Microscopy and Handling a Microscope
A. Pre-Lab Activity- Please read the entire lab before coming to lab, but only complete the prelab activity before lab! The lab will be checked to be sure you have completed all parts before leaving.
Proportions and Scale:
To understand the importance of microscopy and its limitations, one has to understand something of proportion and scale.
Go to the Learn Genetics Website (http://learn.genetics.utah.edu/content/begin/cells/scale/) and use the scroll bar to predict the difference in size of things from a coffee bean to a carbon atom.
How many times greater is the length of a coffee bean compared to a grain of rice?
How many times greater is grain of salt versus an amoeba? What type of organism is an amoeba?
How many times greater is an amoeba versus a skin cell?
How many times greater is an E. coli bacterium than a lysosome?
If you lined-up measles virus particles, how many would you need to have the same length as a lysosome?
Which is bigger a ribosome or tRNA? By how much?
How many times greater is a glucose molecule versus a carbon atom?
Use the internet to define a micron and and angstrom. How do they compare with the width of the human hair in size?
Use the internet to determine the smallest size one can see with the naked eye.
Plant Versus Animal Cells:
Use the Buzzle website (http://www.buzzle.com/articles/plant-and-animal-cell-differences.htm) and explain 7 major differences between plant and animal cells.
Are plants and animals prokaryotes or eukaryotes? Circle one.
What are the major physical obstacles for plant cells when then divide?
B. Lab Activities- Please read the whole lab before lab but do the next sections only during lab.
I. Parts of a Microscope:
Each person should get their own compound microscope with which to work.
You instructor will introduce you to:
1) the proper handling of a microscope
2) how a compound microscope works
3) the parts of the compound microscope for which you will be responsible (circled on Figure 1)
Here are some handling requirements and information for using microscopes. You will be held responsible for this information and for the proper use of the scope.
4) Always pick-up the microscope by the arm AND the base simultaneous.
5) 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.
6) Never touch the lens (ocular 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.
7) Start with the lowest power and move to high power objectives to focus you microscope. ALWAYS carefully watch the lenses move in place above the specimen. You can scratch the lens by rubbing it against the slide!
8) Never leave slides on the stage of the microscope.
9) Some microscopes have lenses designed to use with emersion oil. In this case, you would place a drop of emersion oil on the slide and rotate the lens in place carefully. The idea behind the oil is to reduce the amount of light diffraction as it moves from the specimen to the objective and thus improve the resolution of the sample. ALWAYS use LENS PAPER ONLY to clean the lens when one is finished with oil emersion.
10) ALWAYS use LENS PAPER ONLY to clean the lenses before returning the microscope to the cabinet.
Figure 1- Compound Microscope
(http://www.digitalsmicroscope.com/wp-content/uploads/2011/04/Compound-Light-Microscope-Parts.jpg)
Calculating total magnification- The total magnification is simply the product of the magnification for the objective lens being used with that of the ocular lens. So if the ocular lens had 10X magnification and you were using the 20X objective to observe the specimen, the total magnification would be 10X20=200 times.
The Microscopic Field- When you look through your microscope you will see a circular area that is known as the “microscopic field” once you focus your slide. As your magnification increases the field size decreases because you are literally looking at less of the sample with greater magnification!
Also remember that the sample you will see in the field is a mirror image of the actual sample. Note the direction the sample moves if you move the slide up/down and right/left.
Your instructor will show you how to a wet mount using letters from the papers provided. Cut out the letter “e” from the papers and practice the wet mount technique. Try to keep from getting too many air bubbles underneath your coverslip.
Depth of Field and Planes of Focus- Samples have a measurable thickness, and thus when you focus the microscope there are different depths or layers from which you have to choose to observe (known as “planes of focus”). A thicker sample will have a greater depth of field, so you can’t focus on all planes of focus at once, making the sample harder to focus microscopically. Thus at any one time the other planes of focus will be blurry.
There is one place where using this idea can be helpful. If you are looking at cells on the microscope, you can move the fine focus up and down gently to move through the planes of focus visually and see the outer contours of the cells. Cells that are rounded and smooth are generally healthy. The membrane of dying cells loose the smooth rounded texture visible microscopically.
A Thinking Experiment- You are working with a compound microscope with a 10X ocular lens and using the 4X objective lens. What is the total magnification? Show your calculations.
Then to look more closely at your sample you change to the 60X objective. What is the total magnification? Show your calculations.
Do you see more of the specimen in the field when you used the 4X objective or less compared to when you use the 60X objective? Why?
Remembering the rice grain you saw on the website earlier, do you think the rice grain would have a greater or lesser depth of focus compared to an amoeba? Why?
Using Cells and Microscopy:
1) Compound Microscopy- With many samples, you can simply do a wet mount, perhaps add a little dye to contrast the structures (known as a contrast dye). However, tissue samples have to be handled differently to see the structures in the tissue. They can be simply frozen and sectioned to a few microns in thickness (AKA cryopreserved samples). Or the tissue may be first fixed to cross-link the proteins and help maintain the structures therein, embedded in paraffin (wax) and sectioned to a few microns in thickness (AKA paraffin embedded sections). Embedded sections can be treated with chemicals to remove the paraffin and stained to visualize the structures. Regardless of the type of handling though, all sections must be treated with a nuclear counterstain otherwise it can be very difficult to what exactly in the specimen are actual cells. Often the nuclear counterstain interacts with DNA.
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.
2) Electron Microscopy (EM)- Compound microscopes use light to illuminate an object. The light gows through the sample and is magnified by the objective an ocular lenses. But instead of light 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:
a) 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.
Figure 2- SEM of pollen
(http://en.wikipedia.org/wiki/File:Misc_pollen.jpg)
b) 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)
3) Fluorescence Microscopy- Fluorescence is a high 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.
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)
What is the green and red detecting in the cells of Figure 6?
What is the nuclear counterstain in the cells of Figure 6?
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)
Activity:
Breaking-up into groups of 4-5 and with assistance of your instructor, use the fluorescence microscope to observe the BPAE cells stained similarly to the cells in Figure 6 and 7.
Take some images using the 20X and 60X objectives. What is your total magnification when you use these objectives using our fluorescence microscope?
Thinking Questions:
Could you use immunofluorescence to detect a virus using a compound microscope? Why or why not?
Could you use an antibody to detect a carbon molecule? Why or why not?