Abstract 4884: Toward an annotated digital multiphoton microscopy (MPM) histology atlas of fresh human and rodent tissue for guidance during cancer diagnosis and margin assessments
Authors: Mukherjee Sushmita , Jain Manu , Robinson Brian D. , Sterling Joshua , Scherr Douglas S. , Tewari Ashutosh K. , Maxfield Frederick R. , Zipfel Warren R. , Webb Watt W. 
Published: June 2012

Abstract
Hematoxylin and eosin (H&E)-stained histology from thin sections of tissue obtained from preserved (frozen or formalin-fixed) specimens is the current gold standard for all histopathological diagnoses and intra-surgical margin assessments. While these methods are highly reliable, they have lengthy time requirements (processing, sectioning, staining, and reading by pathologists). Although efforts at digitization of glass slides are in progress, most pathologists still read analog slides; therefore, automated morphometry and real time online consultations are rare. Furthermore, the 2-dimensional nature of histology slides precludes assessment of 3-dimensional tissue architecture without time consuming serial sectioning.
Now, however, Multiphoton microscopy (MPM), a nonlinear imaging technology, allows nearly instant imaging of fresh (unpreserved, unsectioned, and unstained) tissue based on spectrally resolved intrinsic tissue emission (ITE) signals, allowing the generation of 3-dimensional histology of the tissue at sub-micron lateral resolution, and up to 0.5 mm below the surface. The main components of ITE are: (1) autofluorescence arising from various cell cytoplasm components and elastin fibers; and (2) Second Harmonic Generation (SHG), a nonlinear scattering signal from collagen bundles and oriented microtubules. Using a single excitation wavelength and collecting emission signals using wavelength bandpass filters or spectroscopes, SHG and various autofluorescence components can be separately acquired and analyzed, as well as color coded and merged for easy visualization.
Using such approaches, we have analyzed ex vivo tissue from human diagnostic and surgical biopsies, and from sections of excised human and rodent (rats and mice) organs (bladder, prostate, seminal vesicles, vasa deferentia, kidney, testis, colon, thyroid, tonsil, skin, bone and cartilage). In all cases, relevant tissue structures, including cells, collagen, elastin, fat, nerves and blood vessels have been identified. All specimens imaged by MPM have been subsequently subjected to H&E histopathology, and scanned images from the slides have been archived.
Beginning with transurethral biopsy of bladder tumor (TURBT) specimens, we have begun the generation of a pathologist-annotated digital atlas containing both the MPM images and the corresponding H&E histopathology. Blinded diagnostic accuracy studies involving several pathologists and in some cases non-pathologists (e.g., surgeons in the context of margin assessments) are currently in progress, with encouraging preliminary results. Extension of MPM diagnostics to additional organs is being explored successfully by application to selected transgenic mice and to diseased veterinary animal subjects at the College of Veterinary Medicine at Cornell University.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4884. doi:10.1158/1538-7445.AM2011-4884