BIO 7505: Biology: a Molecular Approach, Laboratory

BIO 7505: Biology: A Molecular Approach, Laboratory Microscope Theory and Operation

I. What Microscopy Is:

As your instructor lectures them, write-in the following definitions:

1. Microscopic anatomy

2. Compound light microscope

3. Magnification

4. Resolution

5. Contrast

6. Brightness

II. Proper Care of a Compound Light Microscope:

Read the following instructions for how to care and transport a compound light microscope. After you have read the instructions, go to the microscope cabinet, and safely transport a microscope to your lab bench. Uncover the scope but please do not plug it in or turn it on yet.

 Always pick-up the microscope by the arm AND the base simultaneously.  Always be sure the cored is secured before moving the scope anywhere or using it. Microscopes are literally worth thousands of dollars. The cord needs to be secured to prevent accidents. If someone trips over a cord or catches a cord when they walk by a bench and the microscope falls on the floor it will break.  Never touch the lens (eyepieces or objectives) with your hands. To change lenses, turn the rotating nosepiece (also called a turret) but NEVER touch the lenses themselves. They unscrew and are extremely expensive to replace should one fall on the floor and crack.  Start with the lowest power and move to high power objectives to focus your microscope. ALWAYS carefully watch the lenses move in place above the specimen. You can scratch the lens by rubbing it against the slide!  Never leave slides on the stage of the microscope.  Before you put away your microscope, clean it as described below.  When you put away your microscope, rotate the 4x objective into place, lower the stage to its lowest point, secure the cord, and cover the microscope.

1 III. Identifcation and Function of Microscope Parts:

Use the diagam on the next page to identify the following microscope parts. Then, as the instructor lectures them, write-in their functions.

1. Ocular lens (eyepiece)

2. Interpupillary distance

3. Revolving nosepiece

4. Objective lenses

5. Specimen holder (mechanical stage)

6. Stage

7. Knobs controlling movement of mechanical stage

8. Condenser

9. Iris diaphragm level

10. Illuminator condenser

11. Light intensity control knob

12. Base

13. Arm

14. Coarse focus knob

15. Fine focus knob

2 Figure 1- Compound Microscope (http://www.digitalsmicroscope.com/wp-content/uploads/2011/04/Compound-Light-Microscope- Parts.jpg)

IV. Cleaning Microscope Lenses:

Read the information below. Then, obtain lens paper and isopropyl alcohol, and clean your microscopes eyepieces, objective lenses, condenser, and illuminator condenser. Also, clean those parts before you put your microscope away today.

Microscope lenses, condensers, and illuminators get dusty and smudged and need to be cleaned. To prevent scratching and damage, lenses can be cleaned with only two things: lens paper and/or alcohol. Lens paper is grit free and will not scratch the lenses. Alcohol will dissolve and clean away smudges from fingers and eyes. Do not use any other kind of paper! Do not use any other liquid!

3 V. Total Magnification:

Read the information below. Then, calculate total magnification when viewing specimens for each objective lens.

Most compound microscopes have four objective lenses to give four different levels of magnification. The four different levels of magnification from lowest to highest are scanning, low power, high power, oil immersion. For our microscopes, the scanning is 4x, low power is 10x, high power is 40x and oil is 100x.

The eyepieces also magnify. Our eyepieces are 10x.

The total number of times an object is magnified is called total magnification and is equal to the magnification of the eyepieces times the magnification of the objective lens used.

1. Total Magnification for Scanning Objective

2. Total Magnification for Low Power Objective

3. Total Magnification for High Power Objective

4. Total Magnification for Oil Immersion Objective

VI. Viewing a Specimen, Part 1:

Follow the instructions below:

 Obtain a letter e slide.  Adjust your seat height so can look into the oculars comfortably.  If you wear glasses you do not need to wear them when using the microscope unless they correct a severe astigmatism. Near- and far-sightedness can be corrected by adjusting the microscope focuses. However, if you are comfortable wearing your glasses while looking through the eyepieces, you may leave them on.  Use the coarse focus knob to move the stage down.  Use the revolving nosepiece, not the objectives, to rotate the 4x objective into place. You should feel a click to indicate it is properly positioned.  Use the condenser knob (your instructor will show you where it is) to adjust the condenser to its highest position.  Move the iris diaphragm lever all the way to the left.  Use the clip to insert the e slide into the mechanical stage.  Set the light intensity to its middle setting.  Plug in and turn on the microscope.  Use the mechanical stage knobs to center the e over the light. 4  Look through both eyepieces at the same time. Then, adjust the interpupillary distance. While doing so, gently push apart or pull together the eyepieces to see only one image.  Use the coarse focus knob to move the stage up but do not ram the stage into the objective. Then, bring the e into coarse focus.  Use the fine focus knob to bring the e into clear, sharp focus.  Using the mechanical stage knobs, center the e in the middle of the viewing field.  Use the revolving nosepiece to rotate the 10x objective into place.  Using ONLY the fine focus, bring the e into clear, sharp focus.  Using the mechanical stage knobs, center the e in the middle of the viewing field.  Use the revolving nosepiece to rotate the 40x objective into place.  Using ONLY the fine focus, bring the e into clear, sharp focus.  Change the light intensity setting and observe how that affects the image.  Move the iris diaphragm lever to the right and observe how that affects the image.

VII. Image Orientation, the Iris Diaphragm Lever, and the Effects of Magnification on Image Quality:

As your instructor lectures them, write-in the following definitions:

1. Image orientation

2. Iris diaphragm lever

3. Centering

4. Brightness

5. Field

6. Depth of field

7. Working distance

8. Parfocal

5 VII. Viewing a Specimen, Part 2:

Follow the instructions below to prepare and view a wet mount of your cheek cells:

 Obtain a slide, cover slip, toothpick, and bottle of methylene blue.  Put a drop of methylene blue on a slide.  Gently scrape the inside of your cheek with the flat side of a toothpick. Scrape lightly.  Stir the end of the toothpick into the water and throw the toothpick away into the orange bag in the beaker.  Place a coverslip onto the slide. Put one edge of the coverslip on the slide and then gently drop the other edge into place. See the Figure below. Your goal is to try and not produce air bubbles in the fluid as you lower the coverslip into place.  Place your wet mount into the microscope, and locate cells under scanning or low power. Cells may be hard to see, but they can be found. As you learned above, adjust coarse and fine focus, light intensity, and the iris diaphragm lever.  Once you think you have located a cell, center it in the field, switch to high power, and using only the fine focus knob, bring the cell into clear, sharp focus. You should be able to view the nucleus, cytoplasm, and plasma membrane of the cell.

Coverslip

Methylene blue

Cheek cells Slide

Wet Mount Preparation

VIII. Electron Microscopy:

Read the information below:

The compound microscope is a valuable tool to microscopic things in the lab, but it has its limitations. Most notably, compound microscopes have a maximum magnification of 1000X , beyond which light itself fails to properly resolve structures. To improve on this method several alternatives are possible now.

Electron Microscopy (EM)- Compound microscopes use light to illuminate an object. The light goes through the sample and is magnified by the objective and eyepieces. But instead of light if you use electrons which are tiny, it is possible to see even smaller objects in the specimen. Electron microscopes magnify specimen 1,000,000 times! So you can see ribosomes, proteasomes and many other structures in cells. Typically the sample is coated with an electron dense material like gold. The sample is then bombarded with electrons by the EM and imaged.

There are 2 types of EM: Scanning EM (SEM)- Samples are kept whole, coated and bombarded with electrons in an electron microscope. The electrons scan across the surface of the object so surface structures can be seen. See Figure 2 which shows pollen visualized by a SEM. 6 Figure 2- SEM of pollen (http://en.wikipedia.org/wiki/File:Misc_pollen.jpg)

Transmission EM (TEM)- Samples are sectioned, coated and bombarded with electrons in an electron microscope. The electrons transmit through the specimen and the resulting image is magnified. TEM allows the visualization of internal structures in cells (see the mitochondria in Figure 3).

Figure 3- TEM of Mitochondria. Note the scale is in nanometers. The arrows indicate internal membranes of the mitochondria. (http://www.bing.com/images/search?q=Transmission+Electron+Microscopy+ +and+pic+and+mitochondria&view=detail&id=2E733536D3BAAD4541859FC0CB12E930FF6B14AD)

7 VIII. Fluorescence Microscopy:

Read the information below. Then,if there is time at the end of lab, your instructor will take you to see some flourescent microscopic images.

Fluorescence is a highly sensitive method of tagging things with fluorescent molecules which can be stimulated with one wavelength of light and emit a different wavelength which has an associated color. For example, the molecule Fluorescein Isothiocynate (FITC) is stimulated with 494 nm light and emits 521 nm light which appears green (Figure 4).

Figure 4- Molecular Structure of Fluorescein Isothiocynate (FITC) (http://en.wikipedia.org/wiki/File:FITC-2D-skeletal.png) So if you chemically attach FITC to a protein and treat it with 494nm light, the protein will glow green. One can use this principle in a technique called Immunofluorescence by binding fluorescent molecules like FITC to immunological proteins called antibodies.

Antibodies tag non-self for removal by the immune system. One finds antibodies in many places in an organism, but most people think of antibodies as being in the blood. For example, when someone is infected with bacteria, immune cells produce antibodies to mark the bacteria for removal. They work very well because antibodies are highly specific for the target non-self protein (e.g. in this case bacteria). They will only bind to the specific target to which they were made.

It is thus, possible to inject biomolecules you need to study into an organism. The biomolecules will be recognized as non-self and the organism will induce antibodies against them. Typically the blood containing the antibodies are harvested and used in biotechnology to detect proteins in a cell. In immunofluorescence, they are chemically conjugated with a fluorescent molecule like FITC and used in experiments.

8 Figure 5- FITC-Conjugated Antibodies Binding to One HUGE Cell on a Slide. The pink stars represent the target proteins. The yellow arrows are antibodies and the green bursts are the FITC after it is stimulated with 494 nm light.

The sample is placed on the stage of the fluorescence microscope which is set to treat the samples with the correct wavelength of light to excite the fluorescent molecule on the protein. It is also set to detect the colored emission from the fluorescent molecule and produces a colored image. See Figures 6 and 7.

Figure 6- A Real Example of Immunofluorescence Used to Detect the Cytoskeleton in Bovine Pulmonary Artery Endothelial (BPAE) cells. FluoCells® prepared slide #2 contains BPAE cells, but stained with redfluorescent Texas Red®-X conjugated actin antibodies, mouse monoclonal anti-α-tubulin in conjunction with green-fluorescent BODIPY® FL for labeling microtubules and blue-fluorescent DAPI for labeling the DNA in the nuclei. (www.invitrogen.com) Figure 7- FluoCells® prepared slide #1 contains bovine pulmonary artery endothelial (BPAE) cells stained with a combination of fluorescent dyes. Mitochondria were labeled with red-fluorescent MitoTracker® Red CMXRos, actin was stained using green-fluorescent Alexa Fluor® 488 conjugated to actin antibodies, and blue-fluorescent DAPI was used to label the DNA in the nuclei.

(www.invitrogen.com)