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Necrosis is a form of cell death which is histologically characterized by homogeneous clusters and sheets of dead cells. Although several studies have indicated that the presence of tumor necrosis in pathological specimens may provide adverse prognostic information in solid tumor, the mechanism of necrosis is still unclear. Based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG), multiphoton microscopy (MPM) is commonly used to monitor the morphological changes of biological tissues. In this study, we performed MPM imaging of the breast tissue and found that MPM can be used to rapidly classify the early and late-stage tumor necrosis in invasive breast cancer according to the changes of intracellular proteins. It demonstrated that MPM may provide a new assistant tool for pathologists to quickly and effectively classify the early and late-stage tumor necrosis.

Prolonged exposure of tissues to elevated blood sugar levels lead to the formation of advanced glycation end products (AGEs), thus contributing to diabetic complications. Since the vascular system is in immediate contact with blood, diabetic effects on aorta is expected. In this study, we incubated excised porcine aorta in D-glucose, D-galactose, and D-fructose solutions for different periods. The tissue specimens were imaged using multiphoton microscopy. We found that for Days 4 to 48 incubation, autofluorescence is constant along the radial direction of the aorta sections, suggesting that monosaccharide diffusion is rapid in comparison to the rate of formation of fluorescent AGEs. Moreover, we found that in porcine aorta, the rate of fluorescent AGE formation of D-fructose and D-glucose are factors 2.08 and 1.14 that of D-galactose.

Early diagnosis of Triple Negative Breast Cancer (TNBC) is essential to implementing early, life-saving treatment before the development of metastases. Traditional methods for detecting TNBC is difficult, being both tedious and vulnerable to false positive results. Here we combine optical imaging techniques—deuterium probed resonance Raman spectroscopy (RRS) and multiphoton fluorescence (MPF) to detect TNBC metabolism in early stage. These hallmarks such as glucose and lipid metabolism are revealed through chemical bond vibrational modes with RRS and morphological changes with MPF at subcellular scale.

Breast tumor microenvironment is composed of tumor cells, tumor-related cells, blood vessels and a series of extracellular matrix fibers. Tumor-infiltrating lymphocytes (Tils) in the microenvironment can directly or indirectly influence other components in the microenvironment, thus promoting the occurrence and development of tumors. Multiphoton microscopy (MPM) is based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). And it does not require the use of exogenous probes or staining of tissue. In this study, large-size images with subcellular resolution of the breast tumor tissue was performed using MPM. The results showed that the MPM could clearly distinguish intraepithelial Tils (iTils) and stromal compartments Tils (sTils) by comparing the signal strength and morphological difference. It demonstrated that MPM could be used as a means of pathological diagnosis and in clinical application.

We propose functionalized graphene oxides as an effective cancer cell-targeting agent for multiphoton imaging and near-infrared photothermal therapy. The dual-functional property of functionalized graphene oxides is investigated in human breast cancer cell lines (MDA-MB-231).

There is an increasing need in cosmetic research for non-invasive, high content, skin imaging techniques offering the possibility to avoid performing invasive biopsies and to supply a maximum of information on skin state throughout a study, especially before, during and after product application. Multiphoton microscopy is one of these techniques compatible with in vitro and in vivo investigations of human skin, allowing its three-dimensional (3D) structure to be characterized with sub-μm resolution. Various intra-/extra-cellular constituents present specific endogenous two-photon excited fluorescence and second harmonic generation signals enabling a non-invasive visualization of the 3D structure of epidermal and superficial dermal layers. In association with fluorescence lifetime imaging (FLIM) and specific 3D image processing, one can extract several quantitative parameters characterizing skin constituents in terms of morphology, density and function. Multiphoton FLIM applications in cosmetic research range from knowledge to efficacy evaluation studies. Knowledge studies aim at acquiring a better understanding of appearing skin differences, for example, with aging, solar exposure or between the different skin phototypes. Evaluation studies deal with efficacy assessment of cosmetic ingredients in anti-aging or whitening domains. When using other nonlinear optics phenomena such as CARS (Coherent Anti-Stokes Raman Scattering), multiphoton imaging opens up the possibility of characterizing the cosmetic ingredients distribution inside the skin and founds application in other cosmetic domains such as hydration or antiperspirants. Developments in user-friendly, ultrasensitive, compact, multimodal imaging systems, on-the-fly data analysis and the synthesis of cosmetic ingredients with non-linear optical properties will certainly allow trespassing the todays frontiers of cosmetic applications.

We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mouse. Our images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. At Day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.

Ovarian cancer is the deadliest gynecologic cancer due to predominantly late diagnosis. Early detection of ovarian cancer can increase 5-year survival rates from 40% up to 92%, yet no reliable early detection techniques exist. Multiphoton microscopy (MPM) is a relatively new imaging technique with tremendous potential for clinical diagnosis. A sub-modality of MPM is second harmonic generation (SHG) imaging, which generates contrast from anisotropic structures like collagen molecules, enabling the acquisition of detailed molecular structure maps. As collagen is known to change throughout the progression of cancer, MPM is a promising candidate for ovarian cancer screening. While MPM has shown favorable results in a research environment, it has not yet found broad success in a clinical setting. One major obstacle is the quantitative analysis of the image content. Recently, the application of texture analysis to MPM images has shown success for characterizing the collagen content of the tissue, making it a prime candidate for disease screening. Unfortunately, existing work is limited in its application to ovarian tissue and few texture analysis approaches have been evaluated in this context. To address these challenges, we applied texture analysis to second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) images of a mouse model (TgMISIIR-TAg) of ovarian cancer. Using features from the grey-level co-occurrence matrix, we find that texture analysis of TPEF images of the ovary can differentiate between genotype with high statistical significance (p

The clinical diagnosis of melanocytic lesions is an ongoing medical challenge. Non-invasive tools and technologies can help to distinguish equivocal lesions. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of benign and malignant melanocytic lesions based on combination of three imaging label-free modalities (multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography). Thirty-two melanocytic lesions were studied, using multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography. Multiphoton tomography features of benign melanocytic nevi were characterized by the normal morphology of both the keratinocytes and the nevus cell nests surrounded by collagen. Dysplastic nevi were characterized by their increased intercellular distances and enlarged cell nuclei. Melanomas showed the presence of melanocytes and dendritic structures in all layers of the epidermis. Analysis of the metabolic state revealed that melanomas and dysplastic nevi were characterized by enhanced glycolysis. Optical coherence angiography shows that benign nevi had regular vascular networks and equal numbers of thin and thick vessels. Vascular networks of dysplastic nevi were characterized thin curved vessels. Thick irregular spiral vessels formed a dense microvascular network of melanomas.

Multiphoton imaging commonly relies on laser-scanning setups which quickly image horizontal sections (x y images) by pixelwise scanning a sample region with focused laser pulses. Different horizontal planes are imaged by adjusting the distance relative between focusing optics and sample. However, in many cases actually a visualization of vertical sections is desired that then can only be obtained indirectly from time-consuming acquisition and processing of complete volume scans. We present a modified multiphoton tomograph for clinical in vivo and ex vivo tissue imaging with direct and fast x-z-imaging capability and exemplify different applications spanning from visualizing anatomic structures to substance penetration studies. The fast x-z imaging is realized by synchronizing the scanning-mirror movement with the tuning of the relative distance between sample and focusing optics

The combination of a tunable acoustic gradient (TAG) lens and a resonant mirror were integrated into a multiphoton excited (MPE) microscope that is rapidly and precisely controlled by an embedded field programmable gate array (FPGA) to acquire images at high frame rate.

Hypericin (Hyp) has received attention due to its high phototoxicity against viruses and anti-tumor photoactivity. Using two-photon imaging, we demonstrated that Hyp induced photosensitized modification of collagen fibers in native tissues. Dynamics of photo-processes was monitored by time-lapse multiphoton imaging. We showed that Hyp–mediated processes in collagen tissues may be used for the selective modification of collagen fibers.

Compared with other imaging probes such as fluorescent dyes and quantum dots, second-harmonic radiating imaging probes (SHRIMPs) provide a unique ultrafast, coherent optical contrast that is free of photobleaching and emission intermittency. Using the second-harmonic signal emitted from SHRIMPs, harmonic holography achieves three-dimensional holographic imaging with a color contrast similar to fluorescence microscopy where the uninterested background scattering is efficiently suppressed by an optical filter. The coherent contrast provided by SHRIMPs also enables imaging through turbid media via digital phase conjugation. Here we review the developments and applications of SHRIMPs and harmonic holography.

The vast number of propagating solutions to the wave equation in multimode optical fibers represents a larger information capacity than provided by fiber bundles of the same diameter. Therefore, in the field of imaging, multimode fibers potentially allow the transmission of images with higher resolution. However, image transmission through multimode fibers is not direct as in the fiber bundle case, in which each of the fiber cores can relay a portion of the distal image. In multimode fiber transmission, a distribution of intensity is scrambled in time and space by the propagating modes, leading to a speckle-like pattern that does not resemble the initial distribution. Here, we demonstrate two-photon excitation imaging of fluorescent beads through a multimode optical fiber. We show that our method maintains the advantages of two-photon excitation microscopy compared to single-photon excitation such as reduced photo-bleaching, deeper penetration depth and sectioning capability. Our method is based on time-gated digital phase conjugation, which allows the generation of focused pulses on the other side of a multimode fiber. To acquire an image, the focused femtosecond pulse is scanned in a three-dimensional mesh, producing two-photon excitation on each spatial location of the sample. By collecting the fluorescence through the fiber, a 3D two-photon image is reconstructed.

We present a portable multiphoton system designed for evaluating centimeter-scale surgical margins on surgical breast specimens in a clinical setting. The system is designed to produce large field of view images at a high frame rate, while using GPU processing to render low latency, video-rate virtual H&E images for real-time assessment. The imaging system and virtual H&E rendering algorithm are demonstrated by imaging unfixed human breast tissue in a clinical setting.

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We applied multiphoton microscopic imaging to observe freezing and heating effects in plant- and animal cell samples. The experimental setups consisted of a multiphoton imaging system and a heating and cooling stage which allows for precise temperature control from liquid nitrogen temperature (-196°C; 77 K) up to +600°C (873 K) with heating/freezing rates between 0.01 K/min and 150 K/min. Two multiphoton imaging systems were used: a system based on a modified optical microscope and a flexible mobile system. To illustrate the imaging capabilities, plant leafs as well as animal cells were microscopically imaged in vivo during freezing based on autofluorescence lifetime and intensity of intrinsic molecules. The measurements illustrate the usefulness of multiphoton imaging to investigate freezing effects on animal and plant cells.

We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. Grana and starch are the major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence (2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and starch while 2PF from chlorophyll indicates the identity of grana.

New labeling, imaging, or analysis tools could provide new retrospective insights when applied to archived, paraffin-embedded samples. Deep-tissue multiphoton microscopy of paraffin-embedded specimens is achieved using optical clearing with mineral oil. We tested a variety of murine tissue specimens including skin, lung, spleen, kidney, and heart, acquiring multiphoton autofluorescence and second-harmonic generation, and pump-probe images This technique introduces the capability for non-destructive 3-dimensional microscopic imaging of existing archived pathology specimens, enabling retrospective studies.

We present a multiphoton microscope system which combines the advantages of multiphoton imaging with precise control of the sample temperature. The microscope provides online insight in temperature-induced changes and effects in plant tissue and animal cells with subcellular resolution during cooling and thawing processes. Image contrast is based on multiphoton fluorescence intensity or fluorescence lifetime in the range from liquid nitrogen temperature up to +600°C. In addition, micro spectra from the imaged regions can be recorded. We present measurement results from plant leaf samples as well as Chinese hamster ovary cells.

Multiphoton imaging methods are excellent for non-invasive imaging of living tissue without any need of additional contrast agents. The increasing demand for endoscopic techniques has forced the development of multiphoton endoscopes for imaging of areas with reduced accessibility like chronic wounds. Gradient index (GRIN) lenses can miniaturize the bulky distal focusing optics of conventional tomographs to a diameter of less than 1.4 mm and a numerical aperture (NA) of 0.8. We combined a high NA clinical multiphoton endoscope with existing multiphoton tomographs like the DermaInspect® and the MPTflex® to enable the examination of wound healing processes.

Multiphoton imaging systems are capable of high-resolution 3-D image acquisition of deep tissue. A first commercially available CE-certified biomedical system for subcelluar resolution of human skin has been launched by JenLab company with the DermaInspectR in 2002. The demand for more flexibility caused the development of the MPTflexR, which provides an increased flexibility and accessibility especially for clinical and cosmetic examinations. However the high resolution of clinical multiphoton tomographs are adherent with a small field-of-view (FOV) of about 360×360μm2. Especially time-consuming is the relocation of areas of interest (AOI) like lesions, sweat glands or hair shafts during a multiphoton examination. This limitation can be be overcome by macroscopic large-area (wide-field-ofview) multiphoton tomography, which is tested first within this work.

An Yb fiber laser oscillator with sub-30 fs pulses compressed by MIIPS is tested for multiphoton microscopy. It leads to greatly improved third harmonic generation images. Multiphoton fluorescence, second and third harmonic generation modalities are compared on stained microspheres and unstained biological tissues.

The potential of multiphoton microscopy in providing in-vivo early diagnosis of skin lesions has already been demonstrated, while its capability in therapy follow-up has not been deeply explored so far. Two-photon excited fluorescence and second-harmonic generation microscopy were used in combination to follow-up collagen remodeling after laser micro-ablative rejuvenation. Treated regions of volunteers were imaged with multiphoton microscopy before and after treatment, and we found a strong age-dependence of the treatment effectiveness. In particular, the photorejuvenating effect was negligible in young subjects (< 30 years), whereas a significant production of new collagen was observed in aged subjects (> 70 years). Quantification of the amount of newly produced collagen and its organization were performed by means of visual examination of two-photon images. The obtained results demonstrate the performance of laser fractional micro-ablative rejuvenation without the need of an invasive biopsy as well as the wide applicability range of applications for multiphoton microscopy in clinical dermatology.

In this paper, we present our efforts in improving the penetration depth of MPM and the development of a multiphoton endoscope for imaging intrinsic tissue fluorescence and harmonic generation in vivo, with a main focus on instrument design and optimization.

In this work, we applied multi-photon microscopy to image and characterize the extent of tissue glycation. Specifically, features of multi-photon autofluorescence (MPAF) and second harmonic generation (SHG) images were presented. Our study shows that multiphoton imaging is capable of providing qualitative and quantitativ e information of the extent of tissue glycation in ribose treated samples including bovine sclera and vessel and that this approach may be useful for clinical evaluation of advanced glycati on endproducts (AGEs) formation. Keywords: multiphoton microscopy, spectral imaging, glycation, advanced glycation product 1. INTRODUCTION Glycoxidation of proteins and lipids can lead to the formation of advanced glycation end products (AGEs). 1, 2 The process is initiated by spontaneous reactions between free amino groups on proteins and car bonyl groups of sugars to form a chemically reversible Schiff base adducts which beco me Amadori product. Finally, further reactions result in the formation of AGEs which act as molecula r crosslinks. AGE formation which is increased in diabetic patients as a result of hyperglycaemia, contributes to cardiovascular disease and microvascular complications.

Combining multiphoton microscopy with a newly designed hepatic imaging window, we acquired in vivo images of mice obstructive cholestasis. We observed that in mice with bile duct ligation, bile canaliculi failed to appear during the whole observation period over 100 minutes following carboxyfluorescein diacetate injection, whereas the fluorescence was retained much longer within sinusoids. Furthermore, the fluorescence intensities in sinusoids were persistently higher than in hepatocytes during the course.

Coupling three-photon microscopy with automated stage movement can now produce a live high resolution map of the neurotransmitter serotonin in a single cross section of the whole rat brain. Accurate quantification of these serotonin images demands appropriate spectral filtering. This requires one to consider that the spectral characteristics of serotonin show a remarkable variation as it non-covalently associates with different molecules, as we discuss here. Also it is known that serotonin emission changes when it forms a covalent adduct with para-formaldehyde. This provides a potential route for producing a whole brain serotonin map using multiphoton microscopy in a fixed rat brain. Here we take the initial step showing that multiphoton microscopy of this adduct can quantitatively image chemically induced changes in serotonin distribution.

For secure removal of the basal cell carcinoma tissue, the technique of Mohs' micrographic surgery is often used. However, Mohs' micrographic surgery is time-consuming. In this work, we evaluate the ability of multiphoton fluorescence (MF) and second harmonic generation (SHG) imaging to discriminate the borders of human basal cell carcinoma. Morphologically, basal cell carcinomas are featured by clumps of autofluorecent cells with relatively large nuclei and marked peripheral palisading in the dermis. In contrast, SHG from collagen contributes largely to the multiphoton signal in normal dermis. Within the cancer stroma, SHG signals diminish and are replaced by autofluorescent signals. The results suggest that normal collagen structures responsible for SHG have been altered in the cancer stroma and may reflect an up-regulated collagenolytic activity of cancer cells. To better delineate the cancer cells and cancer stroma from normal dermis, a quantitative MF to SHG index (MFSI) is developed. We demonstrate that this index can be used to differentiate cancer cells and adjacent cancer stroma from normal dermis. Our work shows that MF and SHG imaging can be an alternative for the real-time guidance of the secure removal of basal cell carcinoma.

Near infrared (NIR) femtosecond laser imaging systems represent a novel and very promising diagnostic technology for non-invasive cross-sectional analysis of living biological tissues. In this study 3D multiphoton imaging with submicron resolution has been performed for non-invasive analysis of living native and tissue-engineered (TE) heart valves and blood vessels. High-resolution autofluorescence and second harmonic generation (SHG) images of collagenous structures and elastic fibers were demonstrated using multiphoton excitation at two different wavelengths. Non-invasive optical sections have been obtained without the need of staining or embedding. The quality of the resulting three-dimensional images allowed exact differentiation between collagenous structures and elastic fibers. These experimental results are very encouraging for NIR femtosecond laser scanning microscopy as a useful tool for future non-destructive monitoring and characterization of vital and intact TE cardiovascular structures.

Wound healing is a physiologic process that acts to repair disruptions in the continuity of tissue caused by injury or surgical incision. Keloids and hypertrophic scars are forms of aberrant wound healing, which are characterized by the overproduction of collagen, resulting in an excessive amount of scar tissue. Keloid tumors, by definition, grow outside the boundary of the original tissue damage. Multiphoton microscopy (MPM) is an imaging technique which allows imaging of living specimens, without the use of fixation or stains. Images of collagen fibers are produced by the second harmonic signal intensity generated by endogenous fluorescence through excitation by infrared laser light. A postauricular keloid tumor was excised from a patient. The tissue was dissected, and a portion was imaged using MPM. Normal skin tissue was isolated from a patient undergoing a facelift. A portion of this tissue was also dissected and imaged using MPM. MPM images were taken using a 63X water immersion objective lens on a two-photon microscope and a titanium-sapphire laser. Images were taken beginning at the surface of the tissue and moving in at intervals of 200 nm to a final depth of 30 μm. The two-photon images were used to reconstruct three-dimensional representations of the collagen matrix within the tissues, which are readily contrasted. Density of the collagen within each tissue was also ascertained using depth dependant decay of the image intensity. Multiphoton imaging was successfully used to image the collagen matrix of normal skin and a keloid scar, demonstrating differences in their microstructures.

Multiphoton imaging of the sub-cellular distribution of photosensitizers can provide important clues to their mechanism of action in tumors. We have used this tool to study distribution and pharmacology of photosensitizers in murine hepatoma tumor cells dosed with various photosensitizers. Upon photoactivation, Rose Bengal yields nearly immediate photolytic release of lysomal enzymes, resulting in catastrophic cell destruction within 5-30 minutes. Such marked response is quite different than that observed with other photosensitizer agents, and is consistent with in vivo studies illustrating that Rose Bengal is capable of causing extremely rapid destruction of treated tumors.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.