MICROSCOPY FOR THE DEVELOPMENTAL BIOLOGY STUDENT...

MICROSCOPY FOR THE DEVELOPMENTAL BIOLOGY STUDENT... You will be using two configurations of microscope during the course of the semester to observe specimens and record your results: compound microscopes and stereo (or dissecting) microscopes. The following discussion is meant to serve as a basic introduction to the use of these microscopes and the image capture equipment available in this laboratory. I. FACTORS AFFECTING MICROSCOPE RESOLUTION AND MAGNIFICATION... The total magnification afforded by a microscope is equal to the product of the magnifications provided by the objective used and the oculars. For example, a 100 objective used with 10 oculars gives a total magnification of 1000 : objects at the specimen plane will appear 1000 larger. While it would seem that objectives and oculars could be combined in microscopes to give unlimited magnification, the "useful magnification" of a microscope is limited by the resolution of the objective. Resolution refers to the ability of an optical instrument such as a microscope to distinguish two closely-spaced objects. The theoretical resolution of any optical instrument, including microscopes, telescopes, and cameras, is limited by the diffraction of light as it passes through the lenses (or bounces off mirrors). The resolution of a microscope objective can be expressed as a function of the wavelength of light (λ) and the numerical aperture (NA) of the objective and condenser lenses: R = 0.6 λ / NA As you can see from this formula, objectives with a larger numerical aperture give greater resolving power (they can resolve smaller details). For example: a 10 objective with an NA of 0.25 will resolve details as small as 1.2 micrometer when using green light (λ = 500 nanometer). With a 100 objective (NA = 1.3), you could resolve details as small as 0.23 micrometer using green light. Note that shorter wavelengths of light also provide for greater resolution; smaller details can be resolved with blue light (λ = 480 nanometer) than with red light (λ = 650 nanometer). The NA of dry lenses is limited by the refractive index of air, and the phenomenon of internal reflection, to less than 1.0. By using oil between specimen and objective, numerical apertures as high as 1.4 can be obtained. Thus the resolution of a light microscope equipped with the best diffraction-limited oil immersion objectives is about 0.2 micrometer. Though in some cases smaller details can be seen in light microscopes, they cannot be resolved (i.e.: two small organelles less than 0.2 micrometer apart will be perceived as a single object). The resolution of a microscope objective, in combination with the resolution of your eye (a simple lens), also determines the maximum useful magnification of a microscope. The average human eye under normal conditions can resolve objects as small as 100 micrometer. Dividing the resolution of your eye by the resolution of the microscope objective gives the magnification required to make all details resolved by the objective large enough to see with your eye. Greater magnification is called "empty magnification," because it conveys no additional information. In practice, maximum magnifications used are about two times the theoretical useful magnification, to make details easier to see. Thus a 100 objective (NA 1.3) used with a 10 ocular gives a 1000 magnification, even though the theoretical useful magnification is only about 430. Microscopes and imaging 1

II. USING COMPOUND MICROSCOPES... Despite improvements in optics and introduction of new and powerful computer imaging techniques, the light microscopes used in labs today are little different from those used 50 years ago. The Leitz SM-Lux and Nikon Labophot microscopes you will be using are typical examples of modern upright-compound microscopes. Important parts of the microscope are: 1. A light source, for illuminating the specimen. The light source on the SM-Lux is regulated by a knob on the right side of the base, toward the rear. A field diaphragm, located on the base under the condenser, adjusts the angular spread of the illumination. 2. A condenser, which focuses the illuminating light on the specimen. The condenser of the SM-Lux can be used for low magnification (4x-10x) by removing its upper element (carefully pull the small black knob at the front of the condenser). This condenser contains setting for bright-field microscopy (H) as well as four different phase annuluses for phasecontrast microscopy. A diaphragm in the condenser (also called the sub-stage condenser) can be used to adjust light, but reducing the light with this condenser may also reduce resolution. 3. A stage, upon which the specimen to be observed is mounted. In the simplest case, the stage contains clips on a flat surface for holding a slide or specimen. In more complicated microscopes, the stage contains a graduated carriage for carefully positioning the specimen. Most modern microscopes are focused by moving the stage up or down, using a coarse or fine adjustment. 4. An objective lens, which forms a magnified image of the specimen. Microscope objectives are often described by two characteristics: (1) the magnification factor (typically ranging from 4-100x), and (2) their numerical aperture (NA), which determines their maximum theoretical resolution (see below). The magnification and numerical aperture are engraved on the barrel of most objectives. Low power objectives are often referred to as "dry" lenses, because no immersion oil is used between the coverglass and the objective. Some high power objectives are also used dry, and are often referred to as high-dry objectives. At higher power, a drop of oil is often placed between the coverslip and objective. Such oil-immersion objectives usually have oil or oel engraved on their barrels. Objectives optimized for differential interference microscopy often have DIC engraved on their barrel. Special water immersion lenses are often used for observing living cells, without requiring a coverslip or oil. Modern microscopes often have a turret or nosepiece which contains several different objective lenses. 5. Oculars, or eyepieces, which magnify the image formed by the objective. The eyepieces of most microscopes provide a magnification of 10x or 20x. The spacing of the oculars on binocular microscopes is usually adjustable to account for different interpupillary distance. In most microscopes, the light source and condenser are below the specimen stage, while Microscopes and imaging 2

the objectives and oculars are above. This configuration is called an "upright microscope." Some microscopes, referred to as "inverted microscopes," are built in the opposite orientation. Inverted microscopes are commonly used for the examination of living cells in petri plates, and for electrophysiology experiments where cells must be impaled with microelectrodes from above. III. ADJUSTING COMPOUND MICROSCOPES FOR KOHLER ILLUMINATION... To obtain maximum resolution of cellular detail, the light source and condenser must be adjusted to provide optimal illumination. One configuration, called "Kohler illumination," focuses an image of the field diaphragm at the specimen plane. To adjust a microscope for Kohler illumination: 1. Carefully focus on a specimen, first at 10x, then at 40x. Use a dusty slide, a commercially prepared slide, or a slide of cheek epithelial cells for the alignment procedure. To make a slide of cheek epithelial cells: (a) use a clean toothpick to scrape the inside of your cheek; (b) gently dab the same end of the toothpick in a drop of water on a clean microscope slide; (c) cover with a coverslip. 2. Set the condenser selector to the bright field position (Usually H or J), and open the condenser diaphragm fully (lever on the condenser). 3. Close the field diaphragm completely. 4. Looking through the oculars, you should see a small circle of illumination, or a larger circle of light with a dark center. Carefully focus the condenser (move the condenser up or down relative to the specimen stage) until the illuminated circle is as small as attainable, and the edges are as sharp as possible (there will be some blurred color fringe superimposed on a fairly sharp edge, due to chromatic aberration). 5. Open the aperture diaphragm until the illuminated circle is just smaller than the total field of view. Center the illuminated circle in the field using the knurled adjustment screws on the condenser. 6. Open the aperture diaphragm until the illuminated circle just exceeds the entire field of view. 7. The microscope is now adjusted for optimal ("Kohler") illumination. You should check the condenser focus and alignment when you change objectives. With many phase microscopes (but not the SM-Lux) you must now align the phase annulus in the condenser with the phase ring in the objective. Turn the condenser slider to select the phase annulus number that matches the phase number of the objective in use. Carefully remove one of the oculars, and look down the microscope tube. You should see a dark ring (the phase ring in the objective) superimposed upon a bright ring (the phase annulus in the condenser). A "phase telescope," if available, magnifies the rings making them easy to see. If the rings are not concentric, carefully adjust the condenser following the manufacturers instructions (usually small Microscopes and imaging 3

wrenches are provided for this adjustment). IV. METHODS FOR ENHANCING CONTRAST IN TRANSPARENT SPECIMENS... A. Staining cells for microscopy. Many biological specimens are both transparent and colorless, making them difficult to observe under the microscope. Early biologists used combinations of different stains (usually obtained from the textile industry) to visualize different cellular components. In some cases, living cells will absorb stains from their surrounding. These vital dyes allow the visualization of structures within living cells. However, most staining procedures require killing and preserving the cell or tissue first, a process known to cytologists as fixation. B. Phase contrast microscopy, as pioneered by Zernicke in 1930, is one of the most widely used methods for observing cells and tissues. Phase contrast microscopy takes advantage of the differences in refractive index of otherwise transparent biological samples. The refractive index of an objective is a measure of the speed of light through the object, relative to the speed of light in air. For example, light travels faster through air (N=1.0) than through oil (N ~ 1.5). In a phase contrast microscope, the slight differences in refractive index of cellular components cause small shifts in the phase of the light waves as they pass through cells. Combining the phase-shifted light which passed through different parts of the specimen results in interference, which is visible as differences in brightness. Phase contrast microscopes use a special condenser containing a phase annulus and a matching phase ring in the objective. You can see the phase ring by carefully removing the objective from the microscope and looking through it towards a light (ask the instructor or a TA before removing the objective). The phase annulus in the condenser and the phase ring in the objective must be carefully matched in size. The size of the phase ring is usually engraved on the objective, as a number from 1-4 (ex: phaco 2). Phase condensers usually contain a rotating slider with several different phase annuluses, to match the objectives commonly used on the microscope. To use phase contrast microscopy: 1. First, focus on your specimen and align your microscope for Kohler illumination. 2. Select the appropriate phase objective, and note its phase number (which is engraved on the objective barrel). 3. Turn the condenser selector to choose the appropriate condenser phase ring. 4. Observe your specimen. It should now appear in phase contrast. C. Dark-field microscopy provides a sensitive and strikingly detailed image of living cells. In this technique, cells are illuminated in a manner that makes the cells look bright against a dark background. Dark-field microscopy requires a special condenser. However, many phase microscopes give a reasonable dark field effect. Using the 10 objective ph1 objective, set the condenser to the phase 3 position, and open or close the field diaphragm as necessary. Also try the 40 ph2 Microscopes and imaging 4

objective with the phase 4 position on the condenser. D. Nomarski differential Interference Contrast (DIC) uses polarized light and an interference filter to achieve a shadowed three dimensional effect. It also "sees" a thin plane in focus with relatively little interference from objects not in that plane, so one can view a series of "optical sections" by focusing through the specimen. This method often is better than standard phase contrast for revealing internal details. We have two Nikon microscopes equipped for DIC for your use. These scopes will initially be set up by the instructors. Three things must be set to use them for DIC imaging: 1. Rotate the phase disc to a DIC setting. These are marked to show compatibility with objectives based on "numerical aperture" (NA). This value is printed on the barrel of each objective next to the magnification (e.g., 10 / 0.3 indicates a 10 objective with an NA of 0.3). 2. Slide the polarizing filters into the light path. There are two polarizers: one in the condenser and one above the objective. On our scopes, the top slider is pushed in and the bottom slider is pulled out. 3. Adjust contrast by rotating the bottom polarizer. This is accomplished with a silver knob on the end of the bottom slider. The sub stage iris diaphragm will also affect contrast (see below), but start with it open and try rotating the polarizer first. (On some DIC scopes, contrast is adjusted by turning knurled knob on a small slider inserted just above the objective. This moves a thin glass wedge (you can see these wedges on our scopes, but they are not adjustable). E. Fluorescence microscopy is a very powerful technique for viewing cells and embryos. Fluorescence describes the phenomenon when absorption of light at the excitation wavelength by a molecule (a fluorochrome ) stimulates emission of a longer wavelength of light (the emission wavelength ). Many fluorescent organelle-specific fluorescent dyes, such as rhodamine 123 (stains mitochondria), Hoechst 33258 (stains DNA), and fluorescent phalloidin (stains actin filaments) are commonly used for microscopy. In addition, the conjugation of fluorescent dyes to antibodies allows them to be used for identifying specific proteins within cells or embryos, a technique referred to as immunofluorescence. In modern fluorescent microscopes, the illuminating beam passes though the objective (which thus serves also as the condenser), and matched pairs of emission and excitation filters are mounted in a slider in the body of the microscope. We have several Nikon fluorescence microscopes for examining your embryos. To use them: 1. Turn on the Hg lamp that serves as the light source for fluorescence. 2. Turn on the halogen lamp that serves as the light source for bright field microscopy, and use bright field (or other illumination technique) to locate and identify your sample. 3. To switch to fluorescence, turn off and/or block out the lower light source, move the Microscopes and imaging 5

fluorescence slider to the proper filter combination and open the shutter (a slider on the tube coming from the upper light source) to allow the illuminating beam to reach the specimen. (You should see colored light shining on the specimen.) IV. MISCELLANEOUS STUFF... a. Some other useful tricks for enhancing contrast... 1. Closing down the sub stage diaphragm can increase contrast. Experiment with this control when viewing clear specimens, but remember that you lose resolution. 2. Dark field. Just as one can see dust particles in a beam of sunlight in a dark room, very small objects can be seen against a dark background when they are illuminated from the side. Some microscopes have a dark field (D) setting on the condenser. Alternatively, one can use a phase contrast setting that is "too high" for the objective (e.g., #3 setting on the condenser with a 10 objective). The sub stage diaphragm must be fully open. 3. An effect similar to DIC can be achieved by off axis illumination with our phase contrast microscopes. With the microscope set for bright field and the sub stage diaphragm fully open, begin to move the phase selector disc toward an adjacent setting. At some point before the field becomes dark (roughly 1/3 of the way to the next setting), specimens appear shadowed and reveal features hard to see with normal illumination. Remember that this is not a "legitimate" technique and return the microscope to a conventional setting when finished. b. Calibrating microscope objectives... It is often useful to know the size of objects observed in the microscope. Calibration of research microscopes is usually accomplished by photographing a precision ruled slide, called a stage micrometer. However, even without a camera, it is possible to estimate the size of objects by measuring the field of view obtained with each microscope objective (only needs to be done once per objective): 1. Carefully focus on the stage micrometer with the 4 objective. Note the diameter of the field of view. The larger divisions correspond to 100 micrometers. The smallest divisions are 10 micrometers. 3. Repeat the above procedure for each objective. With the 100 (oil) lens, carefully place a drop of oil on the micrometer before focusing. After your done, clean the oil off the micrometer with a lens tissue moistened with xylene. To estimate the size of a specimen observed in the microscope, estimate its size in relation to the total field diameter. Since you have measured the field diameter for each objective, you can estimate the size of any object. A graduated eyepiece reticle (not available in class) is very helpful in these estimates. Microscopes and imaging 6

V. TIPS FOR USING STEREO (DISSECTING) MICROSCOPES... Stereo microscopes generally provide less magnification and resolution than a compound microscope, but provide a three dimensional image, a larger field of view, greater depth-of-focus, and a larger working space between the objectives and specimen. Many have variable magnification selected by rotating a control ring or knob. Stereomicroscopes often do not have built in illumination or a condenser. Lighting is more "casual" and intuitive if you can work a reading lamp, you're most of the way there. However, it pays to experiment. A few useful tricks: For opaque specimens, try lighting from a fairly high angle above the stage. For pale or transparent subjects, lighting at a shallow angle (almost horizontal) and a dark background will have much of the same effect as dark field. Some microscopes will have a transparent base plate and a mirror below the stage for transmitted light illumination (also for transparent specimens). The mirror can be adjusted to vary contrast. Manipulating objects under the microscope takes some practice. You need to develop a "feel" for the position of your hands and the magnified response to any movement. In general, I find it is best to work at the lowest magnification that provides a clear view of the object being manipulated: greater depth of field and field of view may help more than greater magnification. VI. PROPER CARE OF MICROSCOPES... Microscopes are expensive precision optical instruments. Several precautions will prevent damage: 1. Always carry with two hands: one holding the stand and one under the base. 2. Focus carefully, to avoid crashing the objective through your slides. 3. Use the minimum amount of immersion oil. Wipe up any oil spilled on the stage or other areas of the microscope. Oil collects dirt and grime, which gums up the mechanical parts. Its also bad for the optics. 4. Always clean oil from the objectives when finished for the day. If used frequently with the same brand of immersion oil, simply wipe the excess oil off with a clean, dry lens paper (Do not use kimwipes or kleenix). Use gentle wiping, to avoid scratching the lens. If used infrequently, wipe oil off with a lens tissue moistened with xylenes (or a commercial lens cleaning solution). VII. USING THE CCD CAMERAS AND NIH IMAGE FOR IMAGE CAPTURE... We have six Cohu CCD cameras and MacIntosh work stations for capturing images of your results. The CCD cameras can be attached (with the appropriate adaptors) to either Microscopes and imaging 7

compound or stereo microscopes, depending on the specimen you are imaging. Follow the steps below to set-up, collect, save, and print your images... A. Setting up the camera... 1. Use the procedures described illuminate the specimen, focus, adjust contrast etc. 2. Ask the instructors to attach a video camera to the vertical tube on the trinocular head of the microscope you want to use, via the appropriate adapter. Make sure the CCD camera is connected to the computer (white lead), plugged in, and turned on. Move the beam splitter on the trinocular head to send light to the camera. It is probably best to spilt the light so you can also view through the eyepieces. 3. Switch on the computer monitor. Using the mouse, double click on the microscope icon for NIH image. This loads the imaging program (NIH image). 4. Pull down the "special" menu and click on "start capture". You should now see an image on the monitor (but check the next step if the screen looks blank). In various circumstances, the computer will automatically stop acquiring new images. If you find that the image on the screen no longer changes when you move the specimen or adjust the microscope, select "start capture" again. 5. Adjust the image brightness. First, try adjusting the intensity of the illumination. However, the brightness of the image on the screen may not correspond to what you see in the microscope. At the extreme, it can be all white or all black. If this is the case, try adjusting the shutter speed and gain of the CCD camera. The shutter speed ranges from off (the most sensitive) to 1000" (the least sensitive). Use a small screwdriver to turn the arrow inside the opening. There is also a "gain" setting that gives a continuous adjustment over a narrower range. Note however, that as the gain is increased, the image becomes noisy, and has a salt-and-pepper appearance. This can be reduced by averaging several frames (see below). Although it is best to set up the microscope and camera to collect the best image from the start, it is also possible to adjust image brightness by changing the look-up tables (LUTs) that map the CCD output to the display. Click on "Map" near the bottom left of the screen. This will open a window that allows you to fine tune sensitivity and contrast. The window will display a graph that shows the shape of a light sensitivity curve. You can click and "drag" either end while the other end remains anchored. Steeper means more contrast, flatter means more intermediate shades. You also can click on the middle of the graph and drag right or left without changing slope. Right is brighter, left is darker. Similar effects are obtained with two bars below the graph "B" for brightness and "C" for contrast. Click on the black indicator in either bar and drag left or right. Note that the graph changes as you do this. If you can't get to an appropriate brightness with these controls, check the camera speed setting as described above. Clicking on the miniature graph to the left below the main graph resets to standard values, the other one (step graph) gives maximum contrast. Microscopes and imaging 8

B. Capturing, annotating, and saving images using NIH image... 1. Compose, focus, and adjust the brightness of the image, as described 2. Click on the image, or choose stop capture from the Special drop down menu. This will stop capture, and freeze the display on the last image collected. 3. You can save the file by selecting save as from the Files drop down menu. Save the file to a folder with your name on the hard drive of the local computer (put your folder in the 3235 students stuff folder), or to the server. Since image files can be large, it is often best to crop your image, saving only the portion of interest. This can be done by selecting a region of interest (ROI) with the marquee tool (the dashed box) in the tool panel on the left side of the screen, before saving you image. Only the ROI will be saved. It is best to save a raw image, and add any text or other markings later. 4. A number of typical image processing "tools" can be chosen by clicking the appropriate icons in the tool panel on the left side of the screen. The Text tool ( "A" icon) allows you to add labels. A bar that can be moved with the mouse shows where the lettering will start, and you just type from the keyboard. At a minimum, you probably should add you name, the date, and some specimen identification. Other features will allow you to manipulate images in many ways. If you are familiar with simple image processing, you probably can figure many of these out (or ask the instructors). C. Tips for dealing with image noise and low-light (fluorescence) images... Occasionally, your images may appear noisy, because they required collection at high gain settings. Much of this noise can be eliminated by filtering or averaging several successive frames during image collection; 1. Start capturing. 2. From the Special pull down menu, choose Average images. 3. Enter an integral number of frames to average (try 5-10 for starters). Leave all the other check boxes blank. Start averaging. 4. The hourglass cursor will appear as the frames are averaged and image collected. When the cursor changes back to cross hairs, the image is complete. Save as usual. The camera/image capture board allows you to integrate (add) frames, which is useful for very dim images. 1. Start capturing. Microscopes and imaging 9

2. From the Special pull down menu, choose Average images. 3. Enter an integral number of frames to average (try 5-10 for starters). Check integrate on chip. 4. The hourglass cursor will appear as the frames are collected and added. When the cursor changes back to cross hairs, the image is complete. Save as usual. D. Setting up time-lapse recording...time-lapse or video microscopy has proven to be an important tool in studying many developmental processes. Many of the developmental processes you will be examining are quite amenable to time-lapse imaging. To set up a time-lapse recording: 1. Set up the camera and compose you image, as described above. 2. Use the marquee tool (dotted rectangle) to select the area of interest. Since time-lapse imaging requires a lot of memory and disk space, select the smallest area possible. 3. From the Stacks menu, select Make movie... 4. Enter the number of frames desired, and the interval between (either in frames/sec or seconds/frame). 5. Once you start, the computer will first allocate available memory (if there isn t sufficient memory for the number of frames selected, it will collect all it can). Then, it will begin collecting. Don t interrupt the computer while it is collecting images...all data will be lost. 6. After the time-lapse is complete, SAVE your file. 7. Time-lapse images are saved as multi-image TIF files. You can step through the images by using the > or < keys to navigate. You may view the image as a movie by choosing animate stack from the Stack menu. Hitting the numeric keys adjusts the speed ( 1" is slowest; 9" is fast). You also may choose to save the movie as a Quicktime movie (see the instructors). For large image stacks, the instructors may need to change the virtual memory settings, or memory allocated to NIH image. E. Printing and saving images... When you have the composition you want, pull down the "file" menu and select "print file". This will print what you currently have displayed on the screen (or inside a frame) on a printer in lab. Microscopes and imaging 10