Laboratory 2 Fluorescence Microscopy

Biology 29 Cell Structure and Function Spring, 2009 LABORATORY 2: FLUORESCENCE MICROSCOPY Prior to lab: 1) Read these instructions (p 1-5) 2) Watch the video on “Fluorescence Microscopy” on the course web site (“Laboratory page”, “microscopy videos” sub-page) 3) Read p 586-587 in your textbook. 4) Bring your light microscope lab instructions so you have the protocols for Kohler illumination and phase contrast alignment. Fluorescence microscopy greatly extends the range of information that can be gathered by the light microscope, and has made enormous contributions to our understanding of cellular processes. For example, it allows one to “tag,” or stain, a particular organelle or cellular structure that is normally too small to easily discern by ordinary light microscopy. In some cases, labeling of particular cellular structures with fluorescent dyes can even be done in living cells so that dynamic processes can be observed. Fluorescent labeling is often used to show the precise location of a structure or protein within the cell. If a dye does not exist for a particular protein, fluorescently labeled antibodies (immunofluorescence) can be used for specific staining. Some excellent examples of fluorescent labeling in MDCK cells, using a variety of stains, are shown at Olympus Microscopy resource center, fluorescence digital image gallery (link available on course website): http://www.olympusmicro.com/primer/techniques/fluorescence/gallery/cells/mdck/mdckcells.html This week, you will have a chance to use fluorescence microscopy to visualize some of the architecture of cultured mammalian cells. We have available a variety of fluorescent stains that enable one to tag selectively such cellular components as the Golgi apparatus, mitochondria, nuclei, and the actin cytoskeleton. Your general goals for the week are to: I. Become familiar with the design and use of the fluorescence microscope. II. Carry out, in groups, fluorescence labeling of specific organelles. III. Make detailed observations of your samples in the fluorescence microscope, and use your observations to learn about the architecture of your cells. IV. Capture digital images of your samples. Next week you will present these images to your lab mates, and then you will individually prepare a set of figures and an accompanying figure legend to hand in (more information about how to do this will be provided in a handout in lab). V. Complete worksheet on choice and selection of fluorescence filters. Lab Assignments for this laboratory: The slide show and fluorescence filters worksheet is due in discussion one week after lab, the figure and accompanying figure legend are due two weeks after this lab. 1 I. Becoming familiar with the equipment. Your lab instructor will provide an orientation to the fluorescence microscopes and imaging equipment. You will need to be able to identify the parts of the microscope, know how to manipulate the filters, and understand the various precautions regarding the use of this instrument. II. Fluorescent labeling. Work in groups of four. First, decide with your group which two fluorescent tagging procedures you intend` to use (procedures available are listed in Appendix III on pages 6-8). Then decide upon an order, and divide up the work among your group members. Note that some procedures are used with live cells, while others are applied to chemically fixed cells. The live cells should be observed immediately upon completion of the staining for best results. If you are carrying out such procedures, make sure that a microscope will be available at the time when you need it. Procedures that utilize chemically fixed cells do not require immediate observation. These samples may be stored in the dark for later viewing. The protocols below are designed for use with epithelial cells that are firmly attached to a coverglass. If procedures involve examining live cells, be aware that most cells will die upon sustained illumination from the fluorescence illuminator. Work as quickly as you can, and move to fresh areas of the sample often. III. Observing. Plan on spending some "quality time" with your samples. First remember to properly set up Kohler illumination, you should check phase alignment as well but it should not need adjustment. Look at several fields of view and all the cells within them, and make sure that you get a feeling for the variability of results. You should also practice going back and forth between phase optics (with the correct phase turret setting) and fluorescence optics, so that you get a feeling for which of the structures visible in phase correspond to the fluorescing structures. We suggest that you jot down some sketches and written notes about what you see. These will be useful when you are preparing written descriptions to go with your prepared figure (and they may be substituted for real pictures in the case that something goes wrong with your image capture). It is also important to record which objective lens was used when capturing each image. IV. Capturing images of your samples. Some of these will become part of your individual assignment of a formal figure with figure legend. There will be two fluorescence microscopes, equipped with digital cameras, the digital images can be viewed on a monitor as they are captured and can then be transferred to a computer and stored on your web space. Further information on the digital cameras, the transfer of captured images and the preparing of images for your portfolios will be provided in lab. Choose appropriate cells and magnifications to illustrate your point (most of your images will be taken at 40X magnification). Remember that since fluorescent images show only labeled portions of cells, they should be paired with phase images of the exact same field of view, so that the fluorescing structures can be placed in the context of the whole cell. Some fluorochromes (fluorescent dyes) bleach quickly, so for these dyes you will need to move often to a fresh area and capture the image quickly. V. Choice of filters for fluorescence microscopy. To better understand how to select the proper filters for a particular fluorescent dye, you will be given the excitation and emission information about some fluorochromes, and the transmittance spectra of some filter sets that your lab. Using these data, you will determine which filter set to use for which dye. 2 APPENDIX I: FLUORESCENCE MICROSCOPY Fluorescent dyes (fluorochromes) work by absorbing light at one wavelength, this excites electrons in the dye molecule and causes emission of light at another wavelength. Since the energy associated with a photon of light is inversely proportional to its wavelength, light of shorter wavelengths (greater energy per photon) is absorbed by fluorescent dyes, inducing them to re-emit, or fluoresce, light of longer wavelengths (less energy per photon). Below is an example showing the fluorescence spectra of fluorescein. This fluorochrome absorbs light primarily in the 450-500 nm range (blue) and re-emits it maximally in the 550 nm (green) region of the spectrum, as shown below. Fluorescein is one of many fluorochromes that can be linked to molecules that react with particular compounds or structures in the cell, enabling scientist to label specific structures. Alternatively, fluorochromes can be chemically linked to antibodies and then used in immunofluorescence microscopy. When applied to a sample, these tagged antibodies will recognize their respective antigens (often specific proteins) within cells and light them up when viewed through a fluorescence microscope. The epi-fluorescence microscope, which you will use in the lab, utilizes a mercury lamp as an illuminator. This emits strongly in the ultraviolet and shorter visible wavelengths. As shown in the diagram below, the illumination originates above the specimen, as shown in the figure below. It passes through an excitation filter, which transmits only those wavelengths that are effective in exciting a particular fluorochrome. This filtered illumination is then reflected downward by the chromatic (or dichroic) beam splitter, which is designed to reflect light of wavelength shorter than its cutoff point, but transmit light of wavelength longer than the cutoff point. The light then passes through the objective lens onto a specimen, where it may be absorbed by a fluorochrome bound to the specimen. Fluoresced light is emitted from the fluorochrome in all directions, but a substantial portion travels back up through the same objective. Since the light is now of longer wavelength than the initial light, it can pass through the chromatic beam splitter and through the barrier filter, which allows passage of only the wavelengths of fluorescence. As a result, the viewer observes only those structures or regions that are fluorescing. The non-fluorescing portions of the sample remain dark, as does the background. The three filters involved in this clever system are actually combined into one housing, as you will see demonstrated in the laboratory. 3 Illumination system of epi-fluorescence microscope. In this example, the fluorochrome is excited by wavelengths below 420 nm and fluoresces at wavelengths above 420 nm 4 APPENDIX II: CULTURED MAMMALIAN CELLS To study individual cells, scientists often use cultured cell lines because they are easy to grow to large amounts and the conditions of their growth can be carefully controlled. However, mammalian cells do not naturally grow in culture and so they need some modification in order to survive. Some cell types that are proliferative (dividing) can be grown in culture media for short periods of time, known as primary cultures. For more long term survival, cells need to have undergone some genetic transformation so that they will keep dividing indefinitely, known as cell lines. This generally means that they are somewhat akin to cancerous cells that grow indefinitely. Transformation to create cell lines may include treatment with cancerous agents or tumor-causing viruses, or it may simply be achieved by culturing cells from a tumor. Many cell lines from diverse organs and organisms have been propagated successfully for years, and there exist centers for collection and storage of these for use by the scientific community. One of these centers is the American Type Culture Collection (ATCC) in Rockville, MD; the cultured cells that we are using in this class have been obtained from this center. The cells we are using were originally derived from dog kidney; the name "MDCK" stands for Madin Darby Canine Kidney, and reflects the origin of the cells as well as the names of the scientists who established the line. These are epithelial cells (cells that form linings around all the compartments in the body, such as the gut and the bladder) that prefer to grow attached to and flattened against a surface. Once they have form a layer one-cell thick over the entire culture surface, they are said to be confluent, and there will be no further cell divisions until the cells are diluted and given more space to grow. On the surface these cells spread out and form projections along the surface. They often occur in clusters and form a variety of specialized membrane structures at the points where they touch; these cell junctions provide for such functions as adhesion and communication. The overall shape of the cells is constantly being remodeled, forming various kinds of membranous extensions such as spike-like structures are called filopodia, and tiny extensions called microspikes. More extensive, thin sheet-like extensions are termed lamellipodia and are associated with cell locomotion. 5 APPENDIX III: FLUORESCENT LABELING PROCEDURES Unless noted, carry out each staining procedure on two coverslips simultaneously, but only prepare one slide at a time! Keep the extra coverslips in the coplin jar until you need them; however, when working with live cells, you do not want to hold them in buffer any longer than necessary. Thus, you need to work out the timing of your labeling activities carefully. A. Staining mitochondria with MitoTracker Green FM. This lipid soluble fluorochrome specifically stains mitochondria and does so regardless of the mitochondrial membrane potential, unlike other commonly used mitochondrial markers that depend upon "energized" mitochondria. Once accumulated in the mitochondria, it binds irreversibly to available thiol groups on proteins of this organelle. 1. Place coverglass for 30 minutes in a coplin jar containing 50 nM MitoTracker Green FM in culture medium at 37º C. 2. Transfer the coverglass to a coplin jar containing fresh medium for 30 minutes to allow the dye to accumulate in the mitochondria. 3. Mount the coverglass CELL SIDE DOWN on a slide, in a drop or two of fresh medium. 4. Observe with filters for fluorescein. B. Staining endosomal/lysosomal compartments with acridine orange. Acridine orange is a versatile fluorochrome that under our conditions labels RNA, yielding a green fluorescence. We are also using it to label lysosomes; when it is applied to living cells, acidic vesicles of varying sizes--probably part of the endosomal/lysosomal compartment--give off an orangish fluorescence (that tends to fade quite rapidly). Prepare only one coverslip at a time. 1. Rinse coverglass twice, briefly, in phosphate-buffered saline (PBS) in a coplin jar. 2. Immerse coverglass in acridine orange (5 µg/mL in culture medium) for 5 minutes at room temperature in a coplin jar. 3. Rinse coverglass in PBS briefly, in a coplin jar. 4. Mount coverglass CELL SIDE DOWN on a slide, in a drop of PBS. 5. Examine as quickly as possible. (Cells must be alive in order to retain the dye in the lysosomes.) Use only a fluorescein filter combination that allows both green and red fluorescence to pass through. 6 C. Staining the Golgi apparatus with NBD hexanoic ceramide. This compound is composed of the green-fluorescing fluorochrome NBD, linked to a lipid that tends to accumulate in the Golgi apparatus. It is reported to label a specific part of the Golgi, the trans cisternae, most strongly. Prepare only one coverslip a a time; it doesn’t work to “hold” these cells in buffer. 1. Rinse coverglass two times briefly in PBS (phosphate-buffered saline) in a coplin jar. 2. Place coverglass, CELL SIDE UP, in a moist chamber (on moist filter paper in a petri dish) and place 50 µL of culture medium containing 3 µM NBD-ceramide over the cells. Incubate for 10 minutes, at room temperature. 3. Rinse coverglass two times in PBS (coplin jars). 4. Mount coverglass CELL SIDE DOWN on a slide, in a drop of PBS, and examine with fluorescein filter combination in place. (Remember that the cells are examined LIVE in this procedure. Therefore, you must do this soon after the staining is finished.) D. Staining nuclei with DAPI (4,6-diamidino-2-phenylindole). DAPI stains DNA specifically. It can be applied to either fixed cells (as described here) or live cells. 1. Rinse coverglass once in phosphate-buffered saline (PBS), by just dipping in coplin jar. 2. Fix in a coplin jar for 10 minutes, room temperature, in 4% formaldehyde in PBS (in hood). 3. Rinse twice (1 minute each) with PBS. 4. Stain with DAPI (1 µg/mL in PBS) for 2 minutes in a coplin jar at room temperature. 5. Rinse for one minute in PBS. 6. Mount coverglass CELL SIDE DOWN on a slide in a small drop of PBS. 7. Observe for blue fluorescence with DAPI filter combination. 7 E. Staining actin microfilaments with Alexa Fluor 546-phalloidin. Fluorescent labeling of cytoskeletal components provides informative and often stunning displays of their arrangement in cells. A convenient label for actin filaments (“microfilaments”), one of the major cytoskeletal fibers, is the fungal toxin phalloidin, which specifically labels actin in its polymerized (filamentous) form. 1. Rinse coverglass twice (briefly) with PBS (phosphate-buffered saline), in a coplin jar. 2. Transfer to a coplin jar containing 4% formaldehyde in PBS. Leave for 10 minutes at room temperature. 3. Rinse twice (1 minute each) with PBS. 4. Extract with acetone at -20 °C (freezing compartment in refrigerator) for 3-5 minutes. 5. Air dry the coverglass. 6. Place the coverglass in a moist chamber (petri dish with moist filter paper) CELL SIDE UP and add 50 µL of Alexa Fluor 546-phalloidin solution (0.3-1.0 µM) onto it. Leave for 20 minutes at room temperature, in the dark. 7. Rinse twice, briefly, with PBS. 8. Mount the coverglass on a slide CELL SIDE DOWN in a drop of “SlowFade.” (SlowFade retards the bleaching of fluorochrome that occurs upon exposure to the exciting wavelengths.) 9. Examine with the rhodamine filter combination in place. 8