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

Published online by Cambridge University Press:  04 February 2013

Ufuk Bal ,
Volker Andresen ,
Brenda Baggett  and
Urs Utzinger
Ufuk Bal
Affiliation:
Ege University, Electrical and Electronics Engineering Department, Izmir 35100, Turkey
Volker Andresen
Affiliation:
LaVision BioTec GmbH, Astastrasse 14, Bielefeld, D-33617, Germany
Brenda Baggett
Affiliation:
Biomedical Engineering, University of Arizona, 1127 E James E. Rogers Way, Tucson, AZ 85721, USA
Urs Utzinger*
Affiliation:
Biomedical Engineering, University of Arizona, 1127 E James E. Rogers Way, Tucson, AZ 85721, USA
*
*Corresponding author. E-mail: utzinger@u.arizona.edu
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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.

Keywords

extracellular matrix mimicsconfocal microscopytwo-photon microscopyphotobleachingDsRed2eGFP
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 201 - 212
Copyright
Copyright © Microscopy Society of America 2013

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