Intravital Confocal and Two-Photon Imaging of Dual-Color Cells and Extracellular Matrix Mimics

WOS: 000314426400021PubMed ID: 23380006We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.Turkish government's Higher Education CouncilMinistry of National Education - Turkey; National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [S10RR023737]The authors wish to thank the Turkish government's Higher Education Council for supporting this project with a scholarship. The majority of work was conducted on a National Institutes of Health (NIH) sponsored shared instrument (S10RR023737). The framework to investigate novel 3D imaging techniques was provided by NIH R01HL077683. The authors wish to thank the engineers and scientists at Lavision Biotec for creating a modular intravital microscope and sharing their experience in designing and maintaining state of the art microscopy equipment