10) where R(pCa) represents the column of the measured ratios, and
8. It is easy to perform cryosections on fixed muscles. Because these samples are not well attached on the slide, one can put the fixed
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Chapter 19
Assessment of Population and ECM Production Using Multiphoton Microscopy as an Indicator of Cell Viability
Martin Vielreicher and Oliver Friedrich
Abstract
Multiphoton microscopy allows continuous depth-resolved, nondestructive imaging of scaffold-seeded cells during cell or tissue culture. Spectrally separated images in high resolution can be provided while cells are conserved in their native state. Here we describe the seeding of mesenchymal stem cells to bacterial nanocellulose hydropolymer scaffolds followed by 2-channel imaging of cellular autofluorescence (AF) and collagen-I formation using second harmonic generation (SHG) signals. With this approach the simul- taneous observation of the progression of cell morphology and production of extracellular matrix as hall- marks of viability and cell fitness is possible.
Key words Multiphoton imaging, Cell viability, Extracellular matrix formation, Collagen-I, SHG, Cellular autofluorescence, Scaffold, Bacterial nanocellulose
1 Introduction
Cell viability assessment has been performed in multiple ways mostly using photometric analysis based on colorimetry, fluores- cence, or luminescence and detection of enzymatic activity (mito- chondrial and dehydrogenase activity, e.g., MTT assay) [1–3]. For the purpose of imaging researchers relied on results from cell stain- ing and fluorescence microscopy. Classically, live-dead screenings were carried out using a staining protocol with two viability mark- ers that discriminate between live and dead cells [4, 5]. Live cells, for example, take up Calcein AM and transform it into fluorescing Calcein (green) present in the cytoplasm whereas a second marker passively enters dead cells through ruptured plasma membranes and specifically binds to DNA (blue or red fluorescence depending on the selected marker).
Multiphoton imaging (MPI) became increasingly important through the last decade after its power was demonstrated in the imaging not only of tissue sections but also of living cells [6–10].
Apart from the fact that MPI allows to perform the full spectrum
Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_19, © Springer Science+Business Media LLC 2017
of applications of classical (confocal) fluorescence microscopy it adds on the benefits of noninvasiveness, depth penetration, and higher sensitivity [11, 12] (Fig. 1). Moreover, additional applica- tions are possible like the imaging of certain biological macromol- ecules like collagen-I or -III, myosin-II, or tubulin using SHG [14, 15]. MPI builds upon excitation by two simultaneously incoming near-infrared photons from a pulsed, mode-locked laser.
In the field of tissue engineering the population of scaffolds is a key parameter for successful construct development. Here, on
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folic acid riboflavin NADH
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wavelength (nm)
wavelength (nm)
900 950 1000 φFσ2r(GM)
Fig. 1 Principle of multiphoton microscopy. In (a) the principle of 2-photon fluorescence is explained. The Jablonski diagrams (i) show the energy levels involved and the excitation and emission processes. Other than with classic 1-photon excitation with, for example, 450 nm (blue), 2-photon excitation requires 2 near- simultaneous (ii) photons of 900 nm (half the energy, red). Emission (green) is independent on the excitation process. (iii) Sources of cellular AF. Two-photon action cross sections of various AF cellular molecules are displayed (top) together with the normalized emission spectra (bottom) [13] (copyright 2003 National Academy of Sciences). (b) Energy diagram of SHG, a nonlinear scattering effect (i). No photons are absorbed and no molecular/quantum states are involved in this process. (ii) Two photons of 900 nm simultaneously striking a highly ordered, non-centrosymmetric material may induce a virtual energy state transition and the release of a photon of exactly twice the energy of the incoming photons (450 nm). (c) In classic 1-photon excitation, fluo- rescence is generated all along the light path with maximum intensity in the focal plane (left). A pinhole is required for blocking out-of-focus fluorescence. Two-photon excitation occurs exclusively in a tiny focal volume (~1 μ m3) causing much less photo-bleaching
245 the one hand MPI can visualize cell populations by their natural AF [16]. In addition, as tissue formation goes along with extracel- lular matrix (ECM) formation like the production of collagen-I, ECM imaging is of great benefit for the validation of cell viability (as compared to Fig. 2). Collagen-I is ubiquitously present and the most abundant protein matrix marker in animal tissue [17]. Here, we provide a step-by-step protocol for the preparation, seeding, and growth of mesenchymal stem cells followed by simultaneous high-resolution cell and collagen-I MPI over time (Fig. 3). The described technique is a sophisticated, but highly informative method for cell viability testing of artificial tissues that produce collagen-I (e.g., bone, tendon, ligament, skin, connective tissue, and others).
2 Materials