A "Double-Locked" and Enzyme/pH-Activated Theranostic Agent for Accurate Tumor Imaging and Therapy.

Theranostic agents for concurrent cancer therapy and diagnosis have begun attracting attention as a promising modality. However, accurate imaging and identification remains a great challenge for theranostic agents. Here, we designed and synthesized a novel theranostic agent H6M based on the "double-locked" strategy by introducing an electron-withdrawing nitro group into 1-position of a pH-responsive 3-amino-ß-carboline and further covalently linking the hydroxamic acid group, a zinc-binding group (ZBG), to the 3-position of ß-carboline to obtain histone deacetylase (HDAC) inhibitory effect for combined HDAC-targeted therapy. We found that H6M can be specifically reduced under overexpressed nitroreductase (NTR) to produce H6AQ, which emits bright fluorescence at low pH. Notably, H6M demonstrated a selective fluorescence imaging via successive reactions with NTR (first "key") and pH (second "key"), and precisely identified tumor margins with a high S/N ratio to guide tumor resection. Finally, H6M exerted robust HDAC1/cancer cell inhibitory activities compared with a known HDAC inhibitor SAHA. Therefore, the NTR/pH-activated theranostic agent provided a novel tool for precise diagnosis and efficient tumor therapy.

Combination of oral recombinant methioninase and decitabine arrests a chemotherapy-resistant undifferentiated soft-tissue sarcoma patient-derived orthotopic xenograft mouse model.

Cancer cells are methionine (MET) and methylation addicted and are highly sensitive to MET restriction. The present study determined the efficacy of oral-recombinant methioninase (o-rMETase) and the DNA methylation inhibitor, decitabine (DAC) on restricting MET in an undifferentiated-soft tissue sarcoma (USTS) patient-derived orthotopic xenograft (PDOX) nude-mouse model. The USTS PDOX models were randomized into five treatment groups of six mice: Control; doxorubicin (DOX) alone; DAC alone; o-rMETase alone; and o-rMETase-DAC combination. Tumor size and body weight were measured during the 14 days of treatment. Tumor growth was arrested only in the o-rMETase-DAC condition. Tumors treated with the o-rMETase-DAC combination exhibited tumor necrosis with degenerative changes. This study demonstrates that the o-rMETase-DAC combination could arrest the USTS PDOX tumor suggesting clinical promise.

Fluorescence-Guided Cancer Diagnosis and Surgery by a Zero Cross-Talk Ratiometric Near-Infrared γ-Glutamyltranspeptidase Fluorescent Probe.

The ability to detect cancer early in an accurate and rapid fashion is of critical importance for cancer diagnosis and accurate resection in surgery. γ-Glutamyltranspeptidase (GGT) is overexpressed in several human cancers, while maintaining a low expression in normal microenvironments, and thus is recognized as an important cancer biomarker. To date, rational design of a zero cross-talk ratiometric near-infrared (NIR) GGT fluorescent probe for efficient cancer diagnosis in various biological samples is still a big challenge. In this work, a zero cross-talk ratiometric NIR GGT fluorescent probe named Cy-GSH is developed. Cy-GSH shows high sensitivity to GGT, which is desired for early cancer diagnosis. Upon additional GGT, a large emission shift from 805 to 640 nm is observed, which is suitable for visualizing deeply located cancer in vivo. In addition, successful monitoring of GGT activity in blood, cells, tissues, and in vivo makes Cy-GSH possess great potential for the clinical cancer early diagnosis. Furthermore, accurately visualizing tumors and metastases in mouse models illuminates that the probe may be a convenient tool for fluorescence-guided cancer surgery. To our knowledge, this is the first report to describe the strategy of a zero cross-talk ratiometric NIR GGT fluorescent probe for early cancer diagnosis and fluorescence-guided surgery.

NIR-II Fluorescent Self-Assembled Peptide Nanochain for Ultrasensitive Detection of Peritoneal Metastasis.

Fluorescence-guided cytoreductive surgery is one of the most promising approaches for facile elimination of tumors in situ, thereby improving prognosis. Reported herein is a simple strategy to construct a novel chainlike NIR-II nanoprobe (APP-Ag2 S-RGD) by self-assembly of an amphiphilic peptide (APP) into a nanochain with subsequent chemical crosslinking of NIR-II Ag2 S QDs and the tumor-targeting RGD peptide. This probe exhibits higher capability for cancer cell detection compared with that of RGD-functionalized Ag2 S QDs (Ag2 S-RGD) at the same concentration. Upon intraperitoneal injection, superior tumor-to-normal tissue signal ratio is achieved and non-vascularized tiny tumor metastatic foci as small as about 0.2 mm in diameter could be facilely eliminated under NIR-II fluorescent imaging guidance. These results clearly indicate the potential of this probe for fluorescence-guided tumor staging, preoperative diagnosis, and intraoperative navigation.

Peritoneal lavage with povidone-iodine solution in colorectal cancer-induced rats.

BACKGROUND: Although peritoneal lavage with povidone-iodine (PVPI) is frequently performed after surgery on the gastrointestinal tract, the effects of PVPI on the intestinal epithelial barrier are unknown. The purpose of this study was to investigate the effects of abdominal irrigation with PVPI on the intestinal epithelial barrier in a colorectal cancer (CRC)-induced rat model. MATERIALS AND METHODS: The CRC model was induced in rats with azoxymethane and dextran sodium sulfate. Next, a total of 24 male CRC-induced rats were randomly divided into three groups (n = 8): (1) a sham-operated group, (2) an NS group (peritoneal lavage 0.9% NaCl), and (3) a PVPI group (peritoneal lavage with 0.45%-0.55% PVPI). The mean arterial pressure was continuously monitored throughout the experiment. The levels of plasma endotoxin and D-lactate, blood gases, and protein concentration were measured. The ultrastructural changes of the epithelial tight junctions were observed by transmission electron microscopy. RESULTS: The mean arterial pressure after peritoneal lavage was lower in the PVPI group than that in the NS group. The protein concentration and levels of endotoxin and D-lactate were higher in the PVPI group than they were in the PVPI group. In addition, PVPI treatment resulted in a markedly severe metabolic acidosis and intestinal mucosal injury compared with NS rats. CONCLUSIONS: Peritoneal lavage with PVPI dramatically compromises the integrity of the intestinal mucosa barrier and causes endotoxin shock in CRC rats. It is unsafe for clinical applications to include peritoneal lavage with PVPI in colorectal operations.

Advantages of patient-derived orthotopic mouse models and genetic reporters for developing fluorescence-guided surgery.

Fluorescence-guided surgery can enhance the surgeon's ability to achieve a complete oncologic resection. There are a number of tumor-specific probes being developed with many preclinical mouse models to evaluate their efficacy. The current review discusses the different preclinical mouse models in the setting of probe evaluation and highlights the advantages of patient-derived orthotopic xenografts (PDOX) mouse models and genetic reporters to develop fluorescence-guided surgery.

Experimental study of the potential hazards of surgical smoke from powered instruments.

BACKGROUND: Many surgical instruments have been replaced with powered devices in open gastrointestinal and laparoscopic surgery. The production of smoke as a result of vaporization of surgical tissue is inevitable, and exposure to surgical smoke is a long-standing concern. These vapours are potentially hazardous to patients and surgical teams. The present research was designed to compare various surgical devices to determine whether viable cells exist in their surgical smoke. METHODS: The search for viable cells in surgical smoke was conducted using both in vitro and in vivo experiments. Various cancers were cauterized with electrocautery, radiofrequency ablation and ultrasonic scalpels, and the resulting surgical smoke was aspirated with Transwell(®) membrane; viable cells were sought in the surgical smoke. In an in vivo experiment, samples of SCC7 were cauterized with an ultrasonic scalpel and the sediment from the rinsed Transwell(®) membrane liquid after centrifugation was injected subcutaneously into the lower back of mice. RESULTS: Viable cells were found only in the smoke from ultrasonic scalpels (in all 25 samples taken 5 cm from the cautery; 2 of 25 samples at 10 cm). Viable cells in the surgical smoke from ultrasonic scalpels implanted in mice grew in 16 of 40 injection sites. Histological and biochemical analyses revealed that these cancer cells were identical to the cancer cells cauterized by the ultrasonic scalpel. CONCLUSION: Viable tumour cells are produced in the surgical smoke from tumour dissection by ultrasonic scalpel. Surgical relevance Surgical smoke is a byproduct of dissection using a number of powered devices. Hazards to operating room personnel and patients are unclear. This study has shown that use of an ultrasonic dissection device can produce smoke that contains viable tumour cells. Although the model is somewhat artificial, a theoretical risk exists, and measures to evacuate surgical smoke efficiently are important.

Surgical trauma leads to a shorter survival in a murine orthotopic pancreatic cancer model.

BACKGROUND: Abdominal surgery is frequently followed by immune dysfunction usually lasting for several days. This is especially important in cases with tumour diseases as an intact immune function is essential in this situation. Therefore, we analysed the outcome of tumour-bearing mice in a mouse model of surgically induced immune dysfunction (SID). METHODS: In male C57BL/6 mice, a pancreatic tumour was implanted orthotopically. Following tumour implantation, the model of SID was applied. The control groups were either laparotomised or underwent no surgical procedure. The survival rate was determined by observation for >60 days. The tumour growth progress was imaged by a 7-tesla small animal MRI. RESULTS: On day 60 after tumour implantation, the survival rate in SID mice was reduced to 41%. In the laparotomised group, 81% of mice survived, while the control group had a survival rate of 75%. These differences were significant (SID vs. control: p < 0.02, and SID vs. laparotomy: p < 0.002). The tumour volume was not influenced by the degree of surgical trauma. CONCLUSION: In pancreatic cancer, the SID model is ideally suited to investigate the influence of SID on this tumour entity.

Intraperitoneal chemotherapy (IPC) for peritoneal carcinomatosis: review of animal models.

The development of suitable animal models is essential to experimental research on intraperitoneal chemotherapy (IPC). This review of the English literature (MEDLINE) presents a detailed analysis of current animal models and gives recommendations for future experimental research. Special consideration should be given to cytotoxic drug dose and concentration, tumor models, and outcome parameters.

Water-jet dissector for endoscopic submucosal dissection in an animal study: outcomes of the continuous and pulsed modes.

BACKGROUND: Endoscopic submucosal dissection (ESD) allows en bloc resection of early neoplastic lesions of gastrointestinal tract. Lesions are lifted by submucosal fluid injection before circumferential incision and dissection. High-pressure fluid injection using water jet (WJ) technology is already used for lifting and dissection in surgery. The study was designed to assess WJ for ESD submucosal lifting and dissection. METHODS: An experimental, randomized comparative, "in vivo" nonsurvival animal study on 12 pigs was designed. Stomach mucosal areas were delineated and resected using three ESD techniques: technique A-syringe injection and IT knife dissection; technique B-WJ continuous injection and IT knife dissection; technique C-WJ injection and WJ pulsed dissection. Injection and dissection speeds and complications rates were assessed. RESULTS: Water jet continuous injection is faster than syringe injection (B faster than A, p = 0.001 and B nonsignificantly faster than C, p = 0.06). IT knife dissection is significantly faster after WJ continuous injection (B faster than A, p = 0.003). WJ pulsed dissection is significantly slower than IT knife dissection (C slower than A and B, both p < 0.001). The overall procedure speed was significantly higher and the immediate bleedings rate was significantly lower for technique B than A and C (overall procedure speed p = 0.001, immediate bleedings p = 0.032 and 0.038 respectively). There were no perforations with any technique. CONCLUSIONS: Water jet fluid continuous injection speeds up ESD, whereas pulsed WJ dissection does not.

Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival.

The completeness of tumor removal during surgery is dependent on the surgeon's ability to differentiate tumor from normal tissue using subjective criteria that are not easily quantifiable. A way to objectively assess tumor margins during surgery in patients would be of great value. We have developed a method to visualize tumors during surgery using activatable cell-penetrating peptides (ACPPs), in which the fluorescently labeled, polycationic cell-penetrating peptide (CPP) is coupled via a cleavable linker to a neutralizing peptide. Upon exposure to proteases characteristic of tumor tissue, the linker is cleaved, dissociating the inhibitory peptide and allowing the CPP to bind to and enter tumor cells. In mice, xenografts stably transfected with green fluorescent protein show colocalization with the Cy5-labeled ACPPs. In the same mouse models, Cy5-labeled free ACPPs and ACPPs conjugated to dendrimers (ACPPDs) delineate the margin between tumor and adjacent tissue, resulting in improved precision of tumor resection. Surgery guided by ACPPD resulted in fewer residual cancer cells left in the animal after surgery as measured by Alu PCR. A single injection of ACPPD dually labeled with Cy5 and gadolinium chelates enabled preoperative whole-body tumor detection by MRI, intraoperative guidance by real-time fluorescence, intraoperative histological analysis of margin status by fluorescence, and postoperative MRI tumor quantification. Animals whose tumors were resected with ACPPD guidance had better long-term tumor-free survival and overall survival than animals whose tumors were resected with traditional bright-field illumination only.

Treatment of brain glioblastoma multiforme with pcDNA3.1-Egr. 1p-p16 combined with gamma knife radiation: an experimental study on nude mice.

BACKGROUND: High post-operative recurrence and poor prognosis are likely to be related to the infiltrative growth of the glioblastoma multiforme (GBM). OBJECTIVES: The primary objective of this study is to investigate the possible synergistic effect of the combined treatment of gamma knife radio-surgery (GKRS) and gene therapy for GBM and secondary objective is to explore the role of GKRS for the temporal and spatial regulation of the gene expression. MATERIALS AND METHODS: The study performed on 70 nude mice and randomly divided into seven groups. Subcutaneous injection of human GBM tumor cells (T98G) was carried out to establish the animal models. Various doses of liposome-mediated pcDNA3.1-Egr. 1p-p16 recombinant plasmid were transfected through intra-tumor injection. GKRS was scheduled following the plasmid transfection. Tumor volumes were measured every 4 days after the treatment. Subcutaneous tumor nodule specimens were collected to analyze the cell apoptosis and p16 gene expression using terminal-deoxynucleoitidyl transferase mediated nick end labeling staining and reverse transcription-polymerase chain reaction. Tumor volumes, levels of cell apoptosis and p16 gene expression were compared between groups. RESULTS: Rates of tumor growth were significantly lower in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS groups than that in the remaining groups 28 days following the GKRS management. The p16mRNA expression was noted in both of the pcDNA3.1-Egr. 1p-p16 plasmid group and the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with marginal-dose of 20 Gy group. The level of messenger ribonucleic acid expression was higher in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with the marginal-dose of 20 Gy group, with a markedly increased apoptotic and necrotic cells, than that in the pcDNA3.1-Egr. 1p-p16 plasmid group. CONCLUSIONS: In animal studies, pcDNA3.1-Egr. 1p-p16 in combination with GKRS is a preferable management option for the GBM to the sole use of GKRS or gene therapy. It may be a novel approach for the treatment of human patient with GBM.

[Experimental validation of the use of ND:YAG-interstitial laser coagulation of the kidney].

Interstitial laser coagulation (ILC) is one of the effective methods of minimally invasive destruction of small renal tumors. For the safe use of ILC in clinical practice, it is necessary to select the optimal mode of action of laser radiation on the tissue of target organ and techniques for the procedure. The effects of ILC on kidney tissue in 6 rabbits weighing less than 3 kg and 5 mongrel dogs weighing up to 20 kg were investigated in vivo. The impact of ILC was made intraoperatively. Organ retrieval in experimental animals for the macro- and microscopic examination was performed immediately after the impact of ILC and at various times thereafter during the month in rabbits and 35 days in dogs. Morphological study in different periods after exposure allowed to establish phasing and volumes of changes in the tissues of the kidney, confirming the safety and efficacy of ND:YAG laser irradiation on kidney tissues. The results indicate the possibility of effective application of ILC of kidney tissues for destruction of small size tumors, without impairment of functions of the organ.

Therapeutic effect of genetically modified human neural stem cells encoding cytosine deaminase on experimental glioma.

The aim of this study was to determine the efficacy of neural stem cell-based suicidal gene therapy in rats bearing human glioma. F3 human neural stem cells (NSCs) were transduced to encode cytosine deaminase (CD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Intratumoral or intravenous transplantation of F3.CD human NSCs led to marked reduction in tumor burden and significantly prolonged the survival of brain tumor-bearing rats. The systemic administration of 5-FC with direct intratumoral/intravenous transplantation of F3.CD cells had remarkable therapeutic effect in rats with human glioma cells as compared with transplantation of parental F3 cells. There was 74% reduction in tumor volume in rats receiving direct transplantation of F3.CD cells into tumor site, and 67% reduction in tumor volume in rats receiving intravenous injection of F3.CD cells as compared to control animals transplanted with human glioma U373 cells alone. The combination of F3.CD and 5-FC was a highly effective in the glioma rat model. Our observations suggest that genetically engineered NSCs encoding suicide gene CD could provide clinical application of suicide gene therapy for patients with glioma.

Evaluation of breast tumor margins in vivo with intraoperative photoacoustic imaging.

The use of photoacoustic effect is a promising approach for biomedical imaging in living tissues. Photoacoustic tomography (PAT) has been demonstrated to image breast cancer, brain vasculature, arthritis and seizure focus owing to its rich optical contrast and high resolution in a single imaging modality. Here we report a microelectromechanical systems (MEMS)-based intraoperative PAT (iPAT) technique, and demonstrate its ability to accurately map tumors in three-dimension and to inspect the completeness of tumor resection during surgery in a tumor-bearing mouse model. The MEMS imaging probe is small and has the potential to be conveniently used to guide surgical resection of tumors in the breast.

Electrolysis - a new method of renal ablation?

OBJECTIVE: • To present a novel method of renal ablation using direct current electrolysis, using a porcine model to assess the safety and efficacy of the technique. MATERIALS AND METHODS: • In all, 20 anaesthetised pigs were used, after receiving ethical approval. The pigs were housed and managed in accordance with Institute of Medical and Veterinary Science guidelines. • A single 6 F catheter incorporating cathode and anode was inserted directly into the renal parenchyma via a loin incision. A direct current of 100 mA was applied for varying times. • Several treatments were performed in each pig and a total of 72 treatments were analysed. • The pigs were recovered and observed for 3 days with an analysis of electrolytes and creatinine after ablation. The pigs were humanely killed at 3 days and the kidneys submitted for histological analysis. RESULTS: • Complete tissue destruction was seen in the area of kidney treated with no viable cells. • A sharp line of demarcation was noted between normal renal parenchyma and the area of coagulative necrosis. The area of tissue ablation was reliably predicted from the duration of application of the electrolytic current. • One pig developed a secondary haemorrhage. CONCLUSIONS: • Renal electrolysis is a new method of focal renal ablation. The procedure may be carried out using a fine ablation catheter (6 F) allowing for the development of minimal intervention treatment of small renal masses. • This study shows the technique to be effective and safe. As with other renal ablative techniques there is a small risk of postoperative haemorrhage.

In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation.

Cancer surgery requires the complete and precise identification of malignant tissue margins including the smallest disseminated lesions. Internal green fluorescent protein (GFP) fluorescence can intensely illuminate even single cells but requires GFP sequence transcription within the cell. Introducing and selectively activating the GFP gene in malignant tissue in vivo is made possible by the development of OBP-401, a telomerase-dependent, replication-competent adenovirus expressing GFP. This potentially powerful adjunct to surgical navigation was demonstrated in 2 nude mouse models that represent difficult surgical challenges--the resection of widely disseminated cancer. HCT-116, a model of intraperitoneal disseminated human colon cancer, was labeled by virus injection into the peritoneal cavity. A549, a model of pleural dissemination of human lung cancer, was labeled by virus administered into the pleural cavity. Only the malignant tissue fluoresced brightly in both models. In the intraperitoneal model of disseminated cancer, fluorescence-guided surgery enabled resection of all tumor nodules labeled with GFP by OBP-401. The data in this report suggest that adenoviral-GFP labeling tumors in patients can enable fluorescence-guided surgical navigation.

Intraoperative laparoscopic detection of sentinel lymph nodes with indocyanine green and superparamagnetic iron oxide in a swine gallbladder cancer model.

Mapping of sentinel lymph nodes (SLNs) can enable less invasive surgery. However, mapping is challenging for cancers of difficult-to-access visceral organs, such as the gallbladder, because the standard method using radioisotopes (RIs) requires preoperative tracer injection. Indocyanine green (ICG) and superparamagnetic iron oxide (SPIO) have also been used as alternative tracers. In this study, we modified a previously reported magnetic probe for laparoscopic use and evaluated the feasibility of detecting SLNs of the gallbladder using a laparoscopic dual tracer method by injecting ICG and SPIO into five swine and one cancer-bearing swine. The laparoscopic probe identified SPIO nanoparticles in the nodes of 4/5 swine in situ, the magnetic field counts were 2.5-15.9 µT, and fluorescence was detected in SLNs in all five swine. ICG showed a visual lymph flow map, and SPIO more accurately identified each SLN with a measurable magnetic field quite similar to the RI. We then developed an advanced gallbladder cancer model with lymph node metastasis using recombination activating gene 2-knockout swine. We identified an SLN in the laparoscopic investigation, and the magnetic field count was 3.5 µT. The SLN was histologically determined to be one of the two metastatic lymph nodes. In conclusion, detecting the SLNs of gallbladder cancer in situ using a dual tracer laparoscopic technique with ICG and SPIO was feasible in a swine model.

IRDye800CW labeled uPAR-targeting peptide for fluorescence-guided glioblastoma surgery: Preclinical studies in orthotopic xenografts.

Glioblastoma (GBM) is a devastating cancer with basically no curative treatment. Even with aggressive treatment, the median survival is disappointing 14 months. Surgery remains the key treatment and the postoperative survival is determined by the extent of resection. Unfortunately, the invasive growth with irregular infiltrating margins complicates an optimal surgical resection. Precise intraoperative tumor visualization is therefore highly needed and molecular targeted near-infrared (NIR) fluorescence imaging potentially constitutes such a tool. The urokinase-type Plasminogen Activator Receptor (uPAR) is expressed in most solid cancers primarily at the invading front and the adjacent activated peritumoral stroma making it an attractive target for targeted fluorescence imaging. The purpose of this study was to develop and evaluate a new uPAR-targeted optical probe, IRDye800CW-AE344, for fluorescence guided surgery (FGS). Methods: In the present study we characterized the fluorescent probe with regard to binding affinity, optical properties, and plasma stability. Further, in vivo imaging characterization was performed in nude mice with orthotopic human patient derived glioblastoma xenografts, and we performed head-to-head comparison within FGS between our probe and the traditional procedure using 5-ALA. Finally, the blood-brain barrier (BBB) penetration was characterized in a 3D BBB spheroid model. Results: The probe effectively visualized GBM in vivo with a tumor-to-background ratio (TBR) above 4.5 between 1 to 12 h post injection and could be used for FGS of orthotopic human glioblastoma xenografts in mice where it was superior to 5-ALA. The probe showed a favorable safety profile with no evidence of any acute toxicity. Finally, the 3D BBB model showed uptake of the probe into the spheroids indicating that the probe crosses the BBB. Conclusion: IRDye800CW-AE344 is a promising uPAR-targeted optical probe for FGS and a candidate for translation into human use.

Label-free imaging of human brain tissue at subcellular resolution for potential rapid intra-operative assessment of glioma surgery.

Background: Frozen section and smear preparation are the current standard for intraoperative histopathology during cancer surgery. However, these methods are time-consuming and subject to limited sampling. Multiphoton microscopy (MPM) is a high-resolution non-destructive imaging technique capable of optical sectioning in real time with subcellular resolution. In this report, we systematically investigated the feasibility and translation potential of MPM for rapid histopathological assessment of label- and processing-free surgical specimens. Methods: We employed a customized MPM platform to capture architectural and cytological features of biological tissues based on two-photon excited NADH and FAD autofluorescence and second harmonic generation from collagen. Infiltrating glioma, an aggressive disease that requires subcellular resolution for definitive characterization during surgery, was chosen as an example for this validation study. MPM images were collected from resected brain specimens of 19 patients and correlated with histopathology. Deep learning was introduced to assist with image feature recognition. Results: MPM robustly captures diagnostic features of glioma including increased cellularity, cellular and nuclear pleomorphism, microvascular proliferation, necrosis, and collagen deposition. Preliminary application of deep learning to MPM images achieves high accuracy in distinguishing gray from white matter and cancer from non-cancer. We also demonstrate the ability to obtain such images from intact brain tissue with a multiphoton endomicroscope for intraoperative application. Conclusion: Multiphoton imaging correlates well with histopathology and is a promising tool for characterization of cancer and delineation of infiltration within seconds during brain surgery.