KEYWORDS: 3D image processing, Super resolution, Stereoscopy, Microfabrication, Glasses, Fractal analysis, 3D metrology, Super resolution microscopy, Nanomaterials, Modulation
The potential of micro-and nanofabricated samples as a platform to modulate cell behavior using 3D physical cues has shown tremendous interest in cell differentiation. Investigating cell behavior and organization at the molecular level requires advanced imaging techniques.
We produced multiscale 3D substrates with glass fractal pyramids composed of several generations of octahedra of decreasing sizes, which allow direct observation of cells in 3D SMLM. We show how these samples can be fluorescently labeled and used as a self-referenced sample for calibration and resolution measurements. Moreover, we perform quantitative 3D SMLM on cells growing on such fractal substrates, observing spheroid-like behavior.
We developed a dual-beam optical tweezers setup with video-based force detection to precisely determine the mechanic properties of living cells in suspension with superior sensitivity compared to other techniques like optical stretchers and atomic force microscopy (AFM). With high time resolution we are able to determine both the elastic and the viscous properties of the cells. This novel setup is combined with custom-designed microfluidic cartridges to automatically and reliably pattern cells and beads at specific positions. The beads and cells are trapped and coupled to yield bead-cell-bead complexes.
First results of the elasticity of HEK293 (human embryonic kidney) cells and skin fibroblasts are presented. The latter contain TMEM43-p.S358L mutation, which is linked to arrhythmogenic right ventricular cardiomyopathy.
Quantum dots (Qdots) are nanoparticles exhibiting fluorescent properties that are widely applied for cell staining. We
present here the development of quantum dots for specific targeting of apoptotic cells, for both apoptosis detection and
staining of apoptotic "living" cells. These Qdots are functionalized with Annexin V, a 35-kDa protein that specifically
interacts with the membrane of apoptotic cells: Annexin V recognizes and binds to phosphatidylserine (PS) moieties
which are present on the outer membrane of apoptotic cells and not on this of healthy or necrotic cells. By using Annexin
V, our Qdots probes are made specific for apoptotic cells. For that purpose, Qdots Streptavidin Conjugates are coupled to
biotinylated Annexin V. Staining of apoptotic cells was checked using fluorescence and confocal microscopy techniques
on nonfixed cells. It is shown here that Qdots are insensitive to bleaching after prolonged and frequent exposure as
opposed to organic dyes and this makes them excellent candidates for time-lapse imaging purposes. We illustrate the
application of our Qdots-based probes to continuously follow fast changes occurring on the membrane of apoptotic cells.
Gold nanoparticles exhibit intense and narrow optical extinction bands due to the phenomenon of plasmon resonance
making them useful as contrast agents for light-based imaging techniques. Localized heating results from the absorbed
light energy, which shows potential for these particles in photothermal therapy as well. The bioconjugation of gold
nanoparticles to appropriate antibodies targeted to tumors in vivo, could make highly selective detection and therapy of
tumors possible. We have synthesised gold nanorods based on seed mediated protocols using two methods. The first
method is based on using a mono-surfactant silver assisted method which produces gold nanorods having plasmon peaks
between 670-850 nm within the "optical imaging and therapeutic window". These nanorods have aspect ratios between
2.3 - 3.7. A second method is a silver assisted bi-surfactant method which produce nanorods with peaks in the range of
850-1100 nm having aspect ratios between 5 - 11. Typical concentrations of these particles in aqueous dispersions are in
the range of 1x1010 - 1x1011 particles per mL. We have bioconjugated these gold nanorods with anti-HER2/neu mouse
monoclonal antibodies (MAb). Since the as-prepared CTAB-stabilized nanorods were found to be toxic to SKBR3 cells,
we decided to coat the gold nanorods with polyethylene glycol (PEG). Characterization and size estimation of the
nanoparticles were performed using electron microscopies, optical spectroscopy and confocal microscopy. We present
these results and implications for use of these nanoparticles for in vivo biomedical applications.
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