Human bladder cancer diagnosis using Multiphoton microscopy

Department of Biochemistry, Weill Cornell Medical College, New York, NY.
Proceedings - Society of Photo-Optical Instrumentation Engineers 02/2009; 7161. DOI: 10.1117/12.808314
Source: PubMed


At the time of diagnosis, approximately 75% of bladder cancers are non-muscle invasive. Appropriate diagnosis and surgical resection at this stage improves prognosis dramatically. However, these lesions, being small and/or flat, are often missed by conventional white-light cystoscopes. Furthermore, it is difficult to assess the surgical margin for negativity using conventional cystoscopes. Resultantly, the recurrence rates in patients with early bladder cancer are very high. This is currently addressed by repeat cystoscopies and biopsies, which can last throughout the life of a patient, increasing cost and patient morbidity. Multiphoton endoscopes offer a potential solution, allowing real time, non-invasive biopsies of the human bladder, as well as an up-close assessment of the resection margin. While miniaturization of the Multiphoton microscope into an endoscopic format is currently in progress, we present results here indicating that Multiphoton imaging (using a bench-top Multiphoton microscope) can indeed identify cancers in fresh, unfixed human bladder biopsies. Multiphoton images are acquired in two channels: (1) broadband autofluorescence from cells, and (2) second harmonic generation (SHG), mostly by tissue collagen. These images are then compared with gold standard hematoxylin/eosin (H&E) stained histopathology slides from the same specimen. Based on a "training set" and a very small "blinded set" of samples, we have found excellent correlation between the Multiphoton and histopathological diagnoses. A larger blinded analysis by two independent uropathologists is currently in progress. We expect that the conclusion of this phase will provide us with diagnostic accuracy estimates, as well as the degree of inter-observer heterogeneity.

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Available from: Mohammed Akhtar, Oct 05, 2015
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    • "However, light penetration depth is limited (0.1 to 1 mm) such that firststage bladder cancer cannot be distinguished from the muscle layer. Early and advanced cancer conditions can be detected, but the cancer stages cannot be identified accurately [7]. NBI system is used to detect TCC in the upper urinary tract using two specific light sources (415 and 540 nm) accompanied by the endoscope. "
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    ABSTRACT: Bladder cancer presents a spectrum of different diatheses. A precise assessment for individualized treatment depends on the accuracy of the initial diagnosis. Detection relies on comprehensive and accurate white light cystoscopy. White light cystoscopy has limitations in addition to its invasive nature and the potential risks related to the method. These limitations include difficulties in flat lesion detection, precise tumor delineation to enable complete resection, inflammation and malignancy differentiation, and grade and stage determination. The resolution of these problems depends on the surgeon’s ability and experience with available technology for visualization and resection. In this study, we used multi-spectral imaging technology combined with phase contrast microscopy to analyze bladder cancer cells (BCCs) at various stages using a single-cell array chip. We found from the spectral characteristics of single cell that the cell spectra at the different cancer stages demonstrate a change in the cell’s composition. We cultured 419 normal and diseased bladder cells (BCs). We used principal component analysis and a principal component score map to distinguish the different cancer stages. Diagnosis sensitivity and specificity of this method were 85.7% and 90.2% in 119 stage 0 (normal) cells, 84.3% and 90.8% in 79 stage 2 cancer cells, 87.6% and 92.4% in 151 stage 3 BCCs, and 85.3% and 91.2% in 70 stage 4 BCCs, respectively.
    IEEE Journal of Selected Topics in Quantum Electronics 05/2014; 20(3):68800808. DOI:10.1109/JSTQE.2013.2279804 · 2.83 Impact Factor
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    • "However,, one potential drawback of this approach is the lack of a true morphologic assessment of ganglia in the tissue, since the acquired signal is a spectral wave form rather than an image. MPM is a novel imaging modality that has been investigated for ex-vivo analysis of fresh tumor specimens from the human bladder [18], prostate [19] and the testis [8]. Fig. 2 Combined MPM image from two detection channels combining the SHG signal originating from collagen (360–400 nm, color coded red), and autofluorescence originating from NADH and FAD in cells and elastin in connective tissue (420–490 nm, color coded green). "
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    ABSTRACT: The distribution of ganglion cells in the transition zone of Hirschsprung Disease (HD) colons is extremely variable. Determining the resection margin based on intraoperative biopsies may be imprecise. Multiphoton microscopy (MPM) is a novel imaging technology with the ability to visualize tissues in real time. In this study, we evaluate the potential of MPM to quantify ganglion cells in a murine model of HD. After IACUC approval, formalin-fixed colons from 7 wild type (WT) and 6 Endothelin Receptor B gene (EdnrB) homozygous knockout (KO) mice with distal colonic aganglionosis were assessed by MPM for the presence of myenteric ganglion cells. MPM images were captured starting from the anus progressing proximally at 5mm intervals. Hematoxylin and eosin (H&E) stained biopsies of the imaged were correlated with MPM findings. WT specimens showed normal myenteric plexus ganglia throughout the examined colon. In contrast, distal colons of EdnrB KO animals were devoid of ganglia up to 10mm from the anus. Ganglion cells were visible starting at 20-30mm proximal to the anus. The density of ganglion cells seen by MPM and histology correlated well. MPM can clearly identify the myenteric plexus ganglia in both WT and KO mouse colons. Comparison with the H&E-stained sections showed reproducible correlation. MPM-based real-time imaging of the myenteric plexus may become a useful intraoperative decision-making tool in the future.
    Journal of Pediatric Surgery 06/2013; 48(6):1288-1293. DOI:10.1016/j.jpedsurg.2013.03.025 · 1.39 Impact Factor
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    • "Several recent studies have shown significant progress towards the development of techniques such as confocal microendoscopy [8], [9], [10], allowing acquisition of microscopic images of in-situ tissues including in the GI tract. Additional studies have also demonstrated the promise of two-photon microscopy for imaging cancer in skin, lung, and bladder [11], [12], [13]. These studies suggest that our hyperspectral microscopy technique has significant potential for clinical translation, either for in-situ intraoperative, or real-time bedside ‘instant histology’. "
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    ABSTRACT: Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue's composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents. In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition. These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative 'instant histology' of fresh tissue biopsies.
    PLoS ONE 05/2011; 6(5):e19925. DOI:10.1371/journal.pone.0019925 · 3.23 Impact Factor
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