Chapter 5 Visualizing cells and the csk visible Light microscopy




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Chapter 5


Visualizing cells and the CSK

1. Visible Light microscopy



Ordinary microscopy trans-illuminates the specimen (light shining from above the specimen into the objective lens), whose image contrast is based on differences in optical density, i.e. light absorbance, within the specimen. Improved images can be obtained through phase contrast microscopy, where contrast is based on differences in refractive index, i.e. the optical path of light as it travels through the specimen. Phase contrast gives better definition of cell borders, allowing easier micromanipulation of the cells.


Another type of microscope useful for cytomechanics is differential interference contrast (DIC) that registers the phase shift of light through the specimen. DIC microscopy is much more accurate for metrological measurements, compared with conventional microscopy, as illustrated below (from Fung). In the left panel of Figure 5.1 are phase contrast images of RBCs (transverse and sagittal views) in which the focus was slightly changed from top to bottom. The center panel gives the best view of its biconcave structure, but with slight focus changes, halo effects around the cell perimeter significantly change the cell appearance and apparent size. The 2 DIC images, however, are also taken at different focus adjustments, however the apparent size does not change and measurement would be more reliable.








Figure 5.1 Comparison of phase contrast with differential interference micrscopy. At left are views of an RBC at 3 slightly different focal adjustments. Border definition is difficult. At right, DIC images show a clear border, independent of focal adjustment..




2. Fluorescent Labeling



Fluorescent probes enable researchers to detect particular components of complex biomolecular assemblies, including live cells. Most compounds, ranging from minerals to proteins, can be detected or labeled with a fluorescent dye. Calcium, for example, can be detected quantitatively in its ionic form using dyes whose light output is proportional to [Ca++]. Proteins can be specifically labeled with an antibody carrying a fluorescent dye. The basic plan is schematized below. To look at a specific protein, cells are labeled with a fluorescent dye that is conjugated with an antibody for that protein. This process is sometimes called ‘decorating’ the cell with a dye. Standard techniques and commercial dyes for many different CSK components are available (i.e. , Molecular Probes, Eugene, OR). Once labeled, the protein can be visualized by exciting the dye with a short wavelength light, usually in the ultraviolet range. The dye will then emit (fluoresce) light at a longer wavelength that can be quantified and turned into an image. The mixture of reflected and emitted fluorescent light is focused onto a photodetector (photomultiplier) via a dichroic mirror (beam splitter). The reflected light is deviated by the dichroic mirror while the emitted fluorescent light passes through in the direction of the photomultiplier (PM). The PM can quantify the number of photons, and relate it to concentration of proteins. Cameras can also produce images such as those shown in below:




Figure 5.2

Multiple proteins can be imaged simultaneously, as shown in the cell below that was decorated with Phalloidin, staining F actin green, and with Texas red, staining G actin. Specific antibodies for both F and G actin were attached to those dyes. Note that the exact colorization of different proteins was determined by computer imaging software and thus represents ‘false’ colors. It should be appreciated that the only light seen in this image has come from either F or G actin- all other components of the cell are dark, due to the optics of fluorescent microscopes.




Figure 5.3 Fibroblast

Since the image was obtained from an ordinary fluorescent microscope, the light collected from the proteins represents most or all of the light coming from the entire cell. In other words, while the 2-D resolution is excellent, the 3-D resolution is not, since the cell has a thickness. The consequence of this is that the red coming from the G actin in the nucleus appears more diffuse relative to that coming from F actin. This is because the nucleus is thicker than the rest of cell, and the relative abundance of G actin emits a saturating amount of red light, diffusing its appearance. The nucleus may actually have a lower proportion of G actin for its volume than is indicated. Higher resolution microscopy can be obtained using con-focal optics, as described below.


Dyes that are sensitive to cellular salts, such as Na+, K+ and Ca++ can be introduced into the cell to indicate their respective concentrations. Ca++ is perhaps the most important small-molecule regulator of cell mechanics, and its concentration can be imaged in several ways.. Certain events, such as electrical depolarization, or mechanical stimuli, can release Ca from internal stores, as exemplified below. These image sequences (read left to right) show a temporal wave of Ca++ after an egg cell has been perturbed mechanically at its upper right quadrant (arrow). The experiment started at top left. The wave is indicated by the spectrum of colors that represent calibrated concentrations of Ca++, according to the bar at lower right. Note that the color-coding is done by the computer based on photon intensity, not on color, since Ca++ produces only one color. Thus red represents the highest [Ca++], and blue the lowest (background). These images, taken in near real-time show that a Ca++ wave results from the poke. Fluorescent dyes for Ca++ include Fura-2 and Indo.


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