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Schulmerich M.V.,University of Illinois at Urbana - Champaign | Reddy R.,University of Illinois at Urbana - Champaign | Kodali A.K.,University of Illinois at Urbana - Champaign | Elgass L.J.,University of Illinois at Urbana - Champaign | And 2 more authors.
Analytical Chemistry | Year: 2010

Confocal Raman microscopy is often used for optical sectioning but is problematic when the sample plane of interest has a weak Raman cross-section/signal relative to areas that are out-of-focus. This is especially true for clinical samples in pathology, which consist of a thin tissue (∼5 μm) sample placed on a thick glass slide. Here, we recognize that the problem is the result of the extent of the illumination at the confocal plane being larger than the size of the sample and propose a dark field illumination scheme to efficiently reject substrate signals. The ability of several optical configurations in rejecting out-of-plane signal is investigated for two model systems: SU-8 photo resist over Teflon and SU-8 photo resist over polystyrene. The proposed reflective dark field approach, in which excitation converged to a focal point slightly above the focal plane of the collection optics, was found to be most effective in recording data from the sample. The proposed approach is validated by the rejection of substrate response (fluorescence) in spectra acquired from ∼4 μm of breast tissue on a glass microscope slide. The proposed approach is easy to implement on existing confocal systems, has a straightforward optimization in acquiring data, and is not expected to result in loss of lateral resolution in mapping experiments. © 2010 American Chemical Society. Source


Mir M.,University of Illinois at Urbana - Champaign | Ding H.,University of Illinois at Urbana - Champaign | Wang Z.,University of Illinois at Urbana - Champaign | Reedy J.,Provena Covenant Medical Center | And 2 more authors.
Journal of Biomedical Optics | Year: 2010

Blood smear analysis has remained a crucial diagnostic tool for pathologists despite the advent of automatic analyzers such as flow cytometers and impedance counters. Though these current methods have proven to be indispensible tools for physicians and researchers alike, they provide limited information on the detailed morphology of individual cells, and merely alert the operator to manually examine a blood smear by raising flags when abnormalities are detected. We demonstrate an automatic interferometry-based smear analysis technique known as diffraction phase cytometry (DPC), which is capable of providing the same information on red blood cells as is provided by current clinical analyzers, while rendering additional, currently unavailable parameters on the 2-D and 3-D morphology of individual red blood cells. To validate the utility of our technique in a clinical setting, we present a comparison between tests generated from 32 patients by a state of the art clinical impedance counter and DPC. © 2010 Society of Photo-Optical Instrumentation Engineers. Source


Ali M.Y.,University of Illinois at Urbana - Champaign | Anand S.V.,University of Illinois at Urbana - Champaign | Tangella K.,University of Illinois at Urbana - Champaign | Tangella K.,Provena Covenant Medical Center | And 3 more authors.
Journal of Visualized Experiments | Year: 2015

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity. © 2015 Journal of Visualized Experiments. Source

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