Cell Imaging...Wide-Field Fluorescence

Cell Imaging...Wide Field Fluorescence
Contact Info: Chris Rodesch crodesch@cores.utah.edu Phone: 587-7964 Fax: 585-6364	Back to Cell Imaging Home

Two inverted microscopes, each coupled to cooled, high resolution, low noise CCD cameras
Heated, perfusable stage adaptors for use with live cell cultures
Image acquisition, processing and analysis performed using the Openlab software package (Improvision; Lexington, MA, USA)
- provides a convenient graphic interface programming module for custom design of automated regimes (see below) involving all aspects of image acquisition, processing, and analysis including stage manipulation, focus drive control, excitation filter switching, exposure control, time lapse parameters, etc.
Nearest neighbor and volume deconvolution algorithms for x-y resolution enhancement.
Phase contrast and DIC (Nomarski) imaging modes can be used
Go here to view additional examples of recent analyses

Automations: (Back to Cell Imaging Home)
Openlab provides a convenient graphic programming interface that can be used to build custom automations incorporating any features associated with the hardware and software-driven aspects of an experiment. Hardware control capabilities include x-y stage manipulation, focus drive control, excitation filter switching, exposure control, time lapse imaging parameters, etc. Software-mediated manipulations that can be automated include background subtraction, pseudocoloring, merging, ratio calculations, deconvolution, thresholding, quantitative analysis, and more.

A segment from an automation that incorporates both hardware control and software-mediated image analysis is shown to the right. The automation is designed to determine the proportion of total (DAPI stained) nuclei that express a specific antigen, as identified by fluorescent antibody staining. Images of total and antibody-stained nuclei are captured at multiple fields of view, thresholding is performed to isolate the stained nuclei from any background, and the nuclei in each image are counted. Finally, the overall percentage of stained nuclei is calculated.

Facility staff are available either to assist users with automation development or to fully design and develop automations for users following consultation aimed at determining experimental requirements.

Nearest-neighbor and Volume Deconvolution: (Back to top) (Back to Cell Imaging Home)
These algorithms provide a computational means for enhancing x-y resolution in images. Information related to the imaging system being used and from images collected at different focal planes is utilized to identify and remove blurred information caused by the imaging process and out-of-focus haze contributed by tissue regions away from the plane of focus. The nearest-neighbor algorithm deconvolves a single, central image using one image acquired above and one image acquired below the central focal plane. The volume algorithm deconvolves single images using a user-specified number of images acquired above and below the central focal plane.

Two examples are shown below. The first analysis utilized deconvolved images of a GFP-tagged DNA-binding protein in mutant yeast strains to determine whether cohesion between sister chromatids was correctly established. The second example shows the results from deconvolving images of yeast stained with phalloidin to visualize the actin cytoskeleton in budding cells.

Chromatid Cohesion Assay:

Sister chromatid cohesion was analyzed in mutant strains of the yeast S. cerevisiae. The assay (Ullman and Nasmyth, 1998) utilizes the expression of GFP-tagged tet-repressor in yeast strains that possess a set of tandem tet-operator sequences inserted into a marker chromosome. A green dot can be visualized in the nucleus of the yeast cell upon binding of the tet^r to the tet^o sites. Yeast cells were arrested in G2 of the cell cycle using the microtubule depolymerizing drug nocodozole, enabling analysis of newly replicated sister chromatid cohesion establishment. If cohesion has been successfully established, the GFP signal on sister chromatids will occur adjacent to one another and appear indistinguishable under the microscope. However, if cohesion has not been successfully established, the GFP signal on sister chromatids will not occur adjacent to one another and can be detected by the presence of two distinguishable dots under the microscope.
Representative raw (A) and deconvolved (B) micrographs are shown. Z-series were collected using an Openlab automation, and the resulting image sets were passed through a nearest-neighbor deconvolution algorithm to enhance x-y resolution through removal of out-of-focus haze. The deconvolved images were then examined for the presence of one (arrowheads, A-B) or two (arrows, A-B) dots within individual yeast cells.

Images and protocol courtesy Dr. Mylynda Schlesinger and Dr. Timothy Formosa, Department of Biochemistry.

Actin Staining in S. cerevisiae:

These micrographs present raw (A, C) and deconvolved (B, D) images of actin labeling in S. cerevisiae. Cells were fixed in 4% formaldehyde in medium (10 m at rt), postfixed in 4% formaldehyde in PBS (60 m at rt), stained with Alexa Fluor 488 phalloidin (Molecular Probes; 60 m at rt in the dark), and mounted onto poly-L-lysine coated coverslips. Z-series were collected, and x-y resolution in the images was enhanced using nearest-neighbor deconvolution. The labeled actin, present as tubules in the mother cells and as punctae in the daughter cells, is more clearly resolvable in the deconvolved images.

Images and protocol coutesy Shelly Shiflett, Dr. Diane McVeigh-Ward and Dr. Jerry Kaplan, Department of Pathology.

More Examples of Recent Analyses: (Back to Top)
Further examples of fluorescence analyses recently performed at the facility are presented below. One shows the results from a time lapse experiment that followed the endocytosis and trafficking of sphingomyelin through the endocytic apparatus, to the Golgi, and back to the cell surface. Another shows results from a quantitative fluorescence assay aimed at determining if and how innervation patterns are altered following surgical transplantation of limb buds in chick embryos.

Lipid Tracking Time Lapse:

Human fibroblasts were loaded overnight with a fluorescent dextran conjugate (red labeling, A-D), that, after a chase protocol, is strictly localized within lysosomes. Cells were then labeled with a fluorescent sphingomyelin conjugate (green labeling, A-D) for one hour (A), then chased for an additional hour (B), 4 hours (C), or overnight (D). The sphingomyelin is internalized (A), and transported by the endocytic apparatus to the lysosomes (arrows indicating yellow colocalization areas in inset, B). The sphingomyelin is converted by sphingmyelinases in the lysosomes to ceramide, which is then transported to the Golgi apparatus (arrowheads, B and C). The ceramide is reconverted to sphingomyelin in the Golgi, and transported back to the cell surface (large arrow, C) for additional rounds of internalization. After overnight incubation, labeled sphingomyelin and ceramide again appear in lysosomes (arrows in inset, D) and the Golgi apparatus (arrowhead in main panel, D).

Images and protocol courtesy of Dr. Diane McVeigh-Ward and Dr. Jerry Kaplan, Department of Pathology.

Quantitative Fluorescence Analysis:

In order to investigate whether the somatotopic map of cutaneous projections develops in the appropriate spinal cord segments when sensory axons innervate inappropriate skin regions following rostral limb shifts, the left limb bud of a chick embryo was shifted 2 segments rostrally at St. 17 (embryonic day 3, E3). Central projections were labeled by injecting DiI into a cutaneous nerve (CFL) on the control (right, A) and operated (left, A) sides of the embryo at St. 37 (E11). Transverse sections (100mm in thick) of spinal cord with attached DRGs were then cut with a Vibratome. Sections were systematically imaged, and the extent of innervation from the left and right CFL nerves was evaluated using a
measure (stain index, B) that incorporated both stained area and fluorescence intensity.
A. shows a section from thoracic segment T7. B. shows stain index quantification.

Image and protocol courtesy Dr. Guoying Wang and Dr. Sheryl Scott, Department of Neurobiology and Anatomy.

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