Low-cost, scalable laser scanning module for real-time reflectance and fluorescence confocal microscopy

Confocal laser scanning microscopy (CLSM) has been introduced in clinical settings as a tool enabling a quasi-histologic view of a given tissue, without performing a biopsy. It has been applied to many fields of medicine mainly to the skin and to the analysis of skin cancers for both in vivo and ex vivo CLSM. In vivo CLSM involves reflectance mode, which is based on refractive index of cell structures serving as endogenous chromophores, reaching a depth of exploration of 200 μm. It has been proven to increase the diagnostic accuracy of skin cancers, both melanoma and non-melanoma. While histopathologic examination is the gold standard for diagnosis, in vivo CLSM alone and in addition to dermoscopy, contributes to the reduction of the number of excised lesions to exclude a melanoma, and to improve margin recognition in lentigo maligna, enabling tissue sparing for excisions. Ex vivo CLSM can be performed in reflectance and fluorescent mode. Fluorescence confocal microscopy is applied for “real-time” pathological examination of freshly excised specimens for diagnostic purposes and for the evaluation of margin clearance after excision in Mohs surgery. Further prospective interventional studies using CLSM might contribute to increase the knowledge about its application, reproducing real-life settings.

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Long-Term in Vivo Investigation of Mouse Cerebral Microcirculation by Fluorescence Confocal Microscopy in the Area of Focal Ischemia

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600077 ◽

2005 ◽

Vol 25 (7) ◽

pp. 858-867 ◽

Cited By ~ 85

Author(s):

Yutaka Tomita ◽

Nathalie Kubis ◽

Yolande Calando ◽

Alexy Tran Dinh ◽

Philippe Méric ◽

...

Keyword(s):

Confocal Microscopy ◽

Fluorescence Microscopy ◽

Laser Scanning ◽

Arterial Occlusion ◽

Focal Ischemia ◽

Doppler Flowmetry ◽

Cranial Window ◽

Fluorescence Confocal Microscopy

This study was designed to assess that mouse pial and cortical microcirculation can be monitored in the long term directly in the area of focal ischemia, using in vivo fluorescence microscopy. A closed cranial window was placed over the left parieto-occipital cortex of C57BL/6J mice. Local microcirculation was recorded in real time through the window using laser-scanning confocal fluorescence microscopy after intravenous injection of fluorescent erythrocytes and dextran. The basal velocity of erythrocytes through intraparenchymal capillaries was 0.53 ± 0.30 mm/sec ( n = 121 capillaries in 10 mice). Two branches of the middle cerebral artery were topically cauterized through the window. Blood flow evaluated by laser-Doppler flowmetry in two distinct areas indicated the occurrence of an ischemic core (15.2% ± 5.9% of baseline for at least 2 h) and a penumbral zone. Magnetic resonance imaging and histology were used to characterize the ischemic area at 24 h after occlusion. The infarct volume was 7.3 ± 3.2 mm3 ( n = 6). Microcirculation was repeatedly videorecorded using fluorescence confocal microscopy over the next month. After the decrease following arterial occlusion, capillary erythrocyte velocity was significantly higher than baseline 1 week later, and attained 0.74 ± 0.51 mm/sec ( n = 76 capillaries in six mice, P<0.005) after 1 month, while venous and capillary network remodeling was assessed, with a marked decrease in tortuosity. Immunohistochemistry revealed a zone of necrotic tissue into the infarct epicenter, with activated astrocytes at its border. Such long-term investigations in ischemic cortex brings new insight into the microcirculatory changes induced by focal ischemia and show the feasibility of long-term fluorescence studies in the mouse cortex.

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