A LEVEL Biology - foundations in Biology

What is an electron micrograph

Photograph of an image seen using an electron microscope

What is magnification

The number of times larger (how much bigger) an image appears, compared with the size of the original object.

What are organelles

Small structures within cells, each of which has a specific function

What is resolution

The clarity of an image; the higher the resolution, the clearer the image. It is the ability of an optical instrument to see/produce an image that shows fine detail clearly. i.E 'Ultra high definition' tv has sharp and clear images.

What is the magnification of Optical microscopes?

x1500 to x2000 . Enables us to see some large structures inside cells - but the resolution is limited so they cannot magnify any higher while giving a clear image.

What do Optical microscopes use?

Visible light - wavelength of (400 - 700nm)

How does visible light affect clarity of images?

The wavelength of visible light ranges from 400 - 700nm so structures closer together than 200nm (0.2 micrometres) will appear as one object.

Why can ribosomes not be examined using a light microscope?

Ribosomes are very small, non-membrane-bound cell organelles of about 20nm in diameter, and so they cannot be examined using a light microscope.

Why is it important that Microscopes produce linear magnification?

It means that if a specimen is seen magnified x100, it appears to be 100 times wider and 100 times longer than it really is.

What are the advantages of Optical (light) microscopes, and why are they still used in schools, colleges, hospitals, and research laboratories?

Played a key role in our understanding of cell structure. First sort to be used.

• Relatively cheap.

• Easy to use.

• Portable and able to be used in the field as well as in laboratories.

• Able to be used to study whole living specimens.

Describe the steps to sampling a specimen.

1. The specimen on a slide is placed on the stage and clipped into place.

2. By rotating the nosepiece, the lowest power (smallest) objective lens is placed over the specimen.

3. Adjust the coarse focus knob, while looking into the eyepiece until the image you see is clear and in focus.

4. Whilst viewing the image, adjust the iris diaphragm for optimum light.

5. Make sure that the object you wish to view is directly over the hole in the stage. Now rotate the nosepiece and bring the x10 objective into place over the specimen. Look down the ocular tube and use the fine focus knob to focus the image.

6. Repeat step 5 using the x40 objective lens.

How do you handle a light microscope?

Carry a microscope with its arm in one of your hands whilst having your other hand under its base.

What is the total magnification equation?

total magnification = magnifying power of the objective lens x magnifying power of the eyepiece lens

What is a photomicrograph?

A photograph of an image seen using an optical microscope. Modern digital microscopes display the image on a computer screen.

What is another term for 'Laser scanning microscopes'?

Confocal microscopes

What to confocal microscopes use?

They use laser light to scan an object point by point and assemble (by computer) the pixel information into one image, displayed on a computer screen.

What are the properties of images produced by confocal microscopes?

High resolution and show high contrast.

Why are laser scanning microscopes useful?

They have depth selectivity and can focus on structures at different depths within a specimen. This microscopy can therefore be used to clearly observe whole living specimens, as well as cells.

What are the uses of laser scanning microscopy?

They are used in the medical profession, e.g. to observe fungal filaments within the cornea of the eye of a patient with a fungal corneal infection, in order to give a swift diagnosis and earlier, and therefore more effective, treatment.

They are used in many branches of biology research.

What do electron microscopes use?

They use a beam of fast-travelling electrons within the wavelength of about 0.004nm.

What does the use of a beam of fast-travelling electrons mean?

They have a much greater resolution than optical microscopes and can be used to give clear and highly magnified images.

What is the process of an electron microscope?

The electrons are fired from a cathode and focused, by magnets (rather than glass lenses) on to a screen or photographic plate. .

Why do electron microscopes have a greater resolution than optical microscopes?

The fast-travelling electrons have a wavelength about 125000 times smaller than that of the central part of the visible spectrum. This accounts for an electron microscope's much better resolution compared with an optical microscope.

What is needed to view an image on a Transmission electron microscopes.?

The specimen has to be chemically fixed by being dehydrated and stained.

What is the process of a transmission electron microscope? How is an image formed?

The beam of electrons passes through the specimen, which is stained with metal salts. Some electrons pass through and are focused on the screen or photographic plate. The electrons form a 2D black and white (grey scale) image. When photographed this is called an electron micrograph.

What is the magnification of a transmission electron microscope?

They can produce a magnification of up to 2 million times. A new generation is being developed that can magnify up to 50 million times.

How do scanning electron microscopes work?

Electrons do not pass through the specimen, which is whole, but cause secondary electrons to 'bounce off' the specimen's surface and be focused onto a screen. This gives a 3D image which is black and white, but the computer software programmes can add false colour.

What are the precautions needed for electron microscopes to work?

The specimen has to be placed in a vacuum and is often coated with a fine film of metal.

What is the magnification of a scanning electron microscope?

x15 - x200000.

Disadvantages of electron microscopes (transmission and scanning).

• Very large

• Very expensive

• Need a great deal of skill and training to use.

• Specimens (even whole - for use in SEM) have to be dead as they are viewed in a vacuum.

• The metallic salt stains used for staining specimens may be potentially hazardous to the user.

What are optical instruments

The eye, optical, and electron microscopes. These measure in a logarithmic scale (goes up in steps - i.e. 1 step = 10 fold increase). i.e. atom = 0.1nm and lipids = 1nm

What can you use an optical microscope to view?

• Living organisms (Paramecium & Amoeba)

• Smear preparations of human blood and cheek cells.

• Thin sections of animal, plant, and fungal tissue, such as bone, muscle, leaf, root, or fungal hyphae.

Why do we stain specimens?

Biological structures i.e. single-celled organisms (paramecium) are colourless and transparent.

How do we observe unstained specimens?

Some microscope use light interference, rather than light absorption, in order to produce a clear image without staining. Some use a dark background against which the illuminated specimen shows up.

Why are these particular microscopes useful?

They are useful for studying living specimens.

How can you observe living specimens with a school light microscope?

By adjusting the iris diaphragm to reduce the illumination of the specimen.

What are stains?

Coloured chemicals that bind to molecules in or on the specimen, making the specimen easy to see.

Give an example of an all-purpose stain

Methylene blue

Define differential staining.

Some stains bind to specific cell structures, staining each structure differently so the structures can be easily identified within a single preparation.

Provide examples of differential stains.

• Acetic orcein - binds to DNA and stains chromosomes dark red.

• Eosin - stains cytoplasm; Sudan red stains lipids.

• Iodine in potassium iodide solution stains cellulose in plant cell walls yellow, and starch granules blue/black (look violet under microscope).

How have specimens been prepared and permanently fixed to be observed?

• Dehydration.

• Embedding in wax to prevent distortion during slicing.

• Using special instruments to make very thin slices (sections), which are then stained and mounted in a special chemical to preserve them.

Calculating using magnification

1. Measure the widest part of the specimen (i.e. leaf) on the photomicrograph in mm.

2. Convert the measurement to micrometers by multiplying by 1000.

3. Divide this figure by the magnification - this tells you the actual thickness of the specimen at this point.

Calculating using actual size; IF you are told the actual size of a structure on a photomicrograph (A).

1. Measure its image size on the photomicrograph (I) in micrometers (mm x 1000).

2. Calculate the magnification factor (M) using the formula M = I / A. P.S. There are no units for magnification, but if magnification factor = 1000, rewrite as x1000.

How to make drawings of slides.

1. Use a prepared slide i.e. a transverse section of x. Set it up on the microscope, focusing the specimen under a low power.

2. Use a sharp HB pencil.

3. Use a title that explains exactly what the drawing is and the magnification used.

4. Indicate the scale, e.g. how much bigger your drawing is than the size of the image.

5. Make a low-power plan of the specimen to show where the different tissue areas are - do not draw any individual cells. Use clear unbroken lines - do NOT shade.

6. Label the areas shown on the low power plan.

7. Indicate on the plan a portion of the tissues that you will include on a high-power drawing.

8. Make sure that this area of the specimen on the slide is directly over the hole in the microscope stage.

9. Turn the nosepiece and bring the bigger objective lens into place over it. Make sure that it fully clicks into place.

10. Use the fine-focus knob to bring the specimen into sharp focus.

11. Make a separate drawing of two/three cells from each region that you highlighted in step 5. Draw clear, unbroken lines and do not shade.

12. Label as many structures as you can see and identify. Use a ruler to draw the label lines and make sure that each label points exactly to the structure identified.

What is an eyepiece graticule?

A measuring device placed in the eyepiece of a microscope and acts as a ruler when you view an object under the microscope.

Why is a stage graticule?

A precise measuring device. It is a small scale that is placed on a microscopic stage and used to calibrate the value of eyepiece divisions at different magnifications.

How are eyepiece graticules used?

• A microscope eyepiece can be fitted with a graticule - this graticule is transparent with a small ruler etched on it.

• As the specimen is viewed, the eyepiece graticule scale is superimposed on it, and the dimensions of the specimen can be measured - i.e. just as a large object can be measured by placing a ruler against - in eyepiece units (edu).

How does the scale of the eyepiece graticule differ?

The scale is arbitrary - it represents different lengths at different magnifications. The image of the specimen looks bigger at higher magnifications, but specimen has not increased in size. The eyepiece scale has to be calibrated (its value worked out) for each different objective lens.

What is the function of a stage graticule?

It is used only to calibrate the eyepiece graticule.

How is a stage graticule used to calibrate an eyepiece graticule?

A microscopic ruler on a special slide - the stage graticule - is placed on the microscope stage. The ruler is 1mm long and divided into 100 divisions. Each division is 0.01nm or 10 micrometers.

What are the stages to using a stage graticule to calibrate the eyepiece graticule?

1. Insert an eyepiece graticule into the x10 eyepiece of your microscope. This ruler has a total of 100 divisions.

2. Place a stage graticule on the microscope stage and bring it into focus using the low power (x4) objective. Total magnification is now x40.

3. Align the eyepiece graticule and stage graticule, check the value of one eyepiece division at this magnification on your microscope.

4. e.g. the stage graticule ( = 1mm or 1000 micrometers) correspond to 40 eyepiece divisions.

5. Therefore each eyepiece division equals 1000/40 micrometers = 25 micrometers.

6. Now use the x10 objective lens on your microscope (total magnification = x100) and focus on the stage graticule.

7. Align them both,

8. e.g. 100 eyepiece divisions now correspond with 1mm or 1000 micrometers.

9. Therefore one eyepiece division = 1000/100 micrometers = 10 micrometers.

Values of eyepiece divisions at different magnifications

Magnification of eyepiece lens : x10, x10, x10, x10 Magnification of objective lens: x4, x10, x40, x100 (oil-immersion lens) Total magnification: x40, x100, x400, x1000 Value of one eyepiece division (epu) (micrometers) 25, 10, 2.5, 1.0