Label-free imaging of adipogenesis by coherent anti-stokes Raman scattering microscopy.
Label-free imaging technologies to monitor the events associated with early, intermediate and late adipogenic differentiation in multipotent mesenchymal stromal cells (MSCs) offer an attractive and convenient alternative to conventional fixative based lipid dyes such as Oil Red O and Sudan Red, fluorescent labels such as LipidTOX, and more indirect methods such as qRT-PCR analyses of specific adipocyte differentiation markers such as peroxisome PPARγ and LPL. Coherent anti-Stokes Raman scattering (CARS) microscopy of live cells is a sensitive and fast imaging method enabling evaluation of the adipogenic differentiation with chemical specificity. CARS microscopy is based on imaging structures of interest by displaying the characteristic intrinsic vibrational contrast of chemical bonds. The method is nontoxic, non-destructive, and minimally invasive, thus presenting a promising method for longitudinal analyses of live cells and tissues. CARS provides a coherently emitted signal that is much stronger than the spontaneous Raman scattering. The anti-Stokes signal is blue shifted from the incident wavelength, thus reducing the non-vibrational background present in most biological materials. In this chapter, we aim to provide a detailed approach on how to induce adipogenic differentiation in MSC cultures, and present our methods related to label-free CARS imaging of the events associated with the adipogenesis.
Institute of Biomedicine, Anatomy, University of Helsinki, Helsinki, Finland.
Antti
A
Isomäki
Tarvo
T
Sillat
Mari
M
Ainola
Mikko
M
Liljeström
Yrjö T
YT
Konttinen
Mika
M
Hukkanen
D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D050156:Adipogenesis; D000818:Animals; D001391:Azo Compounds / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D004396:Coloring Agents / Q000032:analysis; D006801:Humans; D007211:Indoles / Q000032:analysis; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D001391:Azo Compounds; D004396:Coloring Agents; D007211:Indoles; C007293:DAPI; C011049:oil red O