Dual Mode Reflectance and Fluorescence Confocal Laser Scanning Microscopy for In Vivo Imaging Melanoma Progression in Murine Skin

Abstract

A system was designed and developed for simultaneous fluorescence and reflectance contrast in vivo confocal imaging of murine skin using 488 nm (fluorescence mode) and 830 nm (reflectance mode) laser light sources. B16 melanoma cells and B16-enhanced green fluorescent protein (EGFP) cells were inoculated intradermally into transgenic C57BL/6-TgN (ACTbEGFP) 10sb and non-transgenic C57BL/6 mice, respectively. The inoculation sites were imaged sequentially over a 20 d period. The in vivo confocal images were correlated with ex vivo conventional microscopy. The combined modality system provided single-cell resolution and adequate image registration. In fluorescence mode, B16 melanoma cells appeared as dark objects in the bright background of the GFP expressing murine cells of the C57BL/6 transgenic mouse, and the B16-EGFP melanoma cells had a bright signal within a dark background in C57BL/6 mice. In the C57BL/6 transgenic mouse, a population of fluorescent dendritic cells was observed in the vicinity of the tumor cells. The reflectance images provide a useful reference for those areas in the dermal tissues lacking a fluorescent signal. Combined reflectance/fluorescence in vivo confocal laser scanning microscopy holds significant promise for studies of tumor progression in murine skin.

The physical and chemical properties of eumelanin

“…As previously mentioned, these are particularly difficult due to scattering effects and background autofluorescence from other fluorophores naturally present in tissue such as skin. Several attempts have been made to use eumelanin fluorescence as a non-invasive diagnostic tool with a particular focus on the diagnosis of malignant melanoma (Laiho et al, 2005;Li et al, 2005;Sterenborg et al, 1996). Unfortunately, it is difficult to assess the spectral quality of the emission from these in vivo studies because of low signal-to-noise.…”

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

“…A number of in vivo high resolution imaging technologies for skin have been reported, but have mainly focused on reflectance mode imaging to reveal tissue microanatomy 32 or on en face features. 33 We have developed a dual-axis confocal (DAC) fluorescence microscope that has the capability of detecting fluorescent signals with high sensitivity to a depth of 150 mm with 488 nm excitation light. 34 Like other confocal microscopes, the DAC system has the ability to optically, noninvasively, section tissues for 3-D reconstruction of images.…”

Real-TimeReflectance Confocal Microscopy, a Noninvasive Tool for in vivo Quantitative Evaluation of Comedolysis in the Rhino Mouse Model

“…In addition, Li et al [28] described the appearance of a similar, but fluorescent, population of dendritic-shaped cells in the vicinity of melanoma cells imaged with a combined reflectance/ fluorescence in vivo confocal laser scanning microscopy system and suggested that those cells were immune dendritic cells.…”