Learning Objectives
In this lab students will:
- Become familiar with the parts of the compound light microscope.
- Know the names and functions of each part of the compound light microscope.
- Become familiar with how to get an image in focus.
- Understand the difference between resolution and contrast .
- Understand the relationship between resolution and contrast and how to achieve the best resolution or contrast.
- Understand the difference between serial sections and whole mounts
- Observe eukaryotic single-celled organisms from pond water.
• Before the invention of the microscope in the 1600’s we were unable to see cells.
• The microscope is an important tool used by biologists.
• 1674– Anton van Leuwenhoek developed microscope lenses that could magnify up to 300 times.
• The microscopes commonly used in a lab today can magnify an image 400-1,000 times.
• There are now microscopes, called electron microscopes, that can magnify 600,000 times!
Locate the following parts of the Compound Light Microscope in Figure 1.1
• Ocular lenses: These eye pieces are lenses that magnify the image of the specimen. The magnification of the ocular lenses is often stamped on its side. Most ocular lenses magnify an image 10 times. You may observe a pointer in the ocular lens. It is used to highlight cells, cellular structures, organisms, etc.
• Arm: This is the structure that supports the head and rotating nosepiece of the microscope and is used to carry the microscope.
• Objective lenses: These are the cylindrical tubes mounted on the rotating nosepiece that contain the lenses that magnify the specimen. Two things are written on the barrel of the lens: The magnification of the objective lenses and the numerical aperture of the lens (more on this later) Most microscopes have the following 4 objective lenses.
1. Scanning power objective lens: Magnifies an image four times. This objective is often referred to as the 4X objective. When this objective is in place with the standard 10X ocular, the total magnification of the specimen is 40 times its size.
2. Low power objective: Magnifies an image ten times. This objective is often referred to as the 10X objective. When this objective is in place with a standard 10X ocular, the total magnification if the specimen is 100 times its size.
-3. High power objective: Magnifies an image 40 times. This objective is often referred to as the 40X objective. When this objective is in place with a standard 10X ocular, the total magnification is 400 times its size.
4. Oil immersion objective: Magnifies an image one hundred times. Oil lubrication must be used with this lens, as the lens can easily be damaged if it makes contact with the glass slide. When this objective is in place with the standard 10X ocular, the total magnification of the specimen is 1,000 times its size.
• Stage: This is the flat, black-colored platform that holds the microscope slides. Notice the hole in the stage. The specimen must always be positioned over the hole for observation.
• Mechanical stage: This is a unit mounted on the stage that includes a pincer-like slide holder and stage controls. The slide holder has a fixed end and a pincer-like moveable end.
• Stage controls: A pair of knobs located along the side of the stage. Turning the top knob moves the slide toward or away from the viewer. Turning the lower knob moves the slide right or left.
• Condenser: this is a cone-like structure located below the stage. It contains a series of lenses that trap and focus incoming light. The condenser knob is used to adjust the position of the condenser. Under ordinary conditions, the condenser must be raised through the stage opening and brought close to the microscope slide without hitting the bottom of the slide. A good estimate of the best height for the condenser is to bring it as high up as it will go and then turn the condenser knob a half turn down away from the stage. If the condenser is not at the proper height you will not get a sharp and clear image of the specimen.
• Iris diaphragm: This is a series of moveable plates that function much like the iris of the human eye. The iris diaphragm regulates the amount of light passing though the microscope lenses, producing varying degrees of image brightness. It is identified by a small iris diaphragm lever under the condenser that moves side to side. It is generally adjusted when changing from one objective to another or from one slide to another in order to maintain optimum light conditions for viewing the specimen. If the image you are viewing looks grainy this means the iris diaphragm is too far closed and you will need to open it more. If the image looks washed out then the iris diaphragm is open too much and you will need to close it at least a little.
• Lamp: This structure rests on the microscope base. It contains a bulb that provides the light source used in viewing the specimen. The on-off switch is located on the front panel of the base. The voltage control dial on the right-side panel of the base is used to adjust the brightness of the light. Rotate the dial toward you to brighten the light.
• Focusing knobs are located on both sides of the microscope on the lower arm near the base.
- Coarse focus knob: The larger knob. It is used to focus the specimen into the field of view only when the scanning objective is in place. It is never used with higher magnifications.
- Fine focus knob: The smaller knob. It is used to make very slight changes in the focus.
• When carrying the microscope you should always use two hands. You should have one hand holding the arm of the microscope and the other hand supporting the base of the microscope.
• If your microscope was stored properly the scanning objective (4X) lens should be in place above the stage and the light should be off with the voltage control dial at its lowest setting.
• Once you plug you microscope in and turn it on you have two ways to control the amount of light
1. the voltage control dial - it is best to start with the light at about half brightness.
2. the Iris diaphragm lever. Open this up until the amount of light coming through to the ocular lenses is comfortable for your eyes. To bright or too dim will make it hard for you to find your specimen.
• Place the slide you wish to view on the stage and clip it in place. Below the stage are the mechanical stage knobs. Use these knobs to align you specimen over the light source coming through the hole in the stage.
• With the scanning (4x) objective in place, use the coarse focus knob to move the stage to its highest position.
• Look through the ocular lenses and slowly, using the coarse focus knob move the stage down until the image comes into focus.
• Center the specimen in the middle of the field of view
• Once the image is in focus and centered you can move to the low power (10X) objective by rotating the nosepiece until it clicks in place.
• Because the objective lenses are parfocal the image should be at or near focus.
• You may need some fine focus to get the image crisp. Use the fine focus knob to achieve this.
• As the magnification increases your working distance ( the distance between the bottom of the lens and the specimen) will decrease. For this reason you should only use the fine focus knob at higher magnification. If you use the coarse focus knob at higher magnifications it is easy to slam the lens into the specimen. This will crack the slide and damage the lens. Be Careful!
• Once you have the specimen in focus and centered at the low power (10x) objective you can safely move the high-power (40X) objective in to place.
• Fine focus to get a nice crisp view of the specimen.
• As the magnification increases you will often need to increase the amount of light. You should first open the iris diaphragm more/ If it is open all the way and you need more light increase the light using the voltage control dial.
Definitions
• Total magnification – is the magnification of the ocular lenses (10x) multiplied by the magnification of the objective lens (4x, 10x, 40x or 100x).
• Field of View –This is the illuminated area you see when you look through the microscope. As your magnification increases, your field of view decreases.
• Working distance –this is the distance between the bottom of the objective lens and the specimen you are viewing when it is in focus.
Parfocal lens - a lens that remains at or near focus when you change the magnification. There will always be some error in focus, but only fine focus will be needed.
• Total magnification - when you view a specimen you are viewing it through two sets of lenses. most ocular lenses magnify an image 10x. The objective lenses magnify the image another 4, 10, 40 or 100 times.
• This means that when you view an image with the scanning (4x) objective the image does NOT appear only 4 times larger than it actually is - but instead it appears 40X larger (10x times 4x = 40x).
• Numerical aperture (NA) - a measure of lenses light gathering capacity. The wider the cone of light rays that can be captured, the higher the NA
• The higher the NA the better the resolution.
• Resolution is the ability to distinguish 2 objects that are very close as separate objects.
• As magnification increases resolution increases
• Contrast in microscopy it is the difference in brightness of the specimen and the brightness if the background.
• Resolution and contrast are inversely related. Increasing resolution means giving up some contrast and vise versa.
• We will now discuss how to know when you should try to achieve the best resolution and the best contrast and how to achieve both.
• You should aim to achieve the best resolution if your specimen is already a very different color from the background (like the types letter "e" we will look at later, or if the specimen is stained (like many of the prepared slides we will look at in lab)
• You should try to achieve best contrast if your specimen is not stained and is a very similar color as the background (like the protists in the pond water we will look at later).
How to achieve the best resolution
• Kohler Illumination is a way to adjust settings on the microscope so the effects of aberrations in lenses are minimized and maximum resolution can be archived.
• Examine the condenser below the stage. It focuses light from the light source onto the specimen.
• Find the knob that moves the condenser up and down. This moves the focal plane of light.
To focus on the correct plane for the specimen do the following:
1. Focus on the specimen
2. Place a piece of cardboard with a hole in it over the light source (lamp) trying to center the hole over the light source. Note - some microscopes have a built in field diaphragm on the base (built into light source). If your microscope has one change the diameter of the cone of light using it instead of the cardboard with a hole in it). A small amount of light will still pass through.
3. Look through the ocular lenses. You should see the edges of the circle of light with edges that are fuzzy rather than crisp.
4. Move the condenser up or down until edges of the circle are in sharp focus. DO NOT change the focus of the microscope with the focus knobs.
5. When the edges are sharp the condenser is at the correct height to give the best resolution for THAT objective.
6. Remove the cardboard with a hole (or open the field diaphragm until the cone of light just fills the field of view. If you open wider, some resolution is lost).
How to achieve best contrast.
• Maximum resolution is not always desired. Often specimens are faintly stained, or unstained.
• You must increase contrast to see anything in this case.
• There are several ways to increase contrast.
1. Stop down the iris diaphragm using the iris diaphragm lever. This gives more grainy texture, but increases contrast.
2. Lower the condenser down to a lower level. This decreases resolution, but will increase the contrast.
Many of the slides we will look at in Developmental Biology will be either serial sections or whole mounts.
It is important to know the difference between these two and how they are made.
Serial section - are slides in which the specimen (often an embryo) has been sliced into 10-μm-thick sections. The sections have been placed in order from anterior to posterior. Not a single section is lost.
The ability to read serial sections and to construct a three-dimensional mental image of the whole organism is an important skill in the lab.
There will be a number of rows of sections on each slide.
By convention, these were laid down starting at the upper left-hand corner. In scanning the slide, you will read it just as you would a book, from left to right.
Start by moving the slide on the stage from left to right along the top row. Notice that when you look through the microscope, your movements look reversed, and the slide appears to be moving from right to left.
To read the next row, you must move the slide down and all the way back to the left.
Whole mounts: is a histological preparation of a specimen in which the whole specimen is mounted.
The specimen is usually mounted on a microscope slide and can be mounted either in a water-based mount such as glycerin jelly or a non-water-based mount such as the plastic mounting media used routinely for stained paraffin sections. Most of these specimens are too large to view with the light microscope. Instead, they are best viewed with the dissection microscope.