Imaging of normal and pathologic joint synovium using nonlinear optical microscopy as a potential diagnostic tool.

An estimated 1.3 million people in the United States suffer from rheumatoid arthritis (RA). RA causes profound changes in the synovial membrane of joints, and without early diagnosis and intervention, progresses to permanent alterations in joint structure and function. The purpose of this study is to determine if nonlinear optical microscopy (NLOM) can utilize the natural intrinsic fluorescence properties of tissue to generate images that would allow visualization of the structural and cellular composition of fresh, unfixed normal and pathologic synovial tissue. NLOM is performed on rabbit knee joint synovial samples using 730- and 800-nm excitation wavelengths. Less than 30 mW of excitation power delivered with a 40×, 0.8-NA water immersion objective is sufficient for the visualization of synovial structures to a maximum depth of 70 µm without tissue damage. NLOM imaging of normal and pathologic synovial tissue reveals the cellular structure, synoviocytes, adipocytes, collagen, vascular structures, and differential characteristics of inflammatory infiltrates without requiring tissue processing or staining. Further study to evaluate the ability of NLOM to assess the characteristics of pathologic synovial tissue and its potential role for the management of disease is warranted.

In vivo three-dimensional imaging of normal tissue and tumors in the rabbit pleural cavity using endoscopic swept source optical coherence tomography with thoracoscopic guidance.

The purpose of this study was to develop a dynamic tunable focal distance graded-refractive-index lens rod-based high-speed 3-D swept-source (SS) optical coherence tomography (OCT) endoscopic system and demonstrate real-time in vivo, high-resolution (10-microm) imaging of pleural-based malignancies in an animal model. The GRIN lens-based 3-D SS OCT system, which images at 39 fps with 512 A-lines per frame, was able to capture images of and detect abnormalities during thoracoscopy in the thoracic cavity, including the pleura, chest wall, pericardium, and the lungs. The abnormalities were confirmed by histological evaluation and compared to OCT findings. The dynamic tunable focal distance range and rapid speed of the probe and SS prototype OCT system enabled this first-reported application of in vivo 3-D thoracoscopic imaging of pleural-based malignancies. The imaging probe of the system was found to be easily adaptable to various sites within the thoracic cavity and can be readily adapted to other sites, including rigid airway endoscopic examinations.

Topographical variations in the polarization sensitivity of articular cartilage as determined by polarization-sensitive optical coherence tomography and polarized light microscopy.

To understand the influence of topographical variations in collagen fibril orientation of articular cartilage on optical phase images of polarization-sensitive optical coherence tomography (PS-OCT), we use polarized light microscopy (PLM) to quantify the orientation and phase retardation of the collagen architecture in cartilage at the same locations imaged by PS-OCT. The PS-OCT experiments demonstrate that articular cartilage has normal variations in polarization sensitivity at different locations over an intact bovine tibial plateau. Articular cartilage is not polarization sensitive along the vertical axis on the medial edge and central areas of the joint surface, but becomes polarization sensitive on the lateral edge of the tibia. This difference in optical phase retardation, as demonstrated by PS-OCT, is verified by PLM to be caused by differences in collagen fibril orientation at different locations of the tibial plateau. This study demonstrates that normal topographical variations in the collagen architecture of articular cartilage within a joint have a profound influence on the optical phase retardation detected by PS-OCT imaging, and therefore must be understood and mapped for specific joints before PS-OCT imaging can be used for the evaluation of the health status of individual joint surfaces.

A comparison of mass removal, thermal injury, and crater morphology of cortical bone ablation using wavelengths 2.79, 2.9, 6.1, and 6.45 microm.

BACKGROUND AND OBJECTIVE: Previous investigations have reported evidence of wavelength dependence on cortical bone ablation. This study used mid-infrared laser wavelengths generated by a free electron laser (FEL) and mass removal measurements to further examine the ablation efficiency of a wavelength (2.79 microm) not previously reported and three wavelengths (2.9, 6.1, and 6.45 microm) previously demonstrated by crater morphology alone to be efficient for cortical bone removal. STUDY DESIGN/MATERIALS AND METHODS: The wavelengths examined were provided by an FEL emitting 4 microseconds macropulses consisting of 1-2 picoseconds duration micropulses delivered at 350 picoseconds intervals. The mass removal measurements were conducted by a microbalance, and the collateral thermal injury and crater morphology of cortical bone were examined by light microscopy following standard histologic processing. RESULTS: The study demonstrated that the highest mass removal was achieved at lambda = 6.1 microm followed by, in order, lambda = 2.9, 6.45, and 2.79 microm. The zones of thermal injury and crater morphology created in cortical bone at the selected wavelengths were examined at the radiant exposure of 28.3 J/cm2. Ablation using lambda = 6.1 microm provided the largest crater size and the least collateral thermal injury. The greatest amount of collateral thermal injury was produced by lambda = 2.79 microm at both the sides and base of the ablation crater. CONCLUSIONS: The mass removal of cortical bone produced by FEL ablation at selected mid-IR wavelengths was measured as a function of incident radiant exposure. The ablation efficiency was found to be dependent upon wavelength. The lambda = 2.79 microm did not offer any improvement over the other wavelengths evaluated, suggesting that a potential shift in the dynamic optical properties of water during tissue irradiance with the FEL does not present an advantage to the cutting of cortical bone. The lambda = 6.1 microm provided the highest ablation efficiency with deepest crater and the least amount of collateral thermal injury.

Determination of characteristics of degenerative joint disease using optical coherence tomography and polarization sensitive optical coherence tomography.

BACKGROUND AND OBJECTIVES: Previous studies have demonstrated that optical coherence tomography (OCT) could be used to delineate alterations in the microstructure of cartilage, and have suggested that changes in the polarization state of light as detected by OCT could provide information on the birefringence properties of articular cartilage as influenced by disease. In this study we have used both OCT and polarization sensitive optical coherence tomography (PS-OCT) technologies to evaluate normal and abnormal bovine articular cartilage according to established structural, organizational, and birefringent characteristics of degenerative joint disease (DJD) in order to determine if this technology can be used to differentiate various stages of DJD as a minimally invasive imaging tool. MATERIALS AND METHODS: Fresh bovine femoral-tibial joints were obtained from an abattoir, and 45 cartilage specimens were harvested from 8 tibial plateaus. Whole ex vivo specimens of normal and degenerative articular cartilage were imaged by both OCT and PS-OCT, then fixed and processed for histological evaluation. OCT/PS-OCT images and corresponding histology sections of each specimen were scored according to a modified Mankin structural grading scale and compared. RESULTS: OCT and PS-OCT imaging allowed structural evaluation of intact articular cartilage along a 6 mm surface length to a depth of 2 mm with a transverse resolution of 12 microm and an axial resolution of 10 microm. The OCT and PS-OCT images demonstrated characteristic alterations in the structure of articular cartilage with a high correlation to histological evaluation (kappa = 0.776). The OCT images were able to demonstrate early to advanced structural changes of articular cartilage while the optical phase retardation images obtained by PS-OCT imaging were able to discriminate areas where disorganization of the cartilage matrix was present, however, these characteristics are much different than those reported where OCT images alone were used to characterize tissue birefringence. No evidence of differences in OCT or PS-OCT images were detected between specimens of similar structural characteristics where proteoglycan was judged present or absent by safranin-O Fast Green staining. CONCLUSIONS: The combined use of OCT and PS-OCT technologies to obtain images from a single system is able to demonstrate and discriminate between characteristics of very early stages of surface irregularities not previously reported for OCT imaging, to deep clefts and collagen matrix disorganization for tissue at depths of up to 2 mm with good correlation to histology. PS-OCT and accumulated optical phase retardation images of articular cartilage as constructed from alterations in Stokes vector parameters appear to give a valuable but different assessment of alterations in tissue birefringence and organization than have been reported for OCT images obtained with the use of polarized or non-polarized light sources. This is the first time that alterations in the polarization state of light reflected from within the tissue have been demonstrated to be consistent with changes observed in the orientation and organization of the collagen matrix in advanced stages of DJD. The degree of phase transformation of light reflected from within the tissue as determined by PS-OCT imaging does not appear to be altered by the presence or absence of proteoglycan.

Mid-IR laser ablation of articular and fibro-cartilage: a wavelength dependence study of thermal injury and crater morphology.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate areas of collateral thermal injury and crater morphology for evidence of wavelength-dependent effects on the ablation of articular cartilage and fibro-cartilage (meniscus) using selected mid-IR wavelengths produced by a free electron laser. STUDY DESIGN/MATERIALS AND METHODS: Two types of cartilage, articular cartilage and fibro-cartilage were used in the study. The wavelengths (lambda) evaluated were 2.79, 2.9, 6.1, and 6.45 microm generated by a free electron laser (FEL) using a 4 microseconds macropulse configuration. The zone of thermal injury and crater morphology produced by laser ablation were examined by light microscopy following standard histologic processing. RESULTS: The zone of thermal injury and crater morphology created in cartilage by the FEL at selected mid-IR wavelengths were examined as a function of incident radiant exposure. Ablation using lambda = 6.1 microm provided the largest crater size for both articular and fibro-cartilage at all radiant exposures. For the zones of collateral thermal injury in articular cartilage, lambda = 6.1 microm produced the least thermal injury at the radiant exposure of 7.6 J/cm2. When the radiant exposure is increased to 20.4 J/cm2, both lambda = 6.1 and 6.45 microm produced less thermal injury than the ablation using lambda = 2.79 and 2.9 microm. The greatest amount of collateral thermal injury was produced by lambda = 2.79 microm for both tissue types. CONCLUSIONS: The results demonstrate that crater depth and collateral thermal injury produced in articular cartilage and fibro-cartilage are wavelength-dependent with 6.1 microm providing the largest craters at all radiant exposures. The least amount of thermal injury was created in articular cartilage using lambda = 6.1 microm at the radiant exposure of 7.6 J/cm2. Both 6.1 and 6.45 microm wavelengths demonstrated similar amount of thermal injury at 20 J/cm2 that was less than lambda = 2.79 and 2.9 microm at similar fluences. These observations are explained based on the absorption by water and protein in the tissue types studied. It is further observed that the use of crater dimensions may not provide a reliable estimate for the amount of tissue removal provided by an ablation procedure.

Use of polarization-sensitive optical coherence tomography to determine the directional polarization sensitivity of articular cartilage and meniscus.

The directional polarization sensitivity of articular cartilage and meniscus is investigated by use of polarization-sensitive optical coherence tomography (PS-OCT) by varying the angle of incident illumination. Experimental results show that when the incident light is perpendicular to the tissue surface, normal articular cartilage demonstrates little polarization sensitivity, while meniscus demonstrates strong polarization sensitivity. Differences in optical phase retardation produced by articular cartilage and meniscus are observed when the incident angle of the scanning light beam is adjusted between 0 and 90 deg relative to the tissue surface. Directional polarization sensitivity of articular cartilage and meniscus as obtained by PS-OCT imaging using variations in the angle of incident illumination can be used to assess the orientation and organization of the collagen matrix of these tissues. The polarization sensitivity as evidenced by the Stokes vector and optical phase retardation images can be explained by the orientation of the angle of illumination relative to the unique structural organization of the collagen fibrils and fibers of articular cartilage and meniscus.

Nonlinear optical microscopy of articular cartilage.

OBJECTIVE: To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage. METHOD: Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination. RESULTS: NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes. CONCLUSION: Thin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Free electron laser ablation of articular and fibro-cartilage at 2.79, 2.9, 6.1, and 6.45 microm: mass removal studies.

BACKGROUND AND OBJECTIVE: The wavelength and tissue-composition dependence of cartilage ablation was examined using selected mid-infrared laser wavelengths. STUDY DESIGN/MATERIALS AND METHODS: The mass removal produced by pulsed laser ablation of articular and fibro-cartilage (meniscus) were measured. The wavelengths examined were 2.79, 2.9, 6.1, and 6.45 microm and provided by a free electron laser (FEL) emitting 4 microsecond macropulses consisting of 1-2 picoseconds duration micropulses delivered at 350 picosecond intervals. The measurement of tissue mass removal was conducted using a microbalance during laser ablation. RESULTS: These studies demonstrated that for articular cartilage the highest mass removal was achieved at lambda = 6.1 microm followed by, in order, lambda = 2.79, 2.9, and 6.45 microm. In comparison, the maximum mass removal for fibro-cartilage was achieved using lambda = 6.1 microm radiation with no statistically significant differences in mass removal provided by the other wavelengths. In evaluation of the comparative influence of each wavelength on tissue type, there was no difference in ablation efficiency between articular and fibro-cartilage at lambda = 6.1 microm. However, the ablation efficiency of articular cartilage was higher than that of fibro-cartilage at both lambda = 2.79 and 2.9 microm. By contrast, lambda = 6.45 microm radiation ablated fibro-cartilage more efficiently than articular cartilage at radiant exposures greater than 12 J/cm2. CONCLUSIONS: The mass removal of articular and fibro-cartilage produced by FEL ablation at selected mid-IR wavelengths was measured as a function of incident radiant exposure. The ablation efficiency was found to depend on both wavelength and tissue type. The 6.1 microm wavelength was found to provide the highest ablation efficiency for both articular and fibro-cartilage.

Use of laser rhinoscopy to treat a nasal obstruction in a captive California sea lion (Zalophus californianus).

Laser rhinoscopy was used to treat a nasal obstruction in a captive California sea lion (Zalophus californianus). The rehabilitated, adult, female sea lion developed mucopurulent, intermittent, bilateral nasal discharge and functional nasal obstruction 20 mo after acquisition by the Aquarium of the Pacific in Long Beach, California. A 3-mm-thick soft tissue structure spanning the region between the soft and hard palates, a deviated nasal septum, and several nasopharyngeal polyps were identified. Biopsies and cultures of the obstructive web showed ulcerative granulation tissue with suppurative inflammation, bacterial infection, and a partial section of an arthropod larva (not speciated). Laser rhinoscopy was performed to relieve the caudal nasopharyngeal obstruction and ablate the polyps. The sea lion appeared to breathe through the nares with lessened nasal discharge for a period of 6 wk after laser therapy, but within 8 wk the mucopurulent nasal discharge returned, the obstruction had reformed, and the sea lion was euthanized. Postmortem examination confirmed antemortem diagnoses of caudal nasopharyngeal obstruction secondary to inflammatory tissue; however, no additional sections of arthropod parasites were located microscopically.

Spectra from 2.5-15 microm of tissue phantom materials, optical clearing agents and ex vivo human skin: implications for depth profiling of human skin.

Infrared measurements have been used to profile or image biological tissue, including human skin. Usually, analysis of such measurements has assumed that infrared absorption is due to water and collagen. Such an assumption may be reasonable for soft tissue, but introduction of exogenous agents into skin or the measurement of tissue phantoms has raised the question of their infrared absorption spectrum. We used Fourier transform infrared spectroscopy in attenuated total reflection mode to measure the infrared absorption spectra, in the range of 2-15 microm, of water, polyacrylamide, Intralipid, collagen gels, four hyperosmotic clearing agents (glycerol, 1,3-butylene glycol, trimethylolpropane, Topicare), and ex vivo human stratum corneum and dermis. The absorption spectra of the phantom materials were similar to that of water, although additional structure was noted in the range of 6-10 microm. The absorption spectra of the clearing agents were more complex, with molecular absorption bands dominating between 6 and 12 microm. Dermis was similar to water, with collagen structure evident in the 6-10 microm range. Stratum corneum had a significantly lower absorption than dermis due to a lower content of water. These results suggest that the assumption of water-dominated absorption in the 2.5-6 microm range is valid. At longer wavelengths, clearing agent absorption spectra differ significantly from the water spectrum. This spectral information can be used in pulsed photothermal radiometry or utilized in the interpretation of reconstructions in which a constant mu(ir) is used. In such cases, overestimating mu(ir) will underestimate chromophore depth and vice versa, although the effect is dependent on actual chromophore depth.

Lasers and laser-tissue interaction.

Light produced by a laser differs from incandescent light in that it is monochromatic, coherent, and intense; and it is these properties that allow lasers to be used as such unique tools in biomedical research and patient care. The effect of a laser beam on tissue is dependent on the optical and mechanical properties of the tissue, and the wavelength, power parameters, and time domains of the laser exposure. Understanding these principles is not only important for the selection of an appropriate laser system for a specific application, but also is essential for that application to be successful.