Confocal Mosaicing Microscopy Research Articles

Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery

Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P < 0·001). The results demonstrate the feasibility of confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours.

Detection of skin cancer margins in Mohs excisions with high-speed strip mosaicing confocal microscopy: a feasibility study

Fluorescence confocal mosaicing microscopy is an emerging technology for rapid imaging of nuclear and morphological detail directly in excised tissue, without the need for frozen or fixed section processing. Basal cell carcinomas (BCCs) can be detected with high sensitivity and specificity in Mohs excisions with this approach. For translation to clinical trials and towards potentially routine implementation, a new and faster approach called strip mosaicing confocal microscopy was recently developed. To perform a preliminary assessment of fluorescence strip mosaicing confocal microscopy for detecting skin cancer margins in Mohs excisions. Tissue samples from 17 Mohs cases were imaged in the form of strip mosaics. Each mosaic was divided into two halves (submosaics) and graded by a Mohs surgeon and a dermatologist who were blinded to the pathology. The 34 submosaics were compared with the corresponding Mohs pathology. The overall image quality was excellent for resolution, contrast and stitching in the 34 submosaics. Components of normal skin including the epidermis, dermis, dermal appendages and subcutaneous tissue were easily visualized. The preliminary measures of sensitivity and specificity were both 94% for detecting skin cancer margins. The new strip mosaicing approach represents another advance in confocal microscopy for imaging of large areas of excised tissue. Strip mosaicing may enable rapid assessment of BCC margins in fresh excisions during Mohs surgery and may serve as an adjunct to frozen pathology.

Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue

Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12 mm² of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called "strip mosaicing," which was demonstrated on a 10-×-10 mm² of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10 mm² of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5 cm² piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

Rapid screening of cancer margins in tissue with multimodal confocal microscopy

BackgroundComplete and accurate excision of cancer is guided by the examination of histopathology. However, preparation of histopathology is labor intensive and slow, leading to insufficient sampling of tissue and incomplete and/or inaccurate excision of margins. We demonstrate the potential utility of multimodal confocal mosaicing microscopy for rapid screening of cancer margins, directly in fresh surgical excisions, without the need for conventional embedding, sectioning, or processing. Materials and methodsA multimodal confocal mosaicing microscope was developed to image basal cell carcinoma margins in surgical skin excisions, with the resolution that shows nuclear detail. Multimodal contrast is with fluorescence for imaging nuclei and reflectance for cellular cytoplasm and dermal collagen. Thirty-five excisions of basal cell carcinomas from Mohs surgery were imaged, and the mosaics analyzed by comparison with the corresponding frozen pathology. ResultsConfocal mosaics are produced in about 9min, displaying tissue in fields of view of 12mm with ×2 magnification. A digital staining algorithm transforms black and white contrast to purple and pink, which simulates the appearance of standard histopathology. Mosaicing enables rapid digital screening, which mimics the examination of histopathology. ConclusionsMultimodal confocal mosaicing microscopy offers a technology platform to potentially enable real-time pathology at the bedside. The imaging may serve as an adjunct to conventional histopathology to expedite screening of margins and guide surgery toward more complete and accurate excision of cancer.

Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.

Detection of basal cell carcinomas in Mohs excisions with fluorescence confocal mosaicing microscopy

Detection of basal cell carcinomas in Mohs excisions with fluorescence confocal mosaicing microscopy

Detection of basal cell carcinomas in Mohs excisions with fluorescence confocal mosaicing microscopy

High-resolution real-time imaging of human skin is possible with a confocal microscope either in vivo or in freshly excised tissue ex vivo. Nuclear and cellular morphology is observed in thin optical sections, similar to that in conventional histology. Contrast agents such as acridine orange in fluorescence and acetic acid in reflectance have been used in ex vivo imaging to enhance nuclear contrast. To evaluate the sensitivity and specificity of ex vivo real-time imaging with fluorescence confocal mosaicing microscopy, using acridine orange, for the detection of residual basal cell carcinoma (BCC) in Mohs fresh tissue excisions. Forty-eight discarded skin excisions were collected following completion of Mohs surgery, consisting of excisions with and without residual BCC of all major subtypes. The tissue was stained with acridine orange and imaged with a fluorescent confocal mosaicing microscope. Confocal mosaics were matched to the corresponding haematoxylin and eosin-stained Mohs frozen sections. Each mosaic was divided into subsections, resulting in 149 submosaics for study. Two Mohs surgeons, who were blinded to the cases, independently assessed confocal submosaics and recorded the presence or absence of BCC, location, and histological subtype(s). Assessment of confocal mosaics was by comparison with corresponding Mohs surgery maps. The overall sensitivity and specificity of detecting residual BCC was 96.6% and 89.2%, respectively. The positive predictive value was 92.3% and the negative predictive value 94.7%. Very good correlation was observed between confocal mosaics and matched Mohs frozen sections for benign and malignant skin structures, overall tumour burden and location, and identification of all major histological subtypes of BCC. Fluorescent confocal mosaicing microscopy using acridine orange enables detection of residual BCC of all subtypes in Mohs fresh tissue excisions with high accuracy. This observation is an important step towards the long-term clinical goal of using a noninvasive imaging modality for potential real-time surgical pathology-at-the-bedside for skin and other tissues.

Confocal mosaicing microscopy in skin excisions: a demonstration of rapid surgical pathology.

Precise micro-surgical removal of tumour with minimal damage to the surrounding normal tissue requires a series of excisions, each guided by an examination of frozen histology of the previous. An example is Mohs surgery for the removal of basal cell carcinomas (BCCs) in skin. The preparation of frozen histology is labour-intensive and slow. Confocal microscopy may enable rapid detection of tumours directly in surgical excisions with minimal need for frozen histology. Mosaicing of images enables observation of nuclear and cellular morphology in large areas of surgically excised tissue. In skin, the use of 10-1% acetic acid as a reflectance contrast agent brightens nuclei in 0.5-5 min and enhances nuclear-to-dermis contrast and detectability of BCCs. A tissue fixture was engineered for precisely mounting surgical excisions to enable mosaicing of 36 x 36 images to create a field of view of 12 x 12 mm. This large field of view displays the excision at 2x magnification, similar to that routinely used by Mohs surgeons when examining frozen histology. Comparison of mosaics to histology demonstrates detectability of BCCs. Confocal mosaicing presently requires 9 min, instead of 20-45 min per excision for preparing frozen histology, and thus may provide a means for rapid pathology-at-the-bedside to expedite and guide surgery.

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.

Sensitivity and specificity for detecting basal cell carcinomas in Mohs excisions with confocal fluorescence mosaicing microscopy

Recent studies have demonstrated the ability of confocal fluorescence mosaicing microscopy to rapidly detect basal cell carcinomas (BCCs) directly in thick and fresh Mohs surgical excisions. Mosaics of confocal images display large areas of tissue with high resolution and magnification equivalent to 2x, which is the standard magnification when examining pathology. Comparison of mosaics to Mohs frozen histopathology was shown to be excellent for all types of BCCs. However, comparisons in the previous studies were visual and qualitative. In this work, we report the results of a semiquantitative preclinical study in which 45 confocal mosaics are blindly evaluated for the presence (or absence) of BCC tumor. The evaluations are made by two clinicians: a senior Mohs surgeon with prior expertise in interpreting confocal images, and a novice Mohs fellow with limited experience. The blinded evaluation is compared to the gold standard of frozen histopathology. BCCs are detected with an overall sensitivity of 96.6%, specificity of 89.2%, positive predictive value of 93.0%, and negative predictive value of 94.7%. The results demonstrate the potential clinical utility of confocal mosaicing microscopy toward rapid surgical pathology at the bedside to expedite and guide surgery.

Confocal mosaicing microscopy in Mohs skin excisions: feasibility of rapid surgical pathology

Mosaicing of confocal images enables observation of nuclear morphology in large areas of tissue. An application of interest is rapid detection of basal cell carcinomas (BCCs) in skin excisions during Mohs surgery. A mosaic is currently created in less than 9 min, whereas preparing frozen histology requires 20 to 45 min for an excision. In reflectance mosaics, using acetic acid as a contrast agent to brighten nuclei, large and densely nucleated BCC tumors were detectable in fields of view of 12 x 12 mm (which is equivalent to a 2x-magnified view as required by Mohs surgeons). However, small and sparsely nucleated tumors remained undetectable. Their diminutive size within the large field of view resulted in weak light backscatter and contrast relative to the bright surrounding normal dermis. In fluorescence, a nuclear-specific contrast agent may be used and light emission collected specifically from nuclei but almost none from the dermis. Acridine orange of concentration 1 mM stains nuclei in 20 s with high specificity and strongly enhances nuclear-to-dermis contrast of BCCs. Comparison of fluorescence mosaics to histology shows that both large and small tumors are detectable. The results demonstrate the feasibility of confocal mosaicing microscopy toward rapid surgical pathology to potentially expedite and guide surgery.