Tumor cell differentiation by label-free fluorescence microscopy

J. Biomed. Opt. 17(10), 101508 (2012)

Petra Weber1, Michael Wagner1, Petra Kioschis2, Waltraud Kessler3, and Herbert Schneckenburger1

 

1Hochschule Aalen, Institut für Angewandte Forschung, Beethovenstr. 1, 73430 Aalen, Germany

2Hochschule Mannheim, Institut für Molekular- und Zellbiologie, Paul-Wittsack-Strasse 10, 68163 Mannheim, Germany

3Steinbeis-Hochschule Berlin, Steinbeis-Institut Multivariate Datenanalyse, Herderstr. 47, 72762 Reutlingen, Germany

 

Abstract

Autofluorescence spectra, images and decay kinetics of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of tumor (control) cells and less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay kinetics. In particular, upon near uv excitation, the fluorescence ratio of the free and protein bound coenzyme nicotinamid adenine dinucleotide (NADH) depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. While larger numbers of fluorescence spectra are evaluated by Principal Component Analysis (PCA), a multivariate data analysis method, additional information on cell metabolism is obtained from spectral imaging and fluorescence lifetime imaging microscopy (FLIM).

 

At a glance:

In comparison with less malignant cell lines tumor (glioblastoma) cells are characterized by

– a higher ratio of free/bound coenzyme NADH (measured around 470 nm and 440 nm);

– shorter fluorescence lifetimes;

– more homogenous cell-substrate distances.

 

Related publication:

Nanotopology of cell adhesion upon variable-angle total internal reflection fluorescence microscopy (VA-TIRFM)

J. Vis. Exp. 68, e4133 (2012).

 

Supplemental figure:

Herbert Schneckenburger-1

This figure shows in its upper part a monolayer of U251-MG glioblastoma cells with tumor suppressor gen PTEN (protein binding parameter PBP increasing from blue to yellow, phase contrast and fluorescence images, effective fluorescence lifetime teff from blue to red corresponding to 1.5-4.0 ns). In the lower part the fluorescence lifetime of protein bound NADH is depicted for U251-MG control (tumor) cells and cells with activated suppressor genes PTEN or TP53.

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