There is an unmet need in tissue engineering for non-invasive, label-free monitoring of cell mechanical behaviour in their physiological environment. Here, we describe a novel optical coherence phase microscopy (OCPM) set-up which can map relative cell mechanical behaviour in monolayers and 3D systems non-invasively, and in real-time. 3T3 and MCF-7 cells were investigated, with MCF-7 demonstrating an increased response to hydrostatic stimulus indicating MCF-7 being softer than 3T3. Thus, OCPM shows the ability to provide qualitative data on cell mechanical behaviour. Quantitative measurements of 6% agarose beads have been taken with commercial Cell Scale Microsquisher system demonstrating that their mechanical properties are in the same order of magnitude of cells, indicating that this is an appropriate test sample for the novel method described.
Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell's morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.
Adipose-derived stem cells (ADSCs) are adult stem cells isolated from lipoaspirates. They are a good candidate for
autologuous cell therapy and tissue engineering. For these applications, label-free imaging could be critical to assess
noninvasively the efficiency of stem cell (SC) differentiation. We report on the development and application of a
multimodal microscope to monitor and quantify ADSC differentiation into osteoblasts and adipocytes.
We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging on which we combine
Coherent Anti-Stokes Raman Scattering (CARS) with Two Photon Excitation Fluorescence (TPEF) using a Nd: YVO4
pump laser. We acquired 2PEF, CARS, and phase contrast images of Multilamellar Vesicles (MLVs) and Giant
Unilamellar Vesicles (GUVs), as well as Raman spectra of the constituent lipids.
A wide range of peptides are harmful to cells by altering the structure of the biological membranes. This effect depends
on the composition of the membrane and the chemical structure of the peptide.
The peptide we studied is the beta amyloid Aβ which is a major component of the amyloid plaques deposited on neuronal
membranes of Alzheimer's disease (AD) patients. AD is neurodegenerative disorder in which the hallmark symptoms
include cognitive decline and dementia[2] and is characterized by the formation of extracellular amyloid fibrils on the
neuronal membranes of the brain. Many questions still remain unanswered concerning the destabilization of cellular
ionic homeostasis due to pores formed during the interactions of lipid membranes with peptides.
In this project, biomimics of cell membranes are used. The structures that best mimic the plasma membranes are MLVs
or GUVs. These vesicles are formed using the gentle hydration technique[3] or the electroformation technique[4]
respectively and are composed of phospholipids such as DOPC, DPPC, D62PPC and their binary mixtures.
The MLVs and GUVs imaging by CARS and TPEF microscopy not only permits the direct imaging of the leakage phenomenon caused by the toxic peptide (Aβ) on the lipid bilayer, but also records simultaneously the lateral structure of the bilayer and peptide distribution in the plane across the membrane.
We report on the construction of a highly flexible system for advanced biological imaging, where all the following
imaging techniques are integrated into the same microscope: Coherent anti-Stokes Raman scattering (CARS), two
photon excitation fluorescence (TPEF), second harmonic generation (SGH), sum frequency generation (SFG),
fluorescence lifetime imaging (FLIM) and differential interference contrast (DIC). The system employs a Nd:YVO4 laser
as pump (7 ps, 1064 nm), and two tunable OPOs (6 ps, 700 - 1000 nm). Our microscope comprises a heater stage and
perfusion cell for imaging of live cells, and features an atomic force microscope (AFM) which enables optical imaging at
10 nm resolution. Multimodal imaging of breast cancer cells and tissue will be demonstrated as well as imaging of anticancer
drugs in living cells.
We have performed Finite Element Electromagnetic modeling of light scattering in 'apertureless' Scanning Probe Optical Microscopy. Metal tips above metallic and transparent surfaces were modeled to determine suitable conditions for Tip-Enhanced Raman Spectroscopy (TERS) and fluorescence. Gold, silicon, and oxidized silicon were evaluated as potential tip materials, as were the widely used flat substrates of gold, mica and silicon. The lateral resolution of optical imaging is calculated, as a function of tip-substrate separation. This resolution can be made significantly smaller than the tip radius for small tip-substrate separations. In order to model biologically relevant samples, aqueous environments are investigated for the first time, yielding some surprising results.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.