Fluorescence confocal microscopy (FCM) is an optical technique that uses laser light sources of different wavelengths to generate real-time images of fresh, unfixed tissue specimens. FCM allows histological evaluation of fresh tissue samples without the associated cryo artifacts after frozen sectioning. The aim of this study was to prospectively evaluate pediatric tumor specimens and assess their suitability for fresh tumor sampling. In addition, we aimed to determine whether tumor cell isolation for stable cell culture is still feasible after FCM imaging. Pediatric tumor specimens were imaged using FCM. Tumor viability and suitability for tissue sampling were evaluated and compared with H&E staining after paraffin embedding. In addition, FCM-processed and non-FCM-processed tissue samples were sent for tumor cell isolation to evaluate possible effects after FCM processing. When comparing estimated tumor cell viability using FCM and H&E, we found good to excellent correlating estimates (intraclass correlation coefficient = 0.891, p < 0.001), as well as substantial agreement in whether the tissue appeared adequate for fresh tissue collection (κ = 0.762, p < 0.001). After FCM, seven out of eight samples yielded passable cell cultures, compared to eight out of eight for non-FCM processed samples. Our study suggests that the use of FCM in tumor sampling can increase the yield of suitable fresh tumor samples by identifying viable tumor areas and ensuring that sufficient tissue remains for diagnosis. Our study also provides first evidence that the isolation and growth of tumor cells in culture are not compromised by the FCM technique.

Fluorescence confocal microscopy (FCM) is an optical technique that uses laser light sources of different wavelengths to generate real-time images of fresh, unfixed tissue specimens. Unlike conventional histologic evaluation methods, FCM is able to assess fresh tissue samples without the associated cryo artifacts typically observed after frozen sectioning. The purpose of this study was to evaluate the utility of FCM imaging in the differential diagnosis of cervical lymphadenopathy. Twenty-two cervical lymph node specimens from patients with lymphadenopathy of unknown origin were imaged by FCM. Two pathologists independently evaluated the scans for suspicion of malignancy and preliminary diagnosis. Malignancy was reliably excluded or confirmed by both pathologists with a sensitivity of 90.9% for pathologist 1 and 100% for pathologist 2. The specificity was 100% for both pathologists. For the preliminary diagnosis, almost perfect agreement with the final diagnosis was observed for both pathologists (κ = 0.94 for pathologist 1 and κ = 1.00 for pathologist 2). This is the first study to investigate lymph node specimens with different diagnoses, including lymphoma, using FCM. Our results indicate that differential diagnosis of lymph node specimens is feasible in FCM images, thus encouraging further exploration of FCM imaging in lymph node specimens to accelerate diagnosis and open the possibility of digitizing diagnosis on fresh, unfixed tissue.

OBJECTIVE: To test the performance of ex vivo fluorescence confocal microscopy (FCM; Vivascope 2500M-G4), as compared to intra-operative frozen section (IFS) analysis, to evaluate surgical margins during robot-assisted radical prostatectomy (RARP), with final pathology as the reference standard.
METHODS: Overall, 54 margins in 45 patients treated with RARP were analysed with: (1) ex vivo FCM; (2) IFS analysis; and (3) final pathology. FCM margins were evaluated by two different pathologists (experienced [M.I.: 10 years] vs highly experienced [G.R.: >30 years]) as strongly negative, probably negative, doubtful, probably positive, or strongly positive. First, inter-observer agreement (Cohen\'s κ) between pathologists was tested. Second, we reported the sensitivity, specificity, positive predictive (PPV) and negative predictive value (NPV) of ex vivo FCM. Finally, agreement between ex vivo FCM and IFS analysis (Cohen\'s κ) was reported. For all analyses, four combinations of FCM results were evaluated.
RESULTS: At ex vivo FCM, the inter-observer agreement between pathologists ranged from moderate (κ = 0.74) to almost perfect (κ = 0.90), according to the four categories of results. Indeed, at ex vivo FCM, the highly experienced pathologist reached the best balance between sensitivity (70.5%) specificity (91.8%), PPV (80.0%) and NPV (87.1%). Conversely, on IFS analysis, the sensitivity, specificity, PPV and NPV were, respectively, 88.2% vs 100% vs 100% vs 94.8%. The agreement between the ex vivo FCM and IFS analyses ranged from moderate (κ = 0.62) to strong (κ = 0.86), according to the four categories of results.
CONCLUSIONS: Evaluation of prostate margins at ex vivo FCM appears to be feasible and reliable. The agreement between readers encourages its widespread use in daily practice. Nevertheless, as of today, the performance of FCM seems to be sub-par when compared to the established standard of care (IFS analysis).

Infiltration of immune cells into the tumor is one of the major drivers of antitumor immune response, which can direct the outcome of anticancer therapies. In mice, implantation of dorsal skinfold window chamber (DSWC) combined with intravital confocal fluorescence microscopy allows real-time observation of splenocyte extravasation and infiltration into tumors. Here, we describe a detailed procedure of the DSWC implantation, splenocyte isolation and fluorescent labeling, intravenous injection of labeled splenocytes, and imaging of splenocyte extravasation into tumors using confocal fluorescence microscopy.

UNASSIGNED: Ex vivo confocal microscopy is a real-time technique that provides high-resolution images of fresh, non-fixed tissues, with an optical resolution comparable to conventional pathology. The objective of this study was to investigate the feasibility of using ex vivo confocal microscopy in fusion mode (FuCM) and the haematoxylin and eosin (H&E)-like digital staining that results for the analysis of basic patterns of lesion in nephropathology.
UNASSIGNED: Forty-eight renal samples were scanned in a fourth-generation ex vivo confocal microscopy device. Samples were subjected to confocal microscopy imaging and were then processed using conventional pathology techniques. Concordance between the techniques was evaluated by means of the percentage of agreement and the κ index.
UNASSIGNED: Agreement between conventional microscopy and H&E-like digital staining was strong (κ = 0.88) in the evaluation of acute tubular damage and was substantial (κ = 0.79) in the evaluation of interstitial fibrosis, interstitial inflammation, arterial and arteriolar lesions. H&E-like digital staining also allows rapid identification of extracapillary proliferation (κ = 0.88), necrosis and segmental sclerosis (κ = .88) in the glomerular compartment, but the results reported here are limited because of the small number of cases with these glomerular findings.
UNASSIGNED: FuCM proved to be as effective as conventional techniques in evaluating the presence of acute tubular necrosis and interstitial fibrosis changes, but in fresh tissue. The ease of acquisition of ex vivo confocal microscopy images suggests that FuCM may be useful for rapid evaluation of kidney biopsies and to restructure the clinical workflow in renal histopathology.

UNASSIGNED: Ex-vivo FCM is a novel digital optical technique that provides images of fresh tissues in a real-time fashion with magnification to subcellular details of a flattened unprocessed sample. Digital images are hematoxylin-eosin-like and can be shared and interpreted remotely. In urology, FCM has been successfully applied for prostate tissue interpretation, either during biopsy and radical prostatectomy. Possible applications of FCM may reflect those of frozen section analysis and can be extended to all fields in which the intra-operative microscopical control is advisable.
UNASSIGNED: This is an investigative prospective case series that aims to explore FCM feasibility in novel surgical settings and provide a depiction of FCM digital images in those fields. The definite purpose is to check the accuracy of surgical specimen during the following interventions: (a) trans-urethral resection of bladder tumors, to confirm the presence of muscular layer; (b) biopsy of a retroperitoneal mass, to check for the location and quality of cores; (c) training in robotic radical prostatectomy, to control surgical margins after a nerve sparing performed by a trainee. To this aim, we collected FCM images during seven surgical procedures. FCM findings were compared to those from the final histopathological analysis and the agreement was assessed.
UNASSIGNED: In all cases, FCM digital images were obtained in the OR. FCM was able to confirm the presence of muscular layer in TURB specimen, the presence of lymphomatous tissue, surgical margins at prostate specimen. FCM intra-operative interpretation was consistent with final histopathology in all cases.
UNASSIGNED: Ex vivo FCM may represent a novel approach to control the quality of specimens, likely to tailor surgical strategy in a real-time fashion. Moreover, digitalization represents a step toward the implementation of telepathology in clinical practice.

Two interesting benzothizolium-based D-π-A type hemicyanine dyes (3a-3b) with a diphenylamine (-NPh2) donor group were evaluated for fluorescence confocal microscopy imaging ability in live cells (MO3.13, NHLF). In sharp contrast to previously reported D-π-A dyes with alkyl amine donor (-NR2) groups (1), 3a and 3b exhibited significantly different photophysical properties and organelle selectivity. Probes 3a and 3b were nearly non-fluorescent in many polar and non-polar solvents but exhibited a bright red fluorescence (λem ≈ 630-640 nm) in stained MO3.13 and NHLF with very low probe concentrations (i.e., 200 nM). Fluorescence confocal microscopy-based co-localization studies revealed excellent lysosome selectivity from the probes 3a-3b, which is in sharp contrast to previously reported D-π-A type benzothiazolium dyes (1) with an alkyl amine donor group (-NR2) (exhibiting selectivity towards cellular mitochondria). The photostability of probe 3 was found to be dependent on the substituent (R\') attached to the quaternary nitrogen atom in the cyanine dye structure. The observed donor-dependent selectivity switching phenomenon can be highly useful in designing novel organelle-targeted fluorescent probes for live-cell imaging applications.

In this prospective observational study, we tested the feasibility and efficacy of a novel one-day PCa diagnosis path based on biparametric magnetic resonance (bpMRI) and digital pathology by fluorescence confocal microscopy (FCM). Patients aged 55-70 years scheduled for PBx due to increased PSA levels (3-10 ng/mL) and/or abnormal digitorectal examination were enrolled. All patients underwent bpMRI and PBx with immediate FCM evaluation of biopsy cores. Patients were asked to fill out a dedicated Patient Satisfaction Questionnaire. Patients\' satisfaction rates and concordance between digital pathology and standard HE evaluation were the outcomes of interest. Twelve patients completed our one-day PCa diagnosis path. BpMRI showed suspicious lesions in 7 patients. Digital pathology by FCM identified PCa in 5 (41.7%) of the 12 patients. Standard pathology confirmed the diagnosis made through digital pathology in all the cases. At a per patient level, high concordance between the methods was achieved in Gleason Grading (4 out of 5 patients). The level of agreement in the number of positive cores was lower but did not affect the choice of treatment in any of the 5 PCa cases. At a per core level, the agreement was very high for the diagnosis of anyPCa (96.2%) and csPCa (97.3%), with a k coefficient of 0.90 and 0.92, respectively (near perfect agreement). In conclusion, one-day PCa diagnosis by FCM represents a feasible, reliable, and fast diagnostic method that provides significant advantages in optimizing time and resources, leading to patients having a higher quality standard of care perception.

Ex vivo Fluorescence Confocal Microscopy (FCM) is a technique providing high-resolution images of native tissues. The method is increasingly used in surgical settings in areas of dermatology and urology. Only a few publications exist about examinations of tumors and non-neoplastic lesions of the liver. We report on the application of FCM in biopsies, surgical specimens and autopsy material (33 patients, 39 specimens) of the liver and compare the results to conventional histology. Our preliminary examinations indicated a perfect suitability for tumor diagnosis (ĸ = 1.00) and moderate/good suitability for the assessment of inflammation (ĸ = 0.4-0.6) with regard to their severity and localization. Macro-vesicular steatosis was reliably detected, micro-vesicular steatosis tended to be underestimated. Cholestasis and eosinophilic granules in granulocytes were not represented in the scans. The tissue was preserved as native material and maintained its quality for downstream histological, immunohistological and molecular examinations. In summary, FCM is a material sparing method that provides rapid feedback to the clinician about the presence of tumor, the degree of inflammation and structural changes. This can lead to faster therapeutic decisions in the management of liver tumors, treatment of hepatitis or in liver transplant medicine.

Bright red to NIR emitting cyanine probes 2-3 were synthesized in very good yields. Probes 2-3 exhibited excellent fluorescent quantum yields (ϕfl ≈ 0.1-0.4) and large Stokes shift (Δλ > 150 nm) due to efficient intramolecular charge transfer (ICT) in the conjugated π system. Organelle specificity of these probes was investigated by live cell fluorescence confocal microscopy studies. Probe 3 exhibited the ability to visualize the cell nucleus and mitochondria simultaneously in live cell samples during imaging experiments. However, in structurally modified probe 2 with different substituents (i.e., benzothiazolium vs benzothiazole), the selectivity of the probe switched entirely toward cellular lysosomes. Spectrometric DNA titration experiments were conducted to confirm the DNA/nucleus selectivity of probe 3. The study further evaluates the role of the substituent toward DNA selectivity. Probe 3 was identified as a valuable fluorescent marker to visually identify and study mitochondrial dysfunction in live cells via fluorescent confocal microscopy.