Haishan Zeng


Relevant Degree Programs


Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2021)
Imaging the dynamics of skin responses to UV challenges using non-invasive in vivo microscopy (2020)

All living cells are dynamic machines that continuously adapt and respond to their local environment. Serial analysis of cellular dynamics over time offers new insights into human skin responses to solar radiation. However, most of the previous studies are based on multiple biopsies and ex vivo analysis, which precludes the monitoring of the same sites and cells over time. In this thesis, a novel methodology based on in vivo real-time multi-modality microscopy is developed to image and quantify cellular dynamics. This imaging system integrates reflectance confocal microscopy (RCM), two-photon excitation fluorescence microscopy (TPF), and second harmonic generation microscopy (SHG) to provide complimentary tissue information. Furthermore, a method for precise serial micro-registration was created for in vivo microscopy imaging of human skin. This method solved the challenges for relocalization repeatedly and efficiently in multiple imaging sessions, enabling quantitative imaging monitoring of the same cells/tissue microstructures over a long period of days and weeks. Concurrently, an automatic segmentation algorithm was established for image analysis; epidermal-dermal junction (DEJ) was delineated in 3D, and epidermal melanin was also segmented. I applied this analysis and precise relocalization method in conjunction with in vivo multimodality multiphoton microscopy to study skin response to UV challenges. The behavior of human skin cells such as cell proliferation, melanin upward migration, blood flow dynamics, and epidermal thickness adaptation can be recorded over time, enabling quantitative cellular dynamics analysis. These results demonstrate that our label-free, automated assessments relying solely on endogenous contrast could be useful for accurate, non-invasive longitudinal skin dynamics study.

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Versatile multimodality microscopy for in vivo skin characterization and skin disease diagnosis (2020)

Various optical imaging technologies have been used to optically biopsy the skin tissue to make an early diagnosis in vivo and noninvasively. However, compared to conventional gold standard- histology, these techniques often have limitations in resolution, contrast, the field of view (FOV), depth correlation, and practicability. This thesis presents a versatile multimodality microscopy system for in vivo and non-invasive skin evaluation at the bedside by providing three-dimensional tissue information with high resolution and multi-contrast in an extended field of view. It integrates reflectance confocal microscopy, two-photon excitation microscopy, second harmonic generation microscopy, and confocal Raman spectroscopy to provide tissue information with high resolution and complementary contrast. Besides horizontal-plane imaging, fast-vertical plane imaging is realized, and it further enabled the development of a motion tolerant y-stacking volumetric imaging method for acquiring three-dimensional tissue information in an extended length. For practical clinical application, the system is integrated with a white light imaging channel using the microscopy objective to take a macro image to guide the microscopic imaging. The use of an articulated mirror arm gives the system flexibility to measure different body sites. The system is tested on healthy skin and skin cancers. With false colour coding of different image modalities, various layers of the healthy skin could be differentiated in the vertical view of the acquired volume while the horizontal plane shows tissue morphology with sub-cellular resolution. For skin cancers, three-dimensional features of the basal cell carcinoma, squamous cell carcinoma and melanoma were described. With volume large enough to cover the cancer margin, we observed the gradual changes of cellular morphology and tissue structure from normal to cancer three-dimensionally. In addition to imaging, the thesis also demonstrates the potential of using the system to do the imaging-guided precise treatment using blood vessel closure on a mouse model as an example. The system is also capable of performing imaging-guided point of interest micro-Raman spectroscopy to provide biochemical information. All the results demonstrate the success of the versatile multimodality microscopy system and its usefulness for skin evaluation and skin disease diagnosis at the bedside. Supplementary materials available at: http://hdl.handle.net/2429/77382.

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Laser Raman spectroscopy as an adjunct in vivo endoscopic device for improving early lung cancer detection (2018)

Lung cancer has an 18% five year survival rate, which is among the lowest in all cancer types. Typically, this low survival rate is due to the detection of the disease at a late stage not amendable to curative therapy. To combat this poor prognosis, many efforts have been made to detect early lung cancers before they become metastatic. Diagnosis of lung cancer involves localization and biopsy but the diagnostic accuracy of small lung lesions is currently sub-optimal. In the central airways autofluorescent bronchoscopy can localize suspicious areas for biopsy with high sensitivity but the specificity is relatively low. In the peripheral airways abnormal tissue is localized through the radial endobronchial ultrasound procedure. The diagnostic yield is relatively low in the sixty percent range, therefore, there remains a need for real time detailed information about benign versus malignant lung tissue for endoscopic diagnosis. Raman spectroscopy, a technique that utilizes the inelastic scattering of light by a molecule, has the ability to show detailed biochemical information that current procedures do not provide. Here we study the feasibility of incorporating Raman spectroscopy into standard clinical procedures. In the central airways we conduct a large scale single centre trial using Raman Spectroscopy as an adjunct to autofluorescence bronchoscopy procedures. It was found that adjunct RS can greatly improve the classification of malignant from benign/normal lesions with a high diagnostic sensitivity (90%) and good specificity (65%). In the peripheral airways, we present the design and development of a novel miniature Raman probe capable of navigating the bronchial architecture into the small airways. To our knowledge, we show the first in vivo clinical test of a fibre bundle peripheral probe and the first Raman spectra of peripheral lung cancer and normal tissue. With follow up clinical testing and validation we believe the opportunity to use Raman spectroscopy as an adjunct device in the entire lung is feasible.

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Multimodality microscopy and micro-raman spectroscopy for in vivo skin characterization and diagnosis (2013)

Accurate and early diagnosis of skin diseases will improve clinical outcomes. Visual inspection alone has limited diagnostic accuracy, while biopsy followed by histopathology examination is invasive and time-consuming. The objective is to design and develop a multimodal optical instrument that provides biochemical and morphological information on human skin in vivo. Raman spectroscopy (RS) is capable of providing biochemical information of tissues. Reflectance confocal microscopy (RCM), which generates micron-level resolution images with capability of optical sectioning, can provide refractive-index-based morphological information of the skin. Multiphoton microscopy (MPM) could simultaneously provide biochemistry-based morphological information from two-photon fluorescence (TPF) and second-harmonic-generation (SHG) images. The thesis hypothesis is that a multimodality instrument combining RS, RCM, and MPM could be developed and provide complementary information in real-time for in vivo skin evaluation and aiding non-invasive diagnosis. A confocal Raman spectroscopy system was initially developed and tested in a study on in vivo mouse skin. Spectral biomarkers (899 and 1325-1330 cm-¹) were found to differentiate tumor-bearing skin from normal skin. A RCM system was then integrated with the spectroscopy system to guide spectral measurements. Noninvasive morphological and biochemical analysis was performed on ex vivo and in vivo human skin. The system was further enhanced by adding an MPM module that can image cellular structures with TPF signals from keratin, NADH, and melanin, as well as image elastic and collaii gen fibers via TPF and SHG signals, respectively. The finalized system was utilized to noninvasively measure a cherry angioma lesion and its surrounding structures on the skin of a volunteer. Confocal Raman spectra from various regions-of-interest acquired under the guidance of MPM and RCM imaging showed different spectral patterns for blood vessels, keratinocytes, and dermal fibers. The system was also successfully used to perform imaging directed two-photon absorption based photothermolysis on ex vivo mouse skin. All the results showed positive evidence, well supporting the overall hypothesis. The developed multimodality system, capable of acquiring co-registered RCM, TPF and SHG images simultaneously at video-rate, and performing image-guided region-of-interest Raman spectral measurements of human skin in vivo, is a powerful tool for non-invasive skin evaluation and diagnosis.

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Master's Student Supervision (2010 - 2020)
Spatially selective photothermolysis guided by reflectance confocal microscopy for precise vessel closure in mouse eye limbus (2020)

Laser photocoagulation of corneal neovascularization (NV) is currently based on conventional selective photothermolysis in which the laser energy can be physically confined in the abnormal blood vessels by choosing appropriate wavelength and pulse duration, and thermally denatures the target vessels. However, because this technique primarily relies on the differential absorption between the targeted chromophore and its surroundings, it may not be sufficiently selective at the microscopic level. For achieving precise microscopic treatment, a spatially selective photothermolysis (SSP) method has been developed by our group utilizing femtosecond laser-based two-photon absorption under the guidance and monitoring of reflectance confocal microscopy (RCM). Since two-photon absorption occurs only at the focal volume of the treatment laser beam, the vascular target can be treated precisely while minimizing the collateral damage to surrounding tissues. In this thesis, we aim to develop an experimental setup that facilitates SSP treatment on the corneal edge of normal mice under RCM guidance. We hypothesize that our RCM imaging-guided two-photon absorption based photothermolysis system could be used for selective vessel closure in mouse limbus with minimal collateral damage to its surroundings. The video rate, high-resolution RCM imaging system was integrated with an adjustable field of view (FOV) white light imaging for target selection. A mouse head holder and an eye stabilizer were developed to reduce the involuntary movement of the mouse eye and achieve stable corneal imaging. The RCM imaging-guided SSP treatment system was developed and demonstrated to perform normal vessel closure in mouse limbus. Multiphoton microscopy (MPM) imaging, including two-photon fluorescence (TPF) imaging and second harmonic generation (SHG) imaging, has been performed before and after SSP treatment for assessing the tissue alternation within and outside the focal volume during laser irradiation. With all the successful results, we believe that the two-photon absorption based SSP method could provide precisely localized blood vessel closure on mice limbus and has great potential to be used for therapeutic applications in the treatment of corneal NV and other eye diseases such as ocular melanoma.

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Anti-Stokes Fluorescence Spectroscopy and Imaging for Cutaneous Porphyrin Detection (2015)

Porphyrins produced by Propionibacterium acnes represent the principal fluorophore associated with acne, and appear as orange-red luminescence under the Wood’s lamp. Assessment of acne based on Wood’s lamp (UV) or visible (VIS) light illumination is limited by photon penetration depth and has limited sensitivity for earlier stage lesions. Inducing fluorescence with near infrared (NIR) excitation may provide an alternative way to assess porphyrin-related skin disorders. The objectives of this thesis are (1) to design and develop an optical instrument that perform anti-Stokes fluorescence spectroscopy and imaging measurements under continuous wave CW laser excitation as well as multiphoton fluorescence spectroscopy and imaging under femtosecond (fs) pulsed laser excitation; and (2) using this system for regular (Stokes) fluorescence measurements to evaluate and compare sebum-associated porphyrin fluorescence properties.A NIR multi-modality fluorescence imaging system with fluorescence spectroscopy capability was constructed. The switchable output from a tunable Ti : Sapphire femtosecond (fs) laser (720-950nm) or a (CW) laser (785nm) was scanned over the objective lens. Co-registered confocal imaging and fluorescence (anti-stokes fluorescence & two-photon fluorescence) imaging were acquired in video rate. Under 785 nm CW laser excitation PpIX powder exhibited anti-Stokes fluorescence with an intensity that was linearly dependent on the excitation power and a red spectral emission of 650-720 nm, while the fs laser excited two-photon excitation fluorescence showed a quadratic dependency. Anti-Stokes fluorescence of psoriasis scale image and ex vivo human nasal skin, skin surface smears from the nose, and ex vivo sebum secretions was obtained by the same system configuration. Regular (Stokes) fluorescence was presented under UV and visible light excitation on ex vivo nasal skin and sebum secretions from non-inflamed acne, but not on skin surface smears from the nose or sebum secretions from inflamed acne. In conclusion, anti-Stokes fluorescence under NIR CW excitation is more sensitive and specific for porphyrins than UV or visible light-excited regular (Stokes) fluorescence and fs laser-excited multi-photon fluorescence. The anti-Stokes fluorescence of porphyrins within sebum could potentially be applied to detecting and targeting acne lesions for treatment via fluorescence image guidance.

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A raman cell based on hollow core photonic crystal fibre for human breath analysis (2013)

Lung cancer is the top cancer killer in Canada and North America. Current lung cancerdetection tools involving X-ray, CT and bronchoscopy are relatively time-consuming andcostly. Breath analyses done by mass spectrometry have shown that certain endogenousvolatile organic compounds (VOCs) are related to lung cancer and revealed the potential ofbreath analysis for lung cancer detection. But mass spectrometry is costly and has slowturnaround times. Raman spectroscopy is a promising candidate for breath analysis because itcan offer unique fingerprint-type signals for molecular identification. Hollow core-photoniccrystal fibre (HC-PCF) is a novel light guide which allows light to be guided in a smallhollow core and it can be filled with a gaseous sample (i.e., human breath) for spectralanalysis. Our objective is to develop a simple, cost-effective and non-invasive tool based onRaman spectroscopy for breath analysis and potentially lung cancer screening.A Raman-gas analyzer was designed, based on photonics technology. A gas supply systemwas built to provide a sealed environment for the loading and unloading of gaseous samples.A laser source at 785 nm was used as the pump for molecular excitation. Stokes Ramansignals generated in the hollow core of the HC-PCF can be guided by collection optics andanalyzed by a Raman spectrometer. Raman spectra have been obtained successfully from air,reference gases (hydrogen gas, oxygen gas, carbon dioxide gas), and human breath. The limitof detection of the system was found to be approximately 15 parts per million by CO²concentration in the ambient air, characterized by the Raman peaks at 1286 cm₋¹ and 1388cm-1. This is more than a 100-fold improvement over the recently reported detection limit with a reflective capillary fibre-based Raman cell. Furthermore the detection limit can be further improved by changes to the optical configurations, optimizing the interaction length of the HC-PCF and possible pre-concentration method to enhance signal-to-noise ratio. This work demonstrated a working prototype of a simple, compact, and cost-effective Raman-gas analyzer based on hollow core photonic crystal fibre, which could potentially be used for lung cancer screening through breath analysis.

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