Blank. ENDOSCOPIC APPROACHES IN SURGERY Watt W. Webb, Applied Physics, Cornell University, Ithaca NY, USA A prototype for Cornell Collaboration of CU-Ithaca.

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ENDOSCOPIC APPROACHES IN SURGERY

Watt W. Webb, Applied Physics, Cornell University, Ithaca NY, USA

•A prototype for Cornell Collaboration of CU-Ithaca with CU-Weill.

•A model for ‘Academic Incubator’ to Technology Transfer Opportunities.

•How does it work: Multiphoton Microscopy (MPM) images fluoresce and SHG with submicroscopic three-

dimensional resolution.

•WE FLUORESCE by MPM and Generate Second Harmonic (SHG), which micro-image tissue interior in real time.

•MPM images appear to be equivalent to pathologists’ subsequent H&E stained images of fixed tissue biopsies.

Single-photon excitation (488 nm)

Two-photon excitation (900 nm)

Because simultaneous absorption of two photons is required for excitation, the emission depends on the square of the intensity, rather than being linearly proportional.

Significant excitation occurs only in the focal volume (at “normal” imaging intensities - mW range).

F

Ix

Femtosecond pulse train at 80 MHz

Two Photon Excitation Is Spatially Localized

0 5 10 15 20 25

WE FLUORESCE - Non-linearlyIntrinsic tissue (MPE) fluorescence (ITF) and Second Harmonic Generation (SHG)

Which components emit and where? Spectroscopy & Image, here in pseudocolor.

Information comes with these signals, including tissue environment, aggregation state, molecular orientation, etc. that is useful for medical diagnostics.How these signals can be detected most efficiently and least obtrusively is our research.

There are appropriate biophysical and biomedical applications we are developing.

Arteriole HeartChoroid Plexus / Pineal Gland

Elastin

Retinoids

NADHCollagenSHG

IndoleaminesCollagen SHG

In Vivo Imaging of Surface Epithelia in Ovary of Conditional P53/rb Knockouts (Mice)

Images: Rebecca M. Williams, Andrea Flesken-Nikitin, Alexander Nikitin, Watt W. Webb and Warren R. Zipfel, Cornell University.

epithelium

breathing artifact

corpus luteum

20 m

Blue autofluorescence (retinoids in hormone producing cells) in transformed ovary.

Ovarian Cancer Detection

MPM Images (A, C, E) compared with conventional hemotoxylin-eosin stained histological pathology (B, D, F).

Normal ovary A vs. BEarly Dysplastic Changes C vs. DInvasive Tumor E vs. F

Note monomorphous polygonal tumor cells at arrow.

Calibration bar: (B -E), 50 m.

Multiphoton Image Pathology Image

Images by Rebecca M. Williams, Douglas Scherr*, Warren R. Zipfel and Watt W. Webb, Cornell University and *Weill Medical College, 2005.

Human Bladder Tumor Biopsy (ex vivo)

Blue autofluorescent cells of low-grade transitional cell carcinoma stage TA, grade 1, showing rounded homogeneous cell array of low nuclear/cytoplasmic ratio and some disorganization of cell layer structure. Some longer wavelength punctate autofluorescent spots not yet identified. Color contrast somewhat degraded by 24 hr delay during shipping after biopsy. Images comparable to Hematoxylin & Eosin pathologist’s stained images. (All scale bars, 20 m)

(A) 20 microns deep. Blue pseudocolor is collagen second harmonic signal; yellow/white pseudocolor represents epithelial tissue of the bladder wall. (B) 120 microns deep. Epithelial cells can be clearly seen in the upper left-hand side. (C) Higher zoom image showing the epithelia and umbrella cells (large yellow cells on the lower edge).

Multiphoton Images of a Mouse Urinary Bladder (ex vivo)

A B C

Images by Rebecca M. Williams, Warren R. Zipfel and Watt W. Webb, Cornell University, 2005.

Scale bar, 10 m Scale bar, 10 m Scale bar, 40 m

CA3

Dentate gyrus

Mossy fibers (red)

Zinc (TSQ) in greenSHG in red

SHG and zinc in the rat hippocampusKarl Kasischke, Harsh Vishwasrao and Dan Dombeck

Dombeck, D. A., K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman and W. W. Webb, "Second Harmonic Generation Microscopy of Uniformly Oriented Microtubules in Native Brain Tissue," PNAS 100, 7081-7086, 2003.

We could see Amyloid deposits with 2P-excited intrinsic fluorescence and polarized microtubules with SHG

1. No obvious alteration in the density and intensity of polarized MTs.

2. At least 6 examples of polarized MTs going through the peripheral regions of deposit.

3. Plaques do not generate SHG.

100m

Inappropriately Cross-linked Molecules (often associated with disease states)

Oxidized monoamines (5HT)

SHG??•Arteriosclerotic plaque•Advanced glycation products•Crystalline and prion deposits

Autofluorescence

(In collaboration with Brad Hyman’s Lab, Harvard Medical School)

NFT lipofuscin

Lipofuscin, Neurofibrillary tangles (NFT’s)

PHF-tau Ab

US Patent 6,839,586Multiphoton Fiber Optic Based Endoscopic Microscope

B) GRIN Lenses for Deep Brain Imaging

Levene, M. J., D. A. Dombeck, R. P. Molloy, K. A. Kasischke, R. M. Williams, W. R. Zipfel and W. W. Webb, "In vivo multiphoton microscopy of deep brain tissue," Journal of Neurophysiology (Online), 10.1152/jn.01007.2003, (December 10, 2003)

Levene, M. J., D. A. Dombeck, R. M. Williams, J. Skoch, G. A. Hickey, K. A. Kasischke, R. P. Molloy, M. Ingelsson, E. A. Stern, J. Klucken, B. J. Backskai, W. R. Zipfel, B. T. Hyman and W. W. Webb. "In vivo multiphoton microscopy of deep tissue with gradient index lenses," Photonics West 2004, San Jose, CA, Proceedings of SPIE, 2004

Cornell Ithaca: Technology Developments•Watt Webb MPM and Biophysics•Harold Craighead Nanobiotechnology•Chris Xu Ultrafast Optics Developments, Biophysics•Alex Gaeta Non-linear Optics – Fibers for Mode-locked Lasers•Rebecca Williams MPM Imaging of Tissue and Cancer•Warren Zipfel MPM Resource Director – Instrumentation, ImagingWeill Medical: Medical MPM Endoscopic Methods (partial list)•Doug Sherr, MD Director of Urological Surgery, Coordinator•Fred Maxfield Chairman Biochemistry, Microscopic Facility

Coordinator, Coordinator of MPM imaging, Coordination with Pathology

•Caputo, Thomas A. OB/GYN oncology•Fahey, Thomas J. Diagnosis and Differentiation of Thyroid Nodules•Jacobson, Ira M. Gastroenterology and Hepatology•Kaner, Robert J. Lung Surgery and Biopsies •Milsom, Jeffrey W. Minimal Access Colon and Rectal Surgery•Pochapin, Mark B. Colon Cancer•Rodeo, Scott A. Orthopedic Surgery•Schwartz, Theodore H. Optical imaging of epilepsy •Tewari, Ashutosh K. Robotic, Nerve-sparing Prostate Surgery•Yankelevitz, David F. Lung Biopsy and Nodule Diagnosis

Initial Collaborators in Developing MPM Endoscopy

Development Stages: Ithaca•Femtosecond lasers OK, plus advancing developments•Optical Fibers for fs lasers [OK, our discovery] plus narrow band, smaller fibers•Laser scanning tools OK for Colonoscope; develop smaller•MPM imaging of cancer in animal models: Williams, Nikitin, Zipfel•Analyze excitation and emission spectral responses of tissues for optical fiber biopsy analyses: Ithaca and Weill•Applicability of MPM Endo in Medical Specialties: Webb with Weill, Plus•Prototype Instrument Developments: Ithaca

Translation to Clinical Setting Stages•Testing in Human Applications: Weill Med MD’s•Development of Proof Test Atlas of Images: Pathology and Maxfield, Weill•Endoscopy Product Developments: All Collaborating•Technology Transfer and Business Strategy: CCTEC +?

Some Technological Background Issues

“Lasers Set Cells Aglow for a Biopsy Without the Knife”

Article by Anne Eisenberg

NY Times, June 26, 2003, Illustration by Bob Scott, copyright NY Times, 2003.

Lasers set Cells Aglow

Zipfel, W.R., R.M. Williams, R.H. Christie, A.Y. Nikitin, B.T. Hyman and W W. Webb, PNAS 100(12), 7075-7080, 2003

Dombeck, D.A., K.A. Kasischke, H.D. Vishwasrao, M. Ingelsson, B.T. Hyman and W.W. Webb, PNAS 100(12), 7081-7086, 2003

The End