Scanning Tunneling Microscopy and Atomic Force Microscopy EEW508 Scanning probe microscopy.
Microscopy
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Transcript of Microscopy
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