MICROSCOPY

3.2 Cell structure and function

Practical Work: The use of the light microscope, preparation of temporary slides, examination of permanent slides using low and high power of the light microscope.

Overview

A) THE LIGHT MICROSCOPE

B) TEMPORARY PREPARATIONS

C) EYEPIECE GRATICULE AND STAGE MICROMETER

Which instrument to use?

Hand lens

Binocular microscope

Compound light microscope

Electron microscope

Depends upon size of object

Sequence: 1

2

3

4

Hand lens

Stereomicroscope/ Binocular microscope

Naked eye

Compound light microscope

5 Electron microscope

specimens are:

Stereomicroscope / binocular microscope is used when:

1. too thick

2. opaque for the light microscope

Parts of the compound light microscope

Arm

Stage

Eyepiece lens Body tube

Revolving nosepiece

Base

Stage clips

Condenser diaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

Correct way to carry microscope

Parts of the compound light microscope

Arm

Stage

Eyepiece lens Body tube

Revolving nosepiece

Base

Stage clips

Condenser diaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

Eyepiece lenses

Parts of the compound light microscope

Arm

Stage

Eyepiece lens Body tube

Revolving nosepiece

Base

Stage clips

Condenser diaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

HP objective lens is close to slide

Objective lenses

Parts of the compound light microscope

Arm

Stage

Eyepiece lens Body tube

Revolving nosepiece

Base

Stage clips Condenser diaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

Stage clips hold the slide

Parts of the compound light microscope

Arm

Stage

Eyepiece lens Body tube

Revolving nosepiece

Base

Stage clips Condenser diaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

A condenser consists of:

2. a variable-aperture diaphragm

1. one or more lenses

The condenser

concentrates light from the illumination source

diaphragm: controls the diameter of the beam of light

poor contrast

poor brightness

Ideal

Parts of the compound light microscope

Arm

Stage

Eyepiece lens Body tube

Revolving nosepiece

Base

Stage clips

Condenser diaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

out of focus

focused

When focusing: First use LP objective Look from the side

rack body tube downwards using the coarse adjustment knob until objective lens is close to slide

Look through eyepiece lens & rack upwards Use fine adjustment knob to bring into focus

Objective collision !!!! Rack UPWARDS to avoid

After focusing on LP, how do you change to HP?

Rotate nosepiece to click HP objective in place

Use fine adjustment knob – rack upwards

Learn:

1. parts of the light microscope

2. procedure to focus

is how much bigger a sample appears to be under the microscope than it is in real life

Magnification

Total magnification =

eyepiece lens X objective lens

total magnification: 10 X 40 = 400X

Example: find total magnification if

Eyepiece lens: 10X

Objective lens: 40X

As magnification increases, detail increases but:

Onion cell 40x

Onion cell 100x

Onion cell 400x

less of the cell is seen

As magnification increases, light intensity decreases

Image appears dimmer Why? X 40 X 400

A smaller area is viewed & so less light is seen

Objective lenses

is the degree of detail which can be seen with a microscope

measures the ability of a microscope to distinguish two objects which are close together

RESOLUTION or RESOLVING POWER

poor resolution

good resolution

High resolution enables viewer to distinguish two objects which are close together

Low resolution

shows two objects as a single one

Resolving power 1/wavelength

short wavelength = great resolution

resolving power of any light

microscope is limited because the wavelength of light has a fixed range

at best it can distinguish two points which are 0.2m apart

maximum possible resolution = half the wavelength of light used

Question Photographs A and B show epithelial cells from the small intestine. One photograph was taken with a light microscope and the other with an electron microscope. Both photographs were

taken at a similar magnification. Which photograph was taken with an electron

microscope? Explain your answer. (2) B;

Better resolution;

Microvilli visible (in B);

Membrane-bound organelles visible (in B);

QUESTION: SEP, 2005 Paper 3

This diagram in Figure 1 represents a light microscope.

1.1 Briefly describe

how an observer may set up this microscope to observe a prepared slide under high power. (5)

Switch on the light source. Check objective lens is low power. Adjust the mirror to let light in. Adjust diaphragm to have enough light. Rack down the tube until it is just above the slide while

looking from the side. Use coarse adjustment knob to rack upwards to focus. Use fine adjustment knob for a sharp image. Turn nosepiece until the high power objective lens clicks

into place. Use fine adjustment knob for focusing. Focus upwards. If focus is still not correct, look at the stage from the side,

lower the tube until the objective lens is almost touching the slide.

Look into the microscope and rack up slowly using the fine adjustment until in focus.

1.2 How may an observer calculate the magnification at which the slide is being viewed? (3)

Magnification = eyepiece lens x objective lens

1.3 How would the brightness of the image observed at low power compare with that observed at high power? (2)

Image is less bright under high power as the field of view is narrower.

QUESTION: SEP, 2005 Paper 3

1.5 Why would the microscope in Figure 1 not be considered suitable for counting the number of stomata on a leaf surface? Name a magnifying instrument that would be suitable for this purpose. (3)

The leaf is too thick to allow light to pass through it and so cannot be viewed by a light microscope.

A binocular microscope / stereoscope.

Note: stomata CAN be seen under the light microscope if leaf is

covered with nail varnish!!

1.6 Why would the microscope in Figure 1 not be considered suitable for counting the number of mitochondria in a cell? Name a magnifying instrument that would be suitable for this purpose. (2)

Mitochondria are not visible under the light microscope due to low resolution.

An electron microscope.

Overview

A) THE LIGHT MICROSCOPE

B) TEMPORARY PREPARATIONS

C) EYEPIECE GRATICULE AND STAGE MICROMETER

Greater resolving power means greater magnification

Magnifies around

500 000 times

Magnifies around

1500 times

Image in an EM:

cannot be detected directly by the naked eye

forms on a screen

black & white photographs can be taken called PHOTOELECTRONMICROGRAPHS

Electron micrograph of soil-bound bacteriophages

Colour-enhanced electron micrograph of an Amoeba

feeding

TWO types of electron microscopes:

Transmission electron microscope [TEM]

Max magnification: 1,000,000 times

Scanning electron microscope [SEM]

Max magnification: up to 300,000 times

TEM

a flat image is created

natural contouring of a specimen cannot be seen

Colour-enhanced bacteriophages attacking a bacterium.

SEM

a 3D image

Scanning electron micrograph of a T4 bacteriophage.

false-colour photos may be produced

Scanning electron micrograph of an insect

head.

How can a SEM give 3D images?

specimen is coated in heavy metals (e.g. gold or platinum) which reflect the electron beam of the surface

in this way the natural contouring of the material may be observed

Spider coated with gold

Freeze etch electron micrograph

Why is freeze etching especially good to detect membrane proteins?

When membrane is fractured, the proteins are either:

1. torn away:

– leave holes

2. stay with the specimen: – seen as bumps

Overview

A) THE LIGHT MICROSCOPE

B) TEMPORARY PREPARATIONS

C) EYEPIECE GRATICULE AND STAGE MICROMETER

Microscope slides & coverslips

Slides may be:

Prepared slides Temporary slides

Temporary preparations of material for light microscopy:

can be made rapidly, unlike permanent preparation

are suitable for quick preliminary investigations

involve fixation, staining and mounting

Mounted needle

Fixation serves to:

"fix" or preserve cell or tissue morphology through the preparation process

e.g. formaldehyde & 70% ethanol

Mounting

involves:

attaching samples to a glass microscope slide

placing thin sections of material to a microscope slide

For temporary slide preparation, maceration may be necessary:

plant section is:

put in acid for a few days

then crushed to separate cells

sclereids

fibres & vessel elements

How to handle microscope slides

Incorrect handling can introduce artefacts.

Correct method for holding slide

Incorrect method

What is an ‘artefact’? something which does not occur in the

undisturbed living cell or organism, but was produced in it during investigation, or during its preparation for investigation.

Artefacts are caused by e.g.:

fingerprints scratches

dust air bubble

Mounting a specimen and lowering a cover-slip on a glass slide.

Trapped air bubbles: artefacts

Why is the specimen covered by a coverslip?

to exclude air and dust

to protect high power microscope objectives

Mount specimen in glycerine, after staining if:

specimen starts to dry out

prolonged examination (longer than 10 minutes) is required

Preparing a slide as a wet mount.

STAINING

The stain usually:

colors one part of the specimen

but not another part

Table 1 Types of stain. Stain Final Colour Suitable for: Permanent stains

Methylene Blue

Blue Nuclei

Stain Final Colour Suitable for: Permanent stains

Safranin Red Nuclei; lignin and suberin of plants

Stain Final Colour

Suitable for: Temporary stains

Iodine solution

Blue-black

Starch

Why is a specimen stained?

many objects do not have distinct, contrasting colors

- this makes it difficult to see details

The use of a biological dye makes the details

visible by creating contrast

Staining therefore usually permits easier identification

study of the different components present in the specimen

No stain – cell contents not visible

Stained onion cells - contents visible

Counterstaining a stain of a contrasting colour is used to colour

the components in a microscopic specimen that are not made visible by the principal stain

Counterstaining of plant tissues. Trifolium (red

clover) stem.

Note that lignified tissue stains red.

Irrigation: an important microscopic technique

the process is used to introduce a dye to a section, which is already mounted under a coverslip

Drop of stain

Filter paper

QUESTIONS 1. The diagram shows a

microscope.

a) Name parts A, B and C. (3)

Eyepiece lens

Objective lens

Light source

b) Calculate the magnification being used. Show your working. (2)

10 x 4 = 40x

c) Explain why the tissue needs to be thin. (1)

To let light pass through.

2. This question concerns the examination of cells through a light microscope.

a) Describe how you would prepare a temporary microscope slide showing epidermal cells from an onion. (4)

Question: SEP 2009, paper 3

A thin inner layer of epidermis is peeled off.

An onion is cut into quarters.

One of the fleshy scale leaves is removed.

Snapping leaf backwards exposes the epidermis.

Epidermis is placed on slide & covered with 2-3 drops of distilled water . Coverslip is lowered.

A drop of stain is put at one end of slide.

1 2 3

5

4

6

7 Stain is drawn over specimen using a small piece of filter paper.

The temporary preparation was subsequently stained with Gram Iodine and photographed at a magnification of 40x (Figure 1).

b) Suggest one reason for staining the preparation with Gram Iodine. (1)

To create contrast making the nuclei and cell walls appear darker than the cytoplasm

Figure 1: Onion epidermal cells stained with Gram Iodine (Adapted from: http://www.lima.ohio-state.edu/academics/biology/archive/organel.html)

2) The diagram shows a microscope slide being prepared.

a) Name A.

Coverslip

b) Describe how the forceps are used when preparing a slide. (1)

To lower the coverslip gently and avoid trapping air bubbles.

c) Explain why the onion tissue is placed in iodine solution. (2)

To stain the cells. Contrast is created making it possible to view the nuclei and cell walls as they appear darker than the cytoplasm.

The drawing shows the appearance of a prepared slide.

d) What causes the ring shapes? (1)

Air bubbles

QUESTION: SEP, 2005 Paper 3

Briefly describe how you would prepare a temporary slide showing a transverse section of a leaf. (4)

A thin transverse section is cut through the leaf using a sharp razor blade.

A drop of water is placed on a clean glass slide and the leaf section is placed on it.

A cover slip is gently lowered using a mounting needle, making sure not to trap air bubbles.

The specimen is stained using the irrigation technique.

Excess stain is wiped off the slide.

Overview

A) THE LIGHT MICROSCOPE

B) TEMPORARY PREPARATIONS

C) EYEPIECE GRATICULE AND STAGE MICROMETER

An eyepiece graticule:

allows the size of a specimen to be measured

is a glass disc with a

scale from 0 to 100 of arbitrary length engraved on it

it is placed inside the

eyepiece of the microscope

An eyepiece graticule needs to be calibrated using a: stage micrometer

A stage micrometer

consists of: a microscope slide on

which is engraved a fine and accurate scale

When an object is viewed through the microscope :

the eyepiece graticule is superimposed on the specimen being viewed

A stage micrometer:

A stage micrometer:

Used to:

calibrate the eyepiece graticule

QUESTION: MAY, 2014 Paper 3

Describe the steps involved in estimating the diameter of a liver lobule using the light microscope. [4]

1. A stage micrometer is used to calibrate the divisions of an eyepiece graticule under high power.

2. The slide having the specimen is placed on the stage and viewed under high power.

3. The number of divisions that fit in the diamter of the lobule are counted.

4. The diameter is calculated by multiplying the size of one division by the number of divisions that fit in the diameter of the lobule.

40 divisions = 1000m 1 division = ? 1 x 1000 = 25 m 40

Stage micrometer

Eyepiece graticule

1mm = 1000m

40 divisions

Liver lobule