13.14: Lab Technique - Light Microscopy

Properties of Microscopy

• Magnification or Magnifying power: indicates how many times (X) larger an object appears to its actual size
  • expressed as total magnification
  • total magnification is calculated as the combined power of the ocular lens and the objective lens (i.e., ocular lens power x objective lens power)
  • e.g. with an ocular of 10X and the 4X objective lens, the total magnifying power or magnification is 40X (4 x 10)
• Resolution or Resolving power: the ability of a microscope to distinguish small objects
  • indicates the sharpness of the image
  • given as a value representing the smallest distance between two points at which they are still distinguishable from each other
  • the higher the resolution, the smaller the resolution distance
  • 0.2 um apart for a compound light microscope
• Depth of Focus: the distance or depth that is in focus at any one time (Figure \(\PageIndex{1}\))
  • sometimes also called "depth of field"
  • objects that are closer or farther away from the lens will go out of focus
  • indicates how far the stage can be moved and still keep the object in focus
  • the higher the total magnification, the thinner the depth of focus
• Field of View (FOV): the illuminated circular area visible under a microscope (Figure \(\PageIndex{1}\))
  • the FOV diameter represents the distance from one edge of the circle to the opposite edge
  • the greater the magnification, the smaller the diameter of the field of view

Prepared Slides

Prepared slides contain organisms, cells, or thin sections of tissues that have been fixed and stained with special dyes (or antibodies) to highlight specific structures. These slides can be prepared in the laboratory prior to viewing or purchased from a manufacturer. Prepared slides that are purchased from companies are typically labeled with the following information:

• specimen name: identifies what is on the slide
  • can contain the species name and/or the common name of the specimen
  • e.g. onion root tip, Euglena sp.
• stain or dye used: identifies the stain or dye used in the preparation (if any)
  • specific stains are used to highlight specific structures
  • common stains:
    • Hematoxylin and Eosin or H&E: hematoxylin (blue/purple stain for nuclei), eosin (pink stain for positively-charged structures like the cytoplasm)
    • Methylene Blue: blue stain for nuclei
    • Iodine: blue/black stain for the cytoplasm
    • Crystal violet: purple stain for Gram-positive bacteria
    • Safranin: red/pink stain for Gram-negative bacteria
• preparation type: explains how the specimen was prepared (Figure \(\PageIndex{2}\))
  • smear - the specimen is spread thinly across a slide to allow for viewing of individual cells
  • squash - the specimen is pressed (i.e., squashed) between the slide and coverslip to spread out the specimen for easier viewing
  • whole mount (‘w.m’) - the entire organism or structure
  • section - a slice of the organism or tissue that has been cut
    • ‘x.s’ or ‘c.s’ for cross section

Wet Mounts

Wet mount are prepared by placing the sample in a drop of liquid onto a clean microscope slide. The liquid can be water, isotonic saline or another type of fluid appropriate for the organism or tissue. A coverslip is then placed over the sample, creating a little “fluid sandwich”. Air bubbles commonly become trapped under the plastic coverslip. These can be distinguished from the sample since they are usually perfect circles with very dark borders. Wet mounts are easy to prepare and allow for the observation of live tissues, cells, or organisms (Figure \(\PageIndex{2}\)).

Basic Protocol:

1. Place the microscope on a flat, clean surface
2. Plug the microscope in and turn on the light course
3. Select the lowest magnifying lens (i.e., 4X or 5X) by rotating the revolving nosepiece
4. Place the slide on the stage and secure it with the stage clips
5. Use the mechanical stage knobs to center the specimen in the slide over the light source
6. While looking through the eyepiece (i.e., the ocular lens), use the coarse focus knob to lift the stage to bring the object into the field of view
   • adjust the light level if needed by using the diaphragm (i.e., the iris)
7. Use the fine focus knob to sharpen the focus
8. Rotate the objective lens nosepiece to change to a higher magnification lens
9. Re-center and re-focus on the specimen
   • higher magnifications will require increasing light levels

Wet Mount Protocol:

1. Place a drop of liquid (e.g., water, saline) on a clean microscope slide using an eyedropper
2. Place the specimen in the drop of liquid
   • alternatively, the specimen may be placed onto the slide first and then the drop of liquid added
3. Place one edge of a coverslip on the microscope slide so that it touches the drop and slowly lower the cover glass onto the slide

Smear Protocol:

1. Place a small drop of the sample on a clean microscope slide
2. Spread the sample evenly across the slide using the edge of another slide (i.e., the spreader slide)
3. Air-dry the smear completely
4. Fix the sample to preserve and kill cells
5. Stain using an appropriate dye
6. Rinse, dry, and view under the microscope

Squash Protocol:

1. Place a small tissue piece on a clean microscope slide
2. Add a drop of stain (if desired) or water
3. Cover with a coverslip
4. Gently press down (or tap) the coverslip with a blunt object to squash the tissue
5. Observe under the microscope

Oil Immersion Protocol:

1. Attempt to focus on the object using the highest power “air” medium lens (i.e. the 40X lens).
2. Lower the stage slowly and carefully.
   • Do not move the stage side to side or front and back or the object will move in the field of view
   • If this is the case, return to the 40X lens and find and focus on the object again
3. With the stage in a lowered position, place a small drop of immersion oil on the area of the slide where the specimen is located.
4. Put the 100X oil immersion lens into place.
5. Slowly raise the stage until the bottom of the lens touches the oil. The moment the lens touches the oil, stop moving the stage.
6. View the specimen object. Use the fine focus to sharpen the focus if needed.
7. When done, spray some lens paper with a lens cleaner solution and gently dab it onto the bottom of the oil immersion lens to remove the oil. To prevent scratching the lens, use only lens paper and do not rub the lens.
8. Clean the prepared slide with dilute soap and water.

Immunofluorescence Protocol:

1. Fix the sample (i.e., cells or tissue section) for 10–15 min at room temperature in Fixative Solution.
2. Wash the sample 3 times for 5 minutes each with Wash Buffer at room temperature.
3. If detecting intracellular targets, permeabilize with Permeabilization Solution for 5–10 min at room temperature and then wash 3 times for 5 minutes each with Wash Buffer
   • if detecting cell surface targets, skip this step.
4. Incubate the sample in Blocking Buffer for 30–60 min at room temperature to reduce non-specific binding.
5. Incubate the sample in Blocking Buffer containing the diluted primary antibody for 1–2 hours at room temperature
   • incubation can be done overnight at 4°C in a humidified chamber (to prevent buffer evaporation).
6. Wash 3 times for 5 minutes each with Wash Buffer at room temperature.
7. Incubate the sample in Blocking Buffer containing the diluted secondary antibody for 1–2 hours at room temperature in the dark.
8. Wash the sample 3 times for 5 minutes each with Wash Buffer at room temperature in the dark.
   • during the final wash, a nuclear stain like DAPI can be added to counterstain the cells.
9. If using a chamber slide, carefully remove the upper chamber and the gasket to yield the glass slide.
10. Mount a coverslip onto the glass slide.
    • mounting can be done using a commercial mounting medium.
    • if using a mounting medium containing a counterstain like DAPI (e.g. Vectashield), do not use DAPI in step #8.
11. Image the cells using a fluorescence microscope with the appropriate filters for the fluorophores used (e.g., FITC, RITC, Alexa Fluor, DAPI).