio-medical imaging in recent times. Among the various applications of NLOI, its utility is the most significant in the field of pre-clinical and clinical cancer research. This review begins by briefly covering the core principles involved in NLOI, such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). Subsequently, there is a short description on the various cellular components that contribute to endogenous optical fluorescence. Later on the review deals with its main theme--the challenges faced during label-free NLO imaging in translational cancer research. While this review addresses the accomplishment of various label-free NLOI based studies in cancer diagnostics, it also touches upon the limitations of the mentioned studies. In addition, areas in cancer research that need to be further investigated by label-free NLOI are discussed in a latter segment. The review eventually concludes on the note that label-free NLOI has and will continue to contribute richly in translational cancer research, to eventually provide a very reliable, yet minimally invasive cancer diagnostic tool for the patient.

Stroma plays an important role during epithelial tumor progression. Probing stroma alteration may become an intrinsic indicator for evaluating epithelial tumor progression. In this review, we summarize our recent works on stromal alterations as quantitative optical biomarkers of epithelial tumor progression by use of nonlinear optical microscopy.

Multiphoton microscopy (MPM), based on advances in the field of nonlinear optics and femtosecond lasers, has been shown to provide detailed real-time information on tissue architecture and cell morphology in live tissue. The purpose of this study was to evaluate the feasibility of using MPM to make real-time optical diagnoses for surgical margins in low rectal cancers.

Thirty fresh, unfixed, and unstained full-thickness surgical margins of low rectal cancers underwent MPM examination and then went through intraoperative frozen procedures and routine pathological procedures. MPM images were compared with the gold standard hematoxylin-eosin (H-E) stained images.

MPM images were acquired by two channels: broadband autofluorescence from cells and second harmonic generation (SHG) from tissue collagen. Peak multiphoton signal intensity was detected in mucosa excited at 800 nm. There were significant differences between negative surgical margins and positive surgical margins under MPM examination. In negative surgical margins, MPM revealed regular tissue architecture and cell morphology, including a typical foveolar pattern with central, round crypt openings, and glands lined by epithelial and goblet cells. SHG signals could be detected around the glands. In positive surgical margins, MPM demonstrated irregular tubular structures, reduced stroma, and cellular and nuclear pleomorphisms. Cancer cells were characterized by an irregular size and shape, enlarged nuclei, and an increased nuclear-cytoplasmic ratio. SHG signals were significantly decreased in positive surgical margins compared with negative surgical margins. MPM images were comparable to H-E stained images.

We demonstrated the feasibility of using MPM to make real-time optical diagnoses for surgical margins in low rectal cancer. With the miniaturization and integration of colonoscopy or probes, MPM has the potential to provide real-time noninvasive optical diagnosis for surgical margins in low rectal cancer in the near future.

Colorectal carcinoma (CRC) has high mortality and increased incidence rates. An early detection of CRC is very important. Multiphoton microscopy (MPM) with high resolution and high sensitivity is used to effectively distinguish the microstructure changes of normal and mucinous adenocarcinoma slices of ex vivo human colonic tissues. In mucinous adenocarcinoma mucosa, the glands are distorted and elongated, the gland cavity is indistinct, and the mesh collagen fibers are diminished. In the submucosa, the collagens are seriously disordered, elongated, pushed aside, and sparsely visible, the content of elastic fibers is also broken and almost disappearing. Many cancer cells, some in cavity-like shape full of mucus surrounded by some collagen fibers, occupied the submucosa, which are comparable to hematoxylin-eosin (HE) stained images. Second harmonic generation and two-photon excitation fluorescence (SHG/TPEF) intensity ratio can be used further to quantitatively evaluate normality and abnormality. The fast Fourier transform (FFT) images show that the normal collagen fibrils are dense and in random order, and the cancerous collagen is certainly organized. The exploratory results show that it has potential for the development of multiphoton mini-endoscopy in real-time early diagnosis of CRC.

Endoscopy is widely used to detect and remove premalignant lesions with the goal of preventing gastrointestinal (GI) cancers. Because current endoscopes do not provide cellular resolution, all suspicious lesions are biopsied and subjected to histologic evaluation. Technologies that facilitate directed biopsies should decrease both procedure-related morbidity and cost. Here we explore the use of multiphoton microscopy (MPM), an optical biopsy tool that relies on intrinsic tissue emissions, to evaluate pathology in both experimental and human GI specimens, using hematoxylin and eosin (H&E)-stained sections from these tissues for comparison. After evaluating the entire normal mouse GI tract, MPM was used to investigate disease progression in mouse models of colitis and colorectal carcinogenesis. MPM provided sufficient histologic detail to identify all relevant substructures in ex vivo normal GI tissue, visualize both acute and resolving stages of colitis, and show the progression of colorectal carcinogenesis. Next, ex vivo specimens from human subjects with celiac sprue, inflammatory bowel disease, and colorectal neoplasia were imaged by MPM. Finally, colonic mucosa in live anesthetized rats was imaged in vivo using a flexible endoscope prototype. In both animal models and human specimens, MPM images showed a striking similarity to the results of H&E staining, as shown by the 100% concordance achieved by the study pathologists' diagnoses. In summary, MPM is a promising technique that accurately visualizes histology in fresh, unstained tissues. Our findings support the continued development of MPM as a technology to enhance the early detection of GI pathologies including premalignant lesions.