Anthony M. D. Lee

Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two-photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique.

Spectroscopic characterization and microscopic imaging of extracted and in situ cutaneous collagen and elastic tissue components under two‐photon excitation

Background/purposes: Understanding the two‐photon excitation spectral characteristics and microscopic morphology of cutaneous collagen and elastic tissue components is important for applying multiphoton microscopy (MPM) in basic skin biology research and for clinical diagnosis.

Frontiers in bronchoscopic imaging

Bronchoscopy is a minimally invasive method for diagnosis of diseases of the airways and the lung parenchyma. Standard bronchoscopy uses the reflectance/scattering properties of white light from tissue to examine the macroscopic appearance of airways. It does not exploit the full spectrum of the optical properties of bronchial tissues. Advances in optical imaging such as optical coherence tomography (OCT), confocal endomicroscopy, autofluorescence imaging and laser Raman spectroscopy are at the forefront to allow in vivo high‐resolution probing of the microscopic structure, biochemical compositions and even molecular alterations in disease states. OCT can visualize cellular and extracellular structures at and below the tissue surface with near histological resolution, as well as to provide three‐dimensional imaging of the airways. Cellular and subcellular imaging can be achieved using confocal endomicroscopy or endocytoscopy. Contrast associated with light absorption by haemoglobin can be used to highlight changes in microvascular structures in the subepithelium using narrow‐band imaging. Blood vessels in the peribronchial space can be displayed using Doppler OCT. Biochemical compositions can be analysed with laser Raman spectroscopy, autofluorescence or multispectral imaging. Clinically, autofluorescence and narrow‐band imaging have been found to be useful for localization of preneoplastic and neoplastic bronchial lesions. OCT can differentiate carcinoma in situ versus microinvasive cancer. Endoscopic optical imaging is a promising technology that can expand the horizon for studying the pathogenesis and progression of airway diseases such as COPD and asthma, as well as to evaluate the effect of novel therapy.

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.

Bronchial thermoplasty in asthma: 2-year follow-up using optical coherence tomography

Bronchial thermoplasty (BT) is a novel, nonpharmacological procedure for treatment of severe asthma. Recently, the Asthma Intervention Research 2 clinical trial demonstrated asthmatics had fewer hospitalisations following BT, which persisted 5 years after therapy [1]. However, it is well recognised that asthma is a heterogeneous disease with distinct asthma phenotypes and, not surprisingly, not all asthmatics in that trial benefited from BT [2]. Optical coherence tomography small airway imaging may be used for better patient phenotyping and selection for BT http://ow.ly/O0Cm2

A high-efficiency fiber-based imaging system for co-registered autofluorescence and optical coherence tomography

We present a power-efficient fiber-based imaging system capable of co-registered autofluorescence imaging and optical coherence tomography (AF/OCT). The system employs a custom fiber optic rotary joint (FORJ) with an embedded dichroic mirror to efficiently combine the OCT and AF pathways. This three-port wavelength multiplexing FORJ setup has a throughput of more than 83% for collected AF emission, significantly more efficient compared to previously reported fiber-based methods. A custom 900 µm diameter catheter ‒ consisting of a rotating lens assembly, double-clad fiber (DCF), and torque cable in a stationary plastic tube ‒ was fabricated to allow AF/OCT imaging of small airways in vivo. We demonstrate the performance of this system ex vivo in resected porcine airway specimens and in vivo in human on fingers, in the oral cavity, and in peripheral airways.

A Method for accurate in vivo micro-Raman spectroscopic measurements under guidance of advanced microscopy imaging

The movement from the subjects during in vivo confocal Raman spectral measurements could change the measurement volume, leading to non-specific signals and inaccurate interpretation of the acquired spectrum. Here we introduce a generally applicable method that includes (1) developing a multimodal system to achieve real-time monitoring of every spectral measurement with reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) imaging; (2) performing region-of-interest measurement by scanning an area of the tissue during spectral acquisition. The developed method has been validated by measuring different micro-structures of in vivo human skin. Our results demonstrated great consistency between RCM images and confocal Raman spectra. The superior quality of the images and spectra allows us to derive blood flow velocity and blood glucose level. We believe this method is valuable for realizing accurate microscopic spectral measurement and have great potential to be adapted into clinic to achieve non-invasive measurement of important biological parameters.

Improving skin Raman spectral quality by fluorescence photobleaching.

Here we present a method for improving Raman spectroscopy signal-to-noise ratio (SNR) based on fluorescence photobleaching. Good SNR is essential to obtain biochemical information about biological tissues. Subtracting high levels of tissue autofluorescence background is a major challenge in extracting weak Raman signals. We found that pre-exposure to laser light significantly reduces tissue autofluorescence, but minimally affects Raman signals, allowing subsequent acquisition of high-SNR Raman spectra. We demonstrated this method with in vivo Raman spectral measurements of human skin. This method will benefit clinical skin Raman measurements of body sites with high autofluorescence background such as the forehead and nose.

In vivo lung microvasculature visualized in three dimensions using fiber-optic color Doppler optical coherence tomography

Abstract. For the first time, the use of fiber-optic color Doppler optical coherence tomography (CDOCT) to map in vivo the three-dimensional (3-D) vascular network of airway segments in human lungs is demonstrated. Visualizing the 3-D vascular network in the lungs may provide new opportunities for detecting and monitoring lung diseases such as asthma, chronic obstructive pulmonary disease, and lung cancer. Our CDOCT instrument employs a rotary fiber-optic probe that provides simultaneous two-dimensional (2-D) real-time structural optical coherence tomography (OCT) and CDOCT imaging at frame rates up to 12.5 frames per second. Controlled pullback of the probe allows 3-D vascular mapping in airway segments up to 50 mm in length in a single acquisition. We demonstrate the ability of CDOCT to map both small and large vessels. In one example, CDOCT imaging allows assignment of a feature in the structural OCT image as a large (∼1  mm diameter) blood vessel. In a second example, a smaller vessel (∼80  μm diameter) that is indistinguishable in the structural OCT image is fully visualized in 3-D using CDOCT.

Wide-field in vivo oral OCT imaging.

We have built a polarization-sensitive swept source Optical Coherence Tomography (OCT) instrument capable of wide-field in vivo imaging in the oral cavity. This instrument uses a hand-held side-looking fiber-optic rotary pullback catheter that can cover two dimensional tissue imaging fields approximately 2.5 mm wide by up to 90 mm length in a single image acquisition. The catheter spins at 100 Hz with pullback speeds up to 15 mm/s allowing imaging of areas up to 225 mm(2) field-of-view in seconds. A catheter sheath and two optional catheter sheath holders have been designed to allow imaging at all locations within the oral cavity. Image quality of 2-dimensional image slices through the data can be greatly enhanced by averaging over the orthogonal dimension to reduce speckle. Initial in vivo imaging results reveal a wide-field view of features such as epithelial thickness and continuity of the basement membrane that may be useful in clinic for chair-side management of oral lesions.