cell & molecular biology

Experiment 1

Microscopes and Lenses

Although cells vary in size, they’re generally quite small. For instance, the diameter of a typical human red blood cell is about eight micrometers (0.008 millimeters). To give you some context, the head of a pin is about one millimeter in diameter, so about 125 red blood cells could be lined up in a row across the head of a pin.

With a few exceptions, individual cells cannot be seen with the naked eye, so scientists must instead use microscopes (micro- = “small”; -scope = “to look at”) to study them. A microscope is an instrument that magnifies objects otherwise too small to be seen, producing an image in which the object appears larger. Most photographs of cells are taken using a microscope, and these pictures can also be called micrographs.

From the definition above, it might sound like a microscope is just a kind of magnifying glass. In fact, magnifying glasses do qualify as microscopes; since they have just one lens, they are called simple microscopes. The fancier instruments that we typically think of as microscopes are compound microscopes, meaning that they have multiple lenses. Because of the way these lenses are arranged, they can bend light to produce a much more magnified image than that of a magnifying glass.

In a compound microscope with two lenses, the arrangement of the lenses has an interesting consequence: the orientation of the image you see is flipped in relation to the actual object you’re examining.

Two parameters are especially important in microscopy: magnification and resolution.

Magnification is a measure of how much larger a microscope (or set of lenses within a microscope) causes an object to appear. For instance, the light microscopes typically used in high schools and colleges magnify up to about 400 times actual size. So, something that was 1 mm wide in real life would be 400 mm wide in the microscope image.

The resolution of a microscope or lens is the smallest distance by which two points can be separated and still be distinguished as separate objects. The smaller this value, the higher the resolving power of the microscope and the better the clarity and detail of the image. If two bacterial cells were very close together on a slide, they might look like a single, blurry dot on a microscope with low resolving power.

Simple Microscope

The simple microscope is generally considered to be the first microscope. It was created in the 17th century by Antony van Leeuwenhoek, who combined a convex lens with a holder for specimens. While this microscope was simple, it was still powerful enough to provide information about biological specimens, including the difference in shapes between red blood cells. Today, simple microscopes are not used often because the introduction of a second lens led to the more powerful compound microscope.

Compound Microscope

With two lenses, the compound microscope offers better magnification than a simple microscope; the second lens magnifies the image of the first. Compound microscopes are bright field microscopes, meaning that the specimen is lit from underneath, and they can be binocular or monocular. These devices provide a magnification of 1,000 times, which is considered to be high, although the resolution is low. This high magnification, however, allows users to take a close look at objects too small to be seen with the naked eye, including individual cells. Specimens are usually small and have some degree of transparency. Because compound microscopes are relatively inexpensive yet useful, they are used everywhere from research labs to high school biology classrooms.

Parts of a Microscope

It consists of mainly three parts:

1. Mechanical part - base, c-shaped arm and stage.

2. Magnifying part - objective lens and ocular lens.

3. Illuminating part - sub stage condenser, iris diaphragm, light source.

Mechanical part

1. Base: It helps in holding the various parts of microscope. It also contains the light source.

2. C-shaped arm: It is used for holding the microscope. And which is connected the eyepiece to the objective lens.

3. Mechanical stage: It is a rigid platform on which specimen to be viewed is placed. It has an aperture at the centre to permit light to reach the object from the bottom. The object on the slide can be moved either sideways or forward and backward with the help of the positioning knobs.

Magnifying part

1. Eyepiece (Ocular lens):

It is the lens where the final image of the object is viewed. Usually; these lenses have a magnification of either 10X or 15X.

2. Objective lens:

There are three types of objective lens: 4X (scanning objective)

1. 1. 10X (Low power objective lens).

   2. 40X (High power objective lens).

   3. 100X (Oil immersion objective lens).

Each objective lens is represented by a particular colour. Here we represents 4X with red band, 10X with yellow, 40X with blue and 100X with white. These objective lenses are fitted on to the revolving nose piece. The working distance of an objective is defined as the distance between the front surface of the lens and the cover glass surface or the specimen when it is in sharp focus.

Illuminating part

1. Sub stage condenser:

It is seen below the stage and made up of a system of convex lenses which focus light from illuminating sources and is used to condense light towards the object. Lowering the condenser diminishes illumination whereas raising the condenser increases the illumination.

2. Iris diaphragm:

It is seen immediately below the condenser. Opening and closing of iris diaphragm controls the light reaching the object.

3. Light source:

Light source is situated at the base of the microscope. It is controlled by an ON /OFF switch and a lamp rheostat. Tungsten-halogen lamps are highly reliable light source used in the light microscope. It generates a continuous distribution of light across the visible spectrum. Adjustments Knobs in the Microscope a) Coarse Adjustment Knob: objective lenses can be moved towards or away from the specimen by using this coarse adjustment knob b) Fine Adjustment Knob: It is used to fine tune the focus on the specimen and also used to focus on various parts of the specimen. commonly one uses the coarse focus first to get close and moves to the fine focus knob for fine tuning.

Focusing On Microscopic Objects

Start with Clean Lenses:

It is important that microscope lenses be very clean. Before viewing through a microscope, use lens paper to gently clean the lenses.

Begin at Low Power Magnification:

Always begin by viewing the object through a low power lens. Depending on how small the object is, start with the scanning or low-power objective.

Using low-power objective lens, get the target object centered in the field-of-view and focus as much as possible, first by using the coarse focus and then fine-tuning the clarity of the image with the fine focus.

Once the object is in focus, switch to the next higher objective power. Do not change the focus or manipulate the focus knobs in any way while changing objectives.

Adjustments for oil immersion objective:

Without changing the adjustment of high power, turn to oil immersion objective. One drop of oil is added into on the slide. The nose piece is turned such that the oil immersion objective touches on the drop of oil. Open the iris diaphragm completely. Use only fine adjustments for focusing.

The Importance of Par focal:

A set of objectives on a microscope are said to be par focal if the viewer can change from one to another and still have the specimen nearly in focus. This is a very convenient feature, because as the magnification increases, even small manipulations of the focus knob can take a specimen far out of focus.

Care of the Microscope

Microscope is a delicate instrument which should be properly used. Fungal growth on the lens or scratches caused by dust can ruin the lenses. So microscope should be handled carefully.

Carry the microscope by holding the C-shaped arm with one hand and other hand under the base. Never swing the microscope while carrying.

Never allow direct light to fall on the microscope. Cover the microscope with a plastic cover when not in use.

While using oil immersion objective, do not adjust the coarse screw.

Oil immersion objective should be cleaned after use by wiping with soft cotton cloth or lens paper.

Dry objective should never come in contact with oil.

At the end of every experiment, clean the lenses with lens paper.

Confocal Microscope

Unlike stereo and compound microscopes, which use regular light for image formation, the confocal microscope uses a laser light to scan samples that have been dyed. These samples are prepared on slides and inserted; then, with the aid of a dichromatic mirror, the device produces a magnified image on a computer screen. Operators can create 3-D images, as well, by assembling multiple scans. Like the compound microscope, these microscopes offer a high degree of magnification, but their resolution is much better. They are commonly used in cell biology and medical applications.

Scanning Electron Microscope (SEM)

The scanning electron microscope, or SEM, uses electrons rather than light for image formation. Samples are scanned in vacuum or near-vacuum conditions, so they must be specially prepared by first undergoing dehydration and then being coated with a thin layer of a conducive material, such as gold. After the item is prepared and placed in the chamber, the SEM produces a 3-D, black-and-white image on a computer screen. Offering ample control over the amount of magnification, SEMs are used by researchers in the physical, medical and biological sciences to examine a range of specimens from insects to bones.

Transmission Electron Microscope (TEM)

Like the scanning electron microscope, the transmission electron microscope (TEM) uses electrons in creating a magnified image, and samples are scanned in a vacuum so they must be specially prepared. Unlike the SEM, however, the TEM uses a slide preparation to obtain a 2-D view of specimens, so it's more suited for viewing objects with some degree of transparency. A TEM offers a high degree of both magnification and resolution, making it useful in the physical and biological sciences, metallurgy, nanotechnology and forensic analysis.

Reference Material

Microscopy: en.wikipedia.org/wiki/Microscopy

Virtual lab of microscopy: vlab.amrita.edu/?sub=3&brch=187&sim=323&cnt=1

Questions

1.Who is known as the father of microscopy?

2. What are the different uses of Microscopes?

3. Draw the Schemetic diagram of compound microscope and label it properly.