title: Tissue-culture light sheet fluorescence microscopy (TC-LSFM) allows long-term imaging of three-dimensional cell cultures under controlled conditions.
abstract: Fluorescence long-term imaging of cellular processes in three-dimensional cultures requires the control of media supply, temperature, and pH, as well as minimal photodamage. We describe a system based on a light sheet fluorescence microscope (LSFM), which is optimized for long-term, multi-position imaging of three-dimensional in-gel cell cultures. The system integrates a stable culture condition control system in the optical path of the light-sheet microscope. A further essential element is a biocompatible agarose container suitable for the LSFM, in which any cell type can be cultured in different gel matrices. The TC-LSFM allows studying any in vitro cultured cell type reacting to, dividing in, or migrating through a three-dimensional extracellular matrix (ECM) gel. For this reason we called it "tissue culture-LSFM" (TC-LSFM). The TC-LSFM system allows fast imaging at multiple locations within a millimeter-sized ECM gel. This increases the number of analyzed events and allows testing population effects. As an example, we show the maturation of a cyst of MDCK (canine kidney epithelial) cells over a period of three days. Moreover, we imaged, tracked, and analyzed MDCK cells during the first five days of cell aggregate formation and discovered a remarkable heterogeneity in cell cycle lengths and an interesting cell death pattern. Thus, TC-LSFM allows performing new long-term assays assessing cellular behavior in three-dimensional ECM-gel cultures. For example migration, invasion or differentiation in epithelial cell systems, stem cells, as well as cancer cells can be investigated.
authors.affiliation: Buchmann Institute for Molecular Life Sciences (BMLS), Institute for Cell Biology and Neurosciences (FB15, IZN), Goethe Universität Frankfurt am Main, Max-von-Laue-Str. 15, D-60438 Frankfurt am Main, Germany. Francesco.pampaloni@physikalischebiologie.de.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pampaloni
authors.forename: Ulrich
authors.initials: U
authors.lastname: Berge
authors.forename: Anastasios
authors.initials: A
authors.lastname: Marmaras
authors.forename: Peter
authors.initials: P
authors.lastname: Horvath
authors.forename: Ruth
authors.initials: R
authors.lastname: Kroschewski
authors.forename: Ernst H K
authors.initials: EH
authors.lastname: Stelzer
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D005109:Extracellular Matrix / Q000502:physiology*; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D018709:Statistics, Nonparametric