MICROSCOPY

3.2 Cell structureand 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.

3.2.2 3.2.2 The fluid mosaic model of cellularmembranes.Structure as revealed by freeze- etching (knowledge of other cytological techniques is not required).

Overview

A) The Light MicroscopeB) The Electron MicroscopeC) Temporary Preparations

Overview

A) The Light MicroscopeB) The Electron MicroscopeC) Temporary Preparations

Which instrument

to use?

Depends upon size of object

Naked eye Hand lens

Stereomicroscope/ binocular microscope

1 2

3 4 Compound light microscope

5 Electron microscope

specimens are:

Stereomicroscope / binocular microscope is used when:

1. too thick2. opaque for the light

microscope

Parts of the compound light microscope

Arm

Stage

Eyepiece lensBody tube

Revolving nosepiece

Base

Stage clipsCondenserdiaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

Correct way to carry microscope

Parts of the compound light microscope

Arm

Stage

Eyepiece lensBody tube

Revolving nosepiece

Base

Stage clipsCondenser diaphragm

Light source

Coarse adjustment knob

Fine adjustment knob

Objective lens

Eyepiece lenses

Parts of the compound light microscope

Arm

Stage

Eyepiece lensBody tube

Revolving nosepiece

Base

Stage clipsCondenser 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 lensBody tube

Revolving nosepiece

Base

Stage clipsCondenser 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 lensBody tube

Revolving nosepiece

Base

Stage clipsCondenser 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 lensBody tube

Revolving nosepiece

Base

Stage clipsCondenserdiaphragm

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

QuestionPhotographs 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) The Electron MicroscopeC) Temporary Preparations

The resolving power of the light microscope is limited. How can the

problem be solved?

using forms of radiation with shorter wavelengths

the electron microscope was developed

Electron & light microscopes work on same principles but:

Light microscope uses light rays (order of wavelength = 500 nm)

Electron microscope uses a beam of electrons of wavelength = 0.005 nm

Greater resolving power means greater magnification

Magnifies around 500 000 times

Magnifies around1500 times

Magnification is controlled by the:

current supplied to the scanning coils

voltage supplied to the deflector plates, and not by objective lens power

OR

[not examinable]

What is used as a focusing devise in

each microscop

e?

LM

Magnetic lens

Magnetic lens

Magnetic lens

Eye

Electrons Lamp

Glass lens

Glass lens

Glass lens

Eye

OcularScreen

LM EM

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 ofsoil-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 seenColour-enhanced bacteriophages attacking

a bacterium.

Why does a TEM not give 3D images?

Structures inside the cell cause electrons to scatter & this pattern

generates an outline of the cell and the cell’s

organelles

Thin specimen

A beam of electrons is passed through thin

specimen

[not examinable]

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

Preparation & Staining of material for

an electron microscope[next 2 slides not

examinable]

material to be observed must be dead as:-• it is observed in a vacuum• is quickly heated up•would be destroyed in the electron

beam in order that biological material, can

withstand the processes it must be treated with a fixative to stabilize it

to achieve this, without the section falling apart, it is embedded in a firm plastic

in this more rigid form,

thin enough sections may be cut using a machine called a MICROTOME

once fixed, the material often requires sectioning a typical section is a mere 50 nm thick

A microtome is a tool used to cut extremely thin slices of material,

known as sectionsFrom the Greek mikros = "small" and temnein = "to cut"

A hand-held microtome & blade

A home-made microtome

Plant stem embedded in wax.

A sharp blade is used to cut thin slices.

is supported on a metal grid: usually of copper

across the mesh is a plastic

film: less than 1 nm thick transmits electrons

Material to be studied by EM

4

View under EM

Glass would scatter the electron beam

Why is the material to be studied placed on a grid and not a glass

slide?

Box for holding grids.Grid

Grid with specimenEmbedded in plastic.

Freeze-etching [Examinable]

technique useful for observing membrane structure

Freeze-etching

Specimen is rapidly frozen in liquid nitrogen.

2Specimen is

fractured with a sharp metal

blade.

The tissue fractures along planes of weakness which often run through membranes.

1

Ice sublimes [solid to gas] away leaving an etched surface.

The specimen is kept: cold in a high vacuum

3

Deep etching:the etching period is extended and a deeper layer of ice can be removed, thereby exposing structures that are located deep within the cell interior

Sublimation

[Etch: to cut into the surface]

4

A replica of this surface is made by depositing a layer of a heavy metal over it & strengthened by carbon.

ShadowingShadowing involves spraying a thin layer of an electron-dense metal such as platinum or gold at an angle across the surface of a biological specimen.

Platinum ‘shadow’

5

The tissue is destroyed with a strong acid.The replica is: placed on a grid examined using an electron microscope.

Platinum ‘shadow’

Grid

Carbon backing

Summary

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

QUESTION: MAY, 2009

Name and briefly describe ONE technique that has been used to prepare membrane material for microscopic study. (3)Freeze-etching

1. Cells are quickly frozen in liquid nitrogen (-196C), which immobilises cell components instantly.

2. Block of frozen cells is fractured. This fracture is irregular and occurs along lines of weakness like the plasma membrane or surfaces of organelles.

3. Surface ice is removed by a vacuum (freeze etching).4. A thin layer of carbon is evaporated vertically onto the

surface to produce a carbon replica.5. Surface is shadowed with a platinum vapour.6. Organic material is digested away by acid, leaving a

replica.7. Carbon-metal replica is put on a grid and examined by

a transmission electron microscope.

Overview

A) The Light MicroscopeB) The Electron Microscope

C) Temporary Preparations

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.:

fingerprintsscratches

dustair 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 stainsMethylene Blue

Blue Nuclei

Stain Final Colour Suitable for:Permanent stainsSafranin Red Nuclei; lignin and

suberin of plants

Stain Final Colour

Suitable for:Temporary stainsIodine 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 specimenNo stain –

cell contents not visibleStained 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

QUESTIONS1. 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 23

5

4

6

7Stain 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.

THE END