Daniel S. Gareau

Feasibility of digitally stained multimodal confocal mosaics to simulate histopathology.

Fluorescence confocal mosaicing microscopy of tissue biopsies stained with acridine orange has been shown to accurately identify tumors and with an overall sensitivity of 96.6% and specificity of 89.2%. However, fluorescence shows only nuclear detail similar to hematoxylin in histopathology and does not show collagen or cytoplasm, which may provide necessary negative contrast information similar to eosin used in histopathology. Reflectance mode contrast is sensitive to collagen and cytoplasm without staining. To further improve sensitivity and specificity, digitally stained confocal mosaics combine confocal fluorescence and reflectance images in a multimodal pseudo-color image to mimic the appearance of histopathology with hematoxylin and eosin and facilitate the introduction of confocal microscopy into the clinical realm.

In vivo volumetric imaging of microcirculation within human skin under psoriatic conditions using optical microangiography.

There is a growing body of evidence suggesting that vascular abnormalities may play crucial role in several dermatologic diseases, such as psoriasis, port wine stain, and skin cancer. To improve our understanding of vascular involvement in these skin conditions, there is a need for a non‐invasive imaging modality capable of assessing 3D microcirculations within skin tissue beds in vivo. This study aims to demonstrate whether ultra‐high sensitive optical microangiography (UHS‐OMAG) is feasible to visualize skin microcirculations in 3D and to quantify microvascular vessel density under normal and psoriatic conditions in vivo.

Confocal fluorescence spectroscopy of subcutaneous cartilage expressing green fluorescent protein versus cutaneous collagen autofluorescence

Optically monitoring the expression of green fluorescent protein (GFP) in the cartilage underlying the skin of a mouse allows tracking the expression of the chondrocyte phenotype. This paper considers how confocal microscopy with spectral detection can sense GFP fluorescence in the cartilage despite light scattering and collagen autofluorescence from the overlying skin. An in vivo experiment tested the abilities of a topical optical fiber measurement and a confocal microscope measurement to detect GFP in cartilage under the skin versus the collagen autofluorescence. An ex vivo experiment tested the ability of a confocal microscope without and with its pinhole to detect a fluorescent microsphere underneath an ex vivo skin layer versus the collagen autofluorescence. In both systems, spectroscopic detection followed by linear analysis allowed spectral discrimination of collagen autofluorescence (M(C)) and the subdermal green fluorescence (M(G)) due to either GFP or the microsphere. Contrast was defined as M(G)/(M(G)+M(C)). The in vivo contrast for GFP using optical fiber and confocal measurements was 0.16 and 0.92, respectively. The ex vivo contrast for a fluorescent microsphere using a confocal system without and with a pinhole was 0.13 and 0.48, respectively. The study demonstrates that a topical optical fiber measurement is affected by collagen autofluorescence, while a confocal microscope can detect subdermal fluorescence while rejecting collagen autofluorescence.

Automated identification of epidermal keratinocytes in reflectance confocal microscopy

Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6±2.8 μm and reflectance gradient b = 3.6±2.1 μm at the nuclear∕cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4 × histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

Optical fiber probe spectroscopy for laparoscopic monitoring of tissue oxygenation during esophagectomies.

Anastomotic complication is a major morbidity associated with esophagectomy. Gastric ischemia after conduit creation contributes to anastomotic complications, but a reliable method to assess oxygenation in the gastric conduit is lacking. We hypothesize that fiber optic spectroscopy can reliably assess conduit oxygenation, and that intraoperative gastric ischemia will correlate with the development of anastomotic complications. A simple optical fiber probe spectrometer is designed for nondestructive laparoscopic measurement of blood content and hemoglobin oxygen saturation in the stomach tissue microvasculature during human esophagectomies. In 22 patients, the probe measured the light transport in stomach tissue between two fibers spaced 3-mm apart (500- to 650-nm wavelength range). The stomach tissue site of measurement becomes the site of a gastroesophageal anastamosis following excision of the cancerous esophagus and surgical ligation of two of the three gastric arteries that provide blood perfusion to the anastamosis. Measurements are made at each of five steps throughout the surgery. The resting baseline saturation is 0.51±0.15 and decreases to 0.35±0.20 with ligation. Seven patients develop anastomotic complications, and a decreased saturation at either of the last two steps (completion of conduit and completion of anastamosis) is predictive of complication with a sensitivity of 0.71 when the specificity equaled 0.71.

Specifying tissue optical properties using axial dependence of confocal reflectance images : confocal scanning laser microscopy and optical coherence tomography

The optical properties of a tissue can be specified by the depth dependence of a reflectance-mode confocal measurement, as the focus is scanned down into a tissue. Reflectance-mode confocal scanning laser microscopy (rCSLM) and optical coherence tomography in focus tracking mode (OCT) are two examples of such confocal measurements. The measurement of reflected signal as a function of the depth of focus, R(z), is expressed as ρe-μz, where ρ [dimensionless] is the local reflectivity from the focus within a tissue and μ [cm-1] is the attenuation of signal as a function of z. The reflectivity of a mirror defines ρ = 1. This paper describes how the experimental ρ and μ map into the optical properties of scattering coefficient, μs [cm-1], and anisotropy of scattering, g [dimensionless]. Preliminary results on tissue for the rCSLM and OCT systems are reported.

Characterizing tissue optical properties using confocal and low- coherence imaging

The signal from a confocal measurement as the focal volume is scanned down into a tissue yields an exponential decay versus depth (z_focus), signal = rho exp(-mu z_focus), where rho [dimensionless] is the local reflectivity and mu [1/cm] is an attenuation coefficient. A simple theory for how rho and mu depend on the optical properties of scattering (mu_s) and anisotropy (g) is presented. Experimental measurements on 5 tissue types from mice (white and gray matter of brain, skin, liver, muscle) as well as 0.1-um-dia. polystyrene microspheres are presented. The tissues have similar mu_s values (about 500 [1/cm]) but variable g values (0.8-0.99). Anisotropy appears to be the primary mechanism of contrast for confocal measurements such as reflectance-mode confocal scanning laser microscopy (rCLSM) and optical coherence tomography (OCT). While fluorescence imaging depends on fluorophores, and absorption imaging depends on chromophores, the results of this study suggest that contrast of confocal imaging of biological tissues depends primarily on anisotropy.

Clinical feasibility of rapid confocal melanoma feature detection

In vivo reflectance confocal microscopy shows promise for the early detection of malignant melanoma. One diagnostic trait of malignancy is the presence of pagetoid melanocytes in the epidermis. For automated detection of MM, this feature must be identified quantitatively through software. Beginning with in vivo, noninvasive confocal images from 10 unequivocal MMs and benign nevi, we developed a pattern recognition algorithm that automatically identified pagetoid melanocytes in all four MMs and identified none in five benign nevi. One data set was discarded due to artifacts caused by patient movement. With future work to bring the performance of this pattern recognition technique to the level of the clinicians on difficult lesions, melanoma diagnosis could be brought to primary care facilities and save many lives by improving early diagnosis.

Full-Pupil Line-Scanning Confocal Microscope for Imaging Weakly Scattering Tissues: Comparison to Divided-Pupil

Confocal reflectance full-pupil and divided-pupil line-scanning microscopes provide optical sectioning of 1-2?m and image nuclear detail in skin. Line-scanning with linear detectors is a simpler alternative to point-scanning for imaging weakly scattering epithelial tissues.