Jianhua Zhao

Real-time Raman Spectroscopy for In Vivo Skin Cancer Diagnosis

Raman spectroscopy is a noninvasive optical technique capable of measuring vibrational modes of biomolecules within viable tissues. In this study, we evaluated the application of an integrated real-time system of Raman spectroscopy for in vivo skin cancer diagnosis. Benign and malignant skin lesions (n = 518) from 453 patients were measured within 1 second each, including melanomas, basal cell carcinomas, squamous cell carcinomas, actinic keratoses, atypical nevi, melanocytic nevi, blue nevi, and seborrheic keratoses. Lesion classification was made using a principal component with general discriminant analysis and partial least-squares in three distinct discrimination tasks: skin cancers and precancers from benign skin lesions [receiver operating characteristic (ROC) = 0.879]; melanomas from nonmelanoma pigmented lesions (ROC = 0.823); and melanomas from seborrheic keratoses (ROC = 0.898). For sensitivities between 95% and 99%, the specificities ranged between 15% and 54%. Our findings establish that real-time Raman spectroscopy can be used to distinguish malignant from benign skin lesions with good diagnostic accuracy comparable with clinical examination and other optical-based methods.

Development and preliminary results of an endoscopic Raman probe for potential in vivo diagnosis of lung cancers

A near-infrared Raman system was developed to collect real-time in vivo human lung spectra. The excitation light and the emission were guided to and from the tissue surface by a reusable fiber catheter passed down the instrument channel of a bronchoscope. Two-stage filtering was used to reduce laser noise, fluorescence, and Raman emissions from the fibers. A second fiber bundle guided the emission to a spectrometer where the fibers, in a round packing geometry, were spread out to form a parabolic arc that improved the signal-to-noise ratio 20-fold, facilitating real-time spectral measurements. Preliminary clinical tests show that clear and reliable Raman spectra can be obtained.

Full range characterization of the Raman spectra of organs in a murine model

Raman spectroscopy is a minimally-invasive optical technique with great potential for in vivo cancer detection and disease diagnosis. However, there is no systematic study of the Raman spectra from different organs to date. We measured and characterized the Raman spectra eighteen naïve mouse organs in a broad frequency range of 700 to 3100 cm⁻¹. The peaks of generic proteins and lipids appeared in Raman spectra of all organs. Some organs like bone, teeth, brain and lung had unique Raman peaks. The autofluorescence was strong in liver, spleen, heart, and kidney. These results suggest that organ specific Raman probe design and specific data processing strategies are required in order to get the most useful information.

In vivo video rate multiphoton microscopy imaging of human skin

We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27 frames per second with 256 pixels × 256 pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.

RAMAN SPECTROSCOPY FOR IN VIVO TISSUE ANALYSIS AND DIAGNOSIS, FROM INSTRUMENT DEVELOPMENT TO CLINICAL APPLICATIONS

Raman spectroscopy is a noninvasive, nondestructive analytical method capable of determining the biochemical constituents based on molecular vibrations. It does not require sample preparation or pretreatment. However, the use of Raman spectroscopy for in vivo clinical applications will depend on the feasibility of measuring Raman spectra in a relatively short time period (a few seconds). In this work, a fast dispersive-type near-infrared (NIR) Raman spectroscopy system and a skin Raman probe were developed to facilitate real-time, noninvasive, in vivo human skin measurements. Spectrograph image aberration was corrected by a parabolic-line fiber array, permitting complete CCD vertical binning, thereby yielding a 16-fold improvement in signal-to-noise ratio. Good quality in vivo skin NIR Raman spectra free of interference from fiber fluorescence and silica Raman scattering can be acquired within one second, which greatly facilitates practical noninvasive tissue characterization and clinical diagnosis. Currently, we are conducting a large clinical study of various skin diseases in order to develop Raman spectroscopy into a useful tool for non-invasive skin cancer detection. Intermediate data analysis results are presented. Recently, we have also successfully developed a technically more challenging endoscopic Laser-Raman probe for early lung cancer detection. Preliminary in vivo results from endoscopic lung Raman measurements are discussed.

Combining field imaging endoscopy with point analysis spectroscopy for improving early lung cancer detection

We propose to combine field imaging endoscopy with point spectral analysis for improving the overall diagnostic accuracy in clinical lung cancer detection. For this purpose, we developed an integrated endoscopy system that uses autofluorescence imaging and white light reflectance imaging to obtain high diagnostic sensitivity, while at the same time uses non-contact point reflectance/fluorescence spectroscopy to reduce false positive biopsies, thus, achieve high diagnostic specificity. A pilot clinical test on 22 lung patients demonstrated that using this system the malignant lung lesions can be differentiated from the benign lesions with both diagnostic sensitivity and specificity of better than 80%. To further reduce the number of false positive diagnosis and allow even higher diagnostic accuracies, we have also developed an endoscopic laser Raman probe for in vivo real-time biochemical analysis of the suspicious tissue areas identified by the field imaging modalities (white light imaging and autofluorescence imaging). Preliminary Raman spectroscopy results will be reported at the conference.

A modular Raman microspectroscopy system for biological tissue analysis

Raman spectroscopy has been used as a sensitive tool for studying biological tissue and evaluating disease. In many applications, microscopic level resolution spectral analysis is desirable. And this has been performed mostly by expensive commercial confocal micro-Raman systems. In this research, we present a simple method for building an economical and modular Raman microspectroscopy system that combines a microscope with a Raman spectrometer using an optical fiber bundle. The bundle with a circular collection end is positioned at an image plane of the microscope to collect Raman signals from the interested micro-location on the sample. The light delivery end is specially configured so that its 37 fibers are arranged along a straight line to fit into the spectrometer entrance slit. This configuration improves light collection efficiency and maintains high spectral resolution. To battle the great background autofluorescence and Raman signals that could originate from the microscope slides and optics due to the non-confocal set-up of our simplified system, conventional normal-incident illumination is replaced by oblique illumination at 45° degrees and the microscope slides are coated with gold. We demonstrated the usefulness of the system by measuring micro-Raman spectra from different skin layers on vertical sections of normal skin tissue samples.

Monitoring Changes of Skin Raman Spectra Induced by Ultrafast Laser Irradiation: a Porcine Skin Model Study

A customized confocal Raman spectroscopy system was used to measure the Raman spectra of porcine skin irradiated by high power ultrafast laser. The changes in Raman spectra indicate that the collagen was denatured and carbonized after high power ultrafast laser irradiation.

Raman spectroscopy for in vivo tissue analysis and diagnosis at the macro- and microscopic levels

We report various technologies developed in our lab for in vivo Raman spectroscopy and related clinical applications. This includes macroscopic probes and endoscopy catheters that interrogate millimeter-scale tissue volumes for skin and lung cancer detection and a confocal microscopy system for depth-resolved Raman measurements of the skin in vivo.

Precise Spatially Selective Photothermolysis Using Modulated Femtosecond Lasers and Real-time Multimodal Microscopy Monitoring.

The successful application of lasers in the treatment of skin diseases and cosmetic surgery is largely based on the principle of conventional selective photothermolysis which relies strongly on the difference in the absorption between the therapeutic target and its surroundings. However, when the differentiation in absorption is not sufficient, collateral damage would occur due to indiscriminate and nonspecific tissue heating. To deal with such cases, we introduce a novel spatially selective photothermolysis method based on multiphoton absorption in which the radiant energy of a tightly focused near-infrared femtosecond laser beam can be directed spatially by aiming the laser focal point to the target of interest. We construct a multimodal optical microscope to perform and monitor the spatially selective photothermolysis. We demonstrate that precise alteration of the targeted tissue is achieved while leaving surrounding tissue intact by choosing appropriate femtosecond laser exposure with multimodal optical microscopy monitoring in real time.