-
[show abstract]
[hide abstract]
ABSTRACT: In spite of the advantages associated with the molecular specificity of fluorescence imaging, there is still a significant need to augment these approaches with label-free imaging. Therefore, we have implemented a form of interference microscopy based upon phase-shifted, laser-feedback interferometry and developed an algorithm that can be used to separate the contribution of the elastically scattered light by sub-cellular structures from the reflection at the coverslip-buffer interface. The method offers an opportunity to probe protein aggregation, index of refraction variations and structure. We measure the topography and reflection from calibration spheres and from stress fibers and adhesions in both fixed and motile cells. Unlike the data acquired with reflection interference contrast microscopy, where the reflection from adhesions can appear dark, our approach demonstrates that these regions have high reflectivity. The data acquired from fixed and live cells show the presence of a dense actin layer located ≈ 100 nm above the coverslip interface. Finally, the measured dynamics of filopodia and the lamella in a live cell supports retrograde flow as the dominate mechanism responsible for filopodia retraction.
Biomedical Optics Express 08/2011; 2(8):2417-37. · 2.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In order to measure the nucleation of nouveau adhesions on the ventral surface of a cell, we have combined phase shifting laser feedback interferometry with a high numerical aperture inverted fluorescence microscope. We use fluorescence to image molecules at the adhesion site and stage scanning interference microscopy in order to measure the distance between the ventral surface of a cell and the substratum with several nanometer precision. Our analytic and Monte Carlo simulations of integrin mediated adhesions predict several features of these nouveau adhesions. An analysis of the energetics of membrane bending and the effects of a composite system of freely diffusing repellers and receptors and a fixed network of ligands on the extracellular matrix predicts that a small bundle of actin filaments should be able to push the membrane down to the extracellular matrix and nucleate a nouveau adhesion with critical radius below the diffraction limit. We have obtained a map of the reflectivity of the ventral surface of fixed metastatic mammary adenocarcinoma cells and we have shown that the data are correlated with markers for a focal adhesion adaptor protein. We are modeling the interference of the incident electric field with the field reflected from the ventral surface so as to obtain the surface topography at focal adhesions from the optical phase data.
Current pharmaceutical biotechnology 09/2009; 10(5):508-14. · 3.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We present a model that provides a mechanistic understanding of the processes that govern the formation of the earliest integrin adhesions ex novo from an approximately planar plasma membrane. Using an analytic analysis of the free energy of a dynamically deformable membrane containing freely diffusing receptors molecules and long repeller molecules that inhibit integrins from binding with ligands on the extracellular matrix, we predict that a coalescence of polymerizing actin filaments can deform the membrane toward the extracellular matrix and facilitate integrin binding. Monte Carlo simulations of this system show that thermally induced membrane fluctuations can either zip-up and increase the radius of a nucleated adhesion or unzip and shrink an adhesion, but the fluctuations cannot bend the ventral membrane to nucleate an adhesion. To distinguish this integrin adhesion from more mature adhesions, we refer to this early adhesion as a nouveau adhesion.
Biophysical Journal 06/2009; 96(9):3555-72. · 3.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is possible to observe gene expression within single cells using a tetracycline inducible promoter for activation. Transcription can be observed by using a fluorescent fusion protein to bind nascent RNA. Ultimately, it is desirable to activate a reporter gene within a single cell with only photons. This is achieved by preparing a chemically altered transcription factor that is functionally unable to activate a reporter gene until it is exposed to photon excitation. We apply two-photon imaging to visualize tumor cells expressing a transgene and ultimately this approach will provide the means to activate a specific gene within a single cell within any tissue to ultimately observe its functional significance in situ.
Journal of Biomedical Optics 10(5):051406. · 3.16 Impact Factor
