title: Intravital confocal and two-photon imaging of dual-color cells and extracellular matrix mimics.
abstract: We 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.
authors: Ege University, Electrical and Electronics Engineering Department, Izmir 35100, Turkey.
authors: Ufuk
authors: U
authors: Bal
authors: Volker
authors: V
authors: Andresen
authors: Brenda
authors: B
authors: Baggett
authors: Urs
authors: U
authors: Utzinger
mesh_terms: D045744:Cell Line, Tumor; D002477:Cells / Q000737:chemistry*; D005109:Extracellular Matrix / Q000737:chemistry*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D008164:Luminescent Proteins
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