The images below show how fluorescence imaging can be used to visualize specific sub-cellular structures. The first image shows microtubules stained with a fluorescent dye; the image was recorded in black and white. The second image shows nuclei stained with a fluorescent compound DAPI; this image was also recorded in black and white but was obtained using excitation light of different wavelength than the microtubule image. To obtain the last image, separate colors were assigned to the two previous images before combining them into one.

One of the experiments in the course involved demonstration and use of microscopes. Student images of various prepared slides (from Carolina Bioscience) acquired with the Leica stereo zoom microscope can be found in this directory. Another of the instruments used was the Olympus BX41 fluorescence microscope at the TEMPO facility at UCSB. The images below show a commercial slide with bovine pulmonary arterial epithelial cells as visualized by three fluorescent stains. The first stain, DAPI, binds to DNA and makes nuclei fluoresce in blue. The second stain, AlexaFluor 488, has been coupled to phalloidin, which binds to actin, allowing visualization of actin filaments of the cytoskeleton. The third dye is MitoTracker Red, which binds to mitochondria. The fluorescence from the sample was resolved with 40X objective and captured with a Nikon CoolPix digital camera. You can click on each image to download the high-resolution file.

The next set of images of bovine pulmonary arterial epithelial cells were recorded using a fluorescence microscope during a last year's visit to UCSB's Microscopy Facility. On the right is a color image obtained by combining three individual images, each showing one component of the cell. Below are three images of the same object, taken with a fluorescence microscope at three different wavelengths. Notice that the light from this sample was resolved with a more powerful onjective and captured with a specialized low-noise CCD camera. An appropriate combination of these three files will give the color image of the cell, similar to the one shown on the right. You can click on each image to download the high-resolution file.

Below is a similar set of images from the year 2005. Notice that this time a different set of filters was used when the recording microtubules such that DAPI fluorescence also shows in this image. Notice the limited depth of field effect in microtubules image where the top right corder is very sharp but the bottom left corner is out of focus. Also notice problems with the actin image, presumably due to overly agressive postprocessing. In this case, three different color images were created to emphasize each of the components in the presence of other two.

The images below are of human cheek epithelial cells as seen through the Olympus Provis microscope in three modes: brightfield, Nomarski interference contrast, and darkfield. The spherical structure seen in the center of the cell in brightfield and Nomarski image is the nucleus; the bright dots throughout the cell in the darkfield image are various granules. While the Nomarski image may appear more real a first sight, the dark and bright areas surrounding the edges of the nucleus and granules are artifacts of this imaging technique.

The images below show how choice of filters allows to improve contrast in brightfield microscopy. The three images show a tissue slice with several cells underging mitosis. The DNA in cells organizes into chromosomes at early stages of mitosis (cell in upper-central part) and the two sets of daugther chromosomes separate in the early anaphase (cells in the lower-central part). The cells are treated with a red dye that binds to DNA. A red dye appears red to our eye in the brightfield microscope because it strongly absorbs blue and green light. The first image shows an unfiltered color view of the tissue. The next two images are recorded in black and white using two different color filters. Notice the improved contrast with an appropriate choice of the filter in the last image.