1.1: Introduction
Introduction
One of the most useful tools in biology is the microscope, which allows you to view organisms, tissues and cells that are too small to be seen with the eyes alone. Resolving power is is the ability of a microscope to distinguish two adjacent structures as separate. The higher the resolution, the better the clarity and detail of the image. We will use a compound microscope in our laboratory course, with occular eye pieces that magnify objects 10x, and objective lenses that magnify 4x, 10x, 40x, and 100x. Our microscopes also have filters that allow us to view specimens in different ways, most commonly we will us the "BF" filter, which stands for bright-field. The bright-field filter focuses simple white light onto the specimen, creating an image where the background will be bright and the sample will be dark. We also have "Ph1" and "Ph2" filters which stands for phase contrast , where the background will appear dark against a light sample. Phase contrast is often used for observations of unstained organisms, where as we typically stain our specimens when we use the bright-field filter. Staining techniques almost always will kill the cells, however, it provides great contrast for observations, and can also be used as tool to detect and differentiate particular structures of interest. For bright field and phase contrast image examples, please see the Prokaryotic Cells lecture slides. You will learn about other, more advanced types of microscopy during lecture and during the Microscopes Part II lab.
A. How to Properly Use the Microscope
1. Always hold the arm of the microscope with one hand and support the base with the other
2. Never drag the microscope across the lab table
3. Before use and after each use
A. The stage should be as low as possible and stage controls centered
B. The lowest objective should be above the center of the stage
C. No immersion oil should be on the microscope (please check all objective lenses)
1) Clean lenses with special lens paper only
2) Use designated lens cleaning solvent if necessary (ask instructor before use)
D. The stage should be free of slides, dimmer at 0, light switch off, cord wrapped neatly
4. To focus a slide
a. Place slide onto stage, right side up, and secure with stage clip
b. Move stage all the way up, towards the scanning or low objective using the coarse adj. knob
c. SLOWLY focus the slide by using the coarse adj. knob to move the stage down
d. Adjust oculars as necessary (width and focus of each ocular lens, if applicable)
e. Once focused, move to the next, higher, objective lens, but don’t move the stage
1) Our microscopes are parfocal – this means that when one lens is in focus, the others are as well; thus, higher magnifications will only require slight adjustment
f. Focus using the FINE adj. knob ONLY for the 40x and 100x objective lenses
g. When finished, rotate the nosepiece until the lowest objective lens clicks into place
h. Move the stage all the way down, as low as possible
i. Clean off any remaining oil
j. Lower the dimmer, turn off the light and unplug the microscope; please grasp the plug, not the cord when unplugging (continued on back)
k. Wrap the cord neatly and replace the microscope cover before storing; be careful not to hit the table or sides of the cabinet when storing the microscope
B. How to Use the Oil Immersion Lens (100x Objective)
At high power (1000X total magnification), the bending of light, or refraction, is magnified and causes the image to be distorted. To remedy this, the space/air between the slide and objective lens is replaced with oil. This will minimize the refraction so we are able to obtain a sharp image at 1000X magnification.
Procedure:
1. Place the prepared slide on the stage and secure with the stage clip
2. Using the 10X objective lens, focus the slide and center the object in the field of view
3. Rotate the nosepiece to the 40X objective lens and focus using the FINE adjustment knob only
4. Rotate the nosepiece until you are at an intermediate position between the 40X and 100X objective lenses (halfway between the 40X and 100X objective lenses)
5. Place one small drop of immersion oil on the center of the viewing area of the slide (use the light from the condenser as a guide). Do NOT move the stage.
6. Rotate the nosepiece until the 100X objective lens clicks into place. Look to make sure the tip of the lens is touching the oil
7. Focus SLOWLY using the FINE adjustment knob only
8. When finished, make certain to clean off any oil from the lenses and slides before returning your microscope to the cabinet and your slide to the designated tray.
C. Hints and common errors
1. If the microscope light will not turn on, check the dimmer, and turn it past 0
2. If the light still seems low or dim, try adjusting the iris diaphragm, make sure it is not closed
3. If you cannot see anything through the oculars even though the lamp is on, try rotating the nosepiece to make sure the objective lens is clicked into place
4. If you are having trouble keeping your object in focus, check to make sure the slide is flush with the edges of the slide holder and stage clip
5. If the image appears clear on the 10X objective lens, but blurry on the 40X objective lens, check to make sure the 40X objective lens is clean and free of oil; if this still does not work, check to make sure your slide is right-side up (specimen on top, facing the objective lenses)
6. Move the condenser to change the intensity of light focused on your sample: in the up position the light intensity increases, while in a lower position, the light is less intense/more diffuse. We generally move the condenser high for stained samples, but lower for unstained samples to improve contrast
7. A diffuser (which looks like an opaque glass ring) should be resting above your light source. If you do not have one, please notify your instructor.
8. Remember, once you use oil, you can’t go back to lower objective unless you clean off all the oil. Only use oil when instructed and be sure your image is clearly in focus on 40x first.