Multicolor probe-based confocal laser endomicroscopy: a new world for in vivo and real-time cellular imaging

Abstract

Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology.The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

Smart imaging of acute lung injury: exploration of myeloperoxidase activity using in vivo endoscopic confocal fluorescence microscopy

“…Up to now, confocal fluorescence embodiments exhibit the best cost/efficacy ratio. Unfortunately, these technologies, although sophisticated, essentially retrieve a "picture" limited mainly to naturally autofluorescent tissues/cells (i.e., elastin, collagens, tobacco tar-loaded macrophages) with or without use of topical optical stain (21,31,32,33). Assuming that missing critical components of this fiber optic ECFM should be observed, "completing the picture" with specific markers was perceived as essential, and we have already published a proofof-principle validation of lung ECFM using an external apparatus with a pen-like probe (Five-1; Optiscan) combined with nondedicated in vivo markers (i.e., antibodies, lectin agglutinins, and nuclear labels) (10).…”

Effects of Fluid Therapy on Mesenteric Microcirculation Using New Probe-Based Confocal Laser Endomicroscopy (Cellvizio®) in a Porcine Model of Endotoxic Shock

“…Cellvizio ® was used at T0, T180, and T360. Images were recorded in vivo in a real-time manner by using the CellViewer software as digital sequences of at least 60-s duration (12 frames per second) [22] and analyzed thereafter using the Cell-Viewer ® software to collect FCD, mean vessel diameter, and total vessel length, according to the cardiovascular and dynamics section of the ESICM latest recommendations [23]. The vessel detection algorithm detects the medial axis and the border of tubular structures represented by FITC-induced contrast in a specified region of interest.…”

In Vivo Microscopy in Neurosurgical Oncology

“…Simultaneous imaging of multiple fluorophores is now possible with several clinical LSCE systems with multichannel imaging capabilities. 30 Flexible Probe Confocal Endomicroscopy: Images obtained utilizing a flexible probe attached to a LSCE have an overall good quality and are comparable to conventional histologic images. 31 The flexible imaging fiber optic extends the imaging field by transmitting the image through a bundle of optical fibers (Fig.…”