laboratory

Assignment 4: Microscopes and Cells

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Work sheet for assignment 4
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Activities:
ACTIVITY 1. MICROSCOPES
Light microscopes
Electron microscopes
ACTIVITY 2. PROKARYOTES: SINGLE-CELLED ORGANISMS
ACTIVITY 3. INTRODUCTION TO EUKARYOTIC CELLS
  Relative size of cells and organelles
  Single-celled eukaryotes
  Structure of an animal cell

Before you attempt this exercise you should have a thorough understanding of the material in the Introduction to Cells and Prokaryotes: Single-celled Organisms topics, plus the first subtopic in Eukaryotes: Animal Cells I. As you should remember from this material, cells are life's basic building blocks. Humans as well as most multi-cellular organisms develop from one cell, a zygote, itself the product of two very different types of cells that have fused together, an egg and sperm. As adults, our cells number into the trillions, including approximately 300 different functional types or specialized cells. Organisms very different from ourselves, such as plants, are also composed of a large number of specialized cells. On the other hand, many organisms consist of only one cell. In single-celled prokaryotes, the cell is structurally very different from the eukaryotic cell of more complex single-celled organisms. Eukaryotic cells also form the tissues and organs of almost all multi-cellular organisms. Thus, we begin our study of life by looking at the basic structure of prokaryotic and eukaryotic cells. Any examination of cells must also consider the most important instrument used to study cells, the microscope.  Since most cells are too small to see with the naked eye, several types of microscopes have been developed to magnify cells. This enables cell structure and function to be studied. 

ACTIVITY 1. THE MICROSCOPE

As you have already learned, there are many different types of microscopes. In most introductory biology courses, students become familiar with two distinct types, the light microscope (found in most class laboratories) and the electron microscope which has been used since the 1930's to examine cellular organelles and substructure.

How does a light microscope work?

The light microscope, as its name implies, uses light with lenses to magnify a image. View the following videos to learn how a simple light microscope works.

Video - animation showing how the light microscope works

Video - a student focusing the light microscope

What are the magnification capabilities of the typical light microscope? 

magnification

The magnification is a function of the lenses in the objective and the eyepiece, so the magnification of the two must be multiplied to obtain the total magnification possible. So, for example, if the objective lens was 4X and the eye piece lens was 10X, the total magnification would be 40. (4 x 10 = 40)

When you understand how to calculate total magnification, answer question 1.


It is becoming a common practice to utilize video microscopy in conjunction with the light microscope. In this system, a video camera is attached to the top of the microscope tube. A lens between the microscope and the camera replaces the eye piece lens so that the image is focused into the camera. The camera is connected to a computer and the image is displayed on the computer screen.

video microscope Most of the virtual microscopy used in this course simulates video microscopy. Note that total magnification is more difficult to calculate in this system, since both the magnification of the upper lens and the additional magnification of the screen display must be known.

What is the relationship between field of view and size at different magnifications?

The size of the field of view using each objective can be estimated by viewing a grid. The image below is a grid as it appears when you look through a microscope using the 4X objective lens. The size of each square on this grid is known to be 1 mm. Thus, the diameter of this field of view is approximately 2.6 mm.

field of vision

The divisions on this grid are too far apart to see at higher magnifications. Thus, we must estimate the field of view at higher magnifications from the data obtained using the 4X objective. This is easily done by using ratios. For example, the ratio of the 4X objection to the 10X objective is 4/10 or 0.4, so we would expect to see 0.4 as much on our slide as we saw with the 4X objective. Thus, the field of view using the 10X objective is approximately
2.6 mm x 0.4 = 1.04 mm. Calculations for other objectives on the microscope are done in the same way, using the appropriate ratio in each case.This method also works for video microscopy as long as observations with all of the objective lenses utilize the same camera and computer screen. Note that many small microscopic structures, such as cells, are so small that it is inconvenient to use millimeters (mm) as the unit of measurement. The micrometer (uM) is commonly used. It is 0.001 of a mm. To convert millimeters to micrometers, multiply by 1,000. Note: another name for micrometer is micron and this term is frequently used.

Table 2. Fields of view for a typical classroom microscope.

Objective lens
4X/Objective used
Field of View (mm)
Field of view (uM)
4X
1
2.60
2,600
10X
0.4
1.04
1,040
40X
0.1
0.26
260
100X
0.04
0.104
104

To see how magnification affects the appearance of cells on a slide, view these images of an onion root tip. Question 2 asks you to arrange them in order from lowest to highest magnification.

When using a light microscope, cell size can be estimated by using the field of view. For example, you can count the number of cells that fit along the diameter of the field of view and then divide the size of the field of view by the number of cells. This is the method you should use to answer question 3 on your assignment sheet.

Another method for measuring size is to lay a grid over the image. For example, a grid can be placed in the eye piece of the microscope. In video microscopy, images of the grid can be captured using each of the objective lenses. A grid with vertical lines at known intervals is often used for this purpose. View the following video to see how this grid (sometimes called a micrometer) appears in video microscopy.

video - viewing a vertical lines grid by video microscopy

If you have a microscopic image of the grid and know which objective lens was used, you can utilize the grid image to determine the size of cells in an image captured at the same magnification. See the following video for an example.

video - determining cell size with a vertical lines grid

When you understand how to use this type of grid (micrometer), answer question 4

How does resolution affect our view of an image?

As you magnify an image on a slide, you will see more detail. However, not all lenses are created equal and one 40x lens may be better able to resolve an image than another. Resolution is a measurement of how well we can distinguish two closely spaced points as two points rather than one. Thus it is an indication of how well the smallest details of an image can be discerned. Some lenses have better resolution because they can bend light waves in ways that prevent them from scattering or interfering with one another. Note that the degree of resolution on the light microscope is limited by the wavelength of light. Most cells are so small that only larger organelles can be resolved by the light microscope, even if the organelles are differentially stained. The small size of organelles is apparent in these light microscopy images.

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