ALC1 Visualising Cells ​ ​CEDB20003 2020

ALC1 Visualising
Cells ​ ​ ​ ​ ​ ​CEDB20003 2020

Learning task
1. Define resolution and factors that affect resolution
Resolution(D) is the minimum distance between two objects that enables them to be distinguished as
two separate objects.
Lambda is the wavelength of light
n is the refractive index of the medium through which the light is passing
Theta is the angular aperture or half the width of the cone of rays collected at the midpoint of the
objective lens


The higher the magnification of the objective, the lager the angular aperture.

The higher the refractive index(n), the higher the resolution

Resolution is impacted by the refractive index of the medium (usually either air, water or oil) through
which the light is passing from above the specimen to the objective lens.



2. Describe the key principles and limitations of light microscopy

Eyepiece DO NOT increase the resolution


The condenser lens improves the illumination of the specimen.
The objective lens resolves details in the specimen. The numerical aperture of this lens determines its
resolution.
The objective and the eyepiece lenses combine to magnify the image to a size greater than the limit of
resolution of the human eye


The limits of resolution of the light microscope is about 0.2 um or 200nm, we can see bacteria,
mitochondria and cell muckiest, maybe large vesicles and granules. But we cannot see virus or
smaller intracellular organeles such as ribosomes.

Transmisssion electron microscopes

Ultra-thin tissue sections are treated with heavy metals that deflect electrons.
Electrons are fired onto the specimen and the image is detected from transmitted electrons.
Structures that bind metals the most (nucleolus, vesicles) show as dark areas.
Structures that do not bind metals (cytoplasm, vacuoles) show as light areas


Similarities and differences between the principal features of a light microscope and a transmission
lectron microscope
The ense in the light microscope are made of glass, those in the electron microscope are magenetic
coils.
The electron microscopes requires that the specimen be placed in a vacuum.

Scanning electron microscope
Used to image surface structure.
The specimen is coated with heavy metals and an image is generated from back-scattered electrons
(not transmitted electrons)

the specimen is scanned by a beam of electrons brought to a focus on the specimen by the
electromagnetic coils that act as lenses.
The detector measures the quantity of electrons scattered or emitted as the beam bombards each
successive point on the surface of the specimen and controls the intensity of successive points in an
image built up on a screen.
The SEM creates striking images of three-dimensional objects with great depth of focus and a
resolution between 3 nm and 20 nm depending on the instrument.



Recognise and compare SEM and TEM images
SEM - Scanning Electron Microscope
Ultra-thin tissue sections are treated with heavy metals that deflect electrons.
Electrons are fired onto the specimen and the image is detected from transmitted electrons.
Structures that bind metals the most (nucleolus, vesicles) show as dark areas.
Structures that do not bind metals (cytoplasm, vacuoles) show as light areas

TEM - Transmission Electron Microcsope
Used to image surface structure.
The specimen is coated with heavy metals and an image is generated from back-scattered electrons
(not transmitted electrons)


Describe the role of thickness in microscopy and methods to visualise thick objects
Thick objects must be sectioned when using transmitted radiation. Making tisssue sections

For reference, a sheet of paper is about 100 µm thick.
For light microscopy we require sections ~1 - 50 µm thick.
For electron microscopy we require sections ~50 - 100 nm thick


Problems with sectioning

Histological processing: Chenical fixation, dehydration and embedding
Rapid freezing
These processes
Killing living things
Can cause changes in cell structure - artefacts
Three-dimensional shape must be recostructed - singal thin sections sometimes give misleading
impression. The true three-dimensional shape can be reconstructed from a complete set of serial
section.

What is contrast?

Contrast is the relative difference in brightness/darkness or colour between an object and its
surroundings, or between different parts of an object


Describe the role of contrast in microscopy and methods to increase contrast
Living cells are usually transparent
lack of contrast
Method to increase contrast
Optical Methods: enhance microscope images
Staining: thin sections of fixed tissue
Selective labeling: The most widely used example is Green Fluorescent Protein (GFP)

The role of light phase of contrast insight microscopy
Light passing through the unstained living cell experiences very little change in amplitude, and the
structural details cannot be seen if the image is highly magnified.
The phase of the light, however, is altered by its passage through either thicker or denser parts of the
cell, and small phase differences can be made visible by exploiting interference effects using a phase-
contrast or a differential-interference-contrast microscope



Differeftial Interference Cotrast (DIC) microscopy
Using without staining can increase contrast
Can observe cell in their nature state
The objective needs to be thin
single-celled organisms and small, transparent multicellular organisms or embryos;
individual cells which can be extracted and spread out on a slide, e.g. blood smear, cervical smear,
analysis of semen (sperm count and motility)

Three typed of light microscopy
Brightfield- light is transmitted straight through the specimen.
Phase contrast - phase alterations of light transmitted through the specimen are translated into
brightness changes.
DIC - which highlights edges where there is a steep change of refractive index.
Light waves passing through parts of an object that differ in thickness and/or refractive index will
become out of phase, as shown in the diagram above. Phase contrast and DIC microscopy convert
these phase differences into differences in light intensity

Disadvantage - Lack sepicificity they cannot be used to highlight just one part of the cell or just one
type of cell in a tissue. Furthermore, they cannot be used to locate where a particular class of
molecules is located in a cell or tissue.

Staining
When we stain tissue, we are able to abserve diffferent colours due to different stains absorbing
specific wavelength of light.
Staining to incresse contrast in light microscopy
Will absorb light of some wavelength

H&E classic staining method
Haematoxylin, a blue/purple dye that binds to negatively charged (i.e. acidic or basophilic, which
means 'base-loving') molecules. This includes nucleic acids (DNA, RNA), so haematoxylin stains the
nucleus and parts of the cell rich in ribosomes.
Eosin, a pink dye that binds to positively charged (i.e. basic or acidophilic, which means 'acid-loving')
molecules. This includes many proteins and therefore eosin stains cell cytoplasm, collagen and
muscle. Red blood cells are almost all cytoplasm and therefore stain uniformly pink with eosin

Compare methods of examining live cells/tissues versus fixed cells/tissues
Demonstrate basic light microscopy skills to visualise cell and tissue structure

Fluorescent dyes
Excited by light at one wavelength and emit light at longer wavelength

Fluorescence microscopy
Provide very high contrast images . This enable objects to be visualise that contain only a small
number of dye molecules. Thus fluorescent dyes are more sensitive.
The fluorochrme emit light of a particular wavelength while also ensuring that litttle or no light from
unlabeled reons is seen.

How does fluorescence work?
Excitation - cause by a specific narrow band of wavelength
Emission - a different definite colour always have a longer wavelength

Green Fluorescent Protein (GFP)
Naturally occur protein with blue light
The gee coding of GFP can be attache to the gene coding of any protein normally made by cell
GFP- labeled proteins can simply be visualized using fluorescence microscopy
Non-toxic to cell
Does not affect the function of the protein which it attach


Describe the key principles of fluorescent microscopy
Optical filters are used to select an appropriate wavelength to excite the fluorescent dye in use
Ensure that only the wavelength of light emitted by the dye pass to the viewer. This achieve the high
contrast typical of fluorescence microscopy.
epi-illumination- The separation of the excitation and emission wavelengths that is the goal of
fluorescence microscopy is best achieved if the excitation light shines on the specimen from above


Fluorescence immunohistochemistry
Use for staining specific cells, parts of cells or molecules in cells
The techniques use antibodies o perform specific labeling (antibodies are promade by the immune
system in vertebrates, they enable the body’s defenses to recognize foreign proteins in invading virus,
bacteris or other microorganism - these protein targets are antigens.)
Casting the protein we wish to investigate as the antigen.
Inject the antibody in protein into the mammal.
Apply a solution of this ntibody to the cells we are studying
The antibodies will bind tightly only to the antige if and where it is present.
A fluorescence microscopy is used to visualise where the antibody is bound in the cell or tissue.

Direct VS indirect immunohistochemistry
Direct immunohistochemistry - chemically couple the fluorescent label to the antibody molecule (the
primary antibody) that recognises the antigen of interest. ( not very convenient because a fluorescent
tag has to be added to each and every type of primary antibody that a researcher wishes to use.)
Indirect immunohistochemistr
- the primary antibody does not carry the fluorescent marker. It marker on another type of antibody,
the secondary antibody which recognise binds to the primary antibody. ( very sensitive )


Demonstrate basic light microscopy skills to visualise cell and tissue structure

Resolution and magnification
What is resolution? Write a definition and then discuss this with the rest of your group.

What is the resolving power of a typical young person with normal vision?

Look closely at the skin on one of your fingers. What must the resolution of your optical instrument
be, approximately, if you wish to:
Determine the arrangement of cells in skin?
Identify organelles in skin cells? e.g. differentiate Golgi body from mitochondria.
Visualise details of the internal structure of organelles of skin cells? e.g. examine what is inside a
mitochondrion.
Build a microscope activity























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Practical microscopy skills
How thick or thin is the tissue section?
Is it translucent or opaque?
Are the colours you see in the sections natural?

Are the cells in the tissue still alive, or at least fairly fresh?

Is there any aspect of the tongue tissue you can more readily see with the naked eye than with a
microscope?

More generally, in what situations would it be worthwhile looking at a microscope slide with the
naked eye before examining the slide under the microscope?


What does trans-illumination mean?


Why does this method of illumination require the tissue section to be relatively thin?


How do you calculate the overall magnification obtained?


What is the role of the condenser?


Why is the condenser position adjustable?


What is the working distance of an objective?


For what reason would you need a reasonably long working distance?




What sort of information can you obtain with the 4x objective?


What extra information about the structure of cells and the tissue can you obtain with the 40×
objective that was not seen at lower power?


What sorts of things (organelles) can't you see well even at this level of magnification? For example,
can you see the cell membranes between two adjacent cells? Does using a 100× objective help?



What are the relative advantages of low- and high-power magnification?
Low-powerHigh-power
Magnification e.g. 4x e.g. 40x
Working distance
Field of view
Readily distinguish individual cells?
List some likely uses or situations


Define resolution (again)



Has your understanding of resolution changed since you wrote a definition of resolution above? If so,
write a more comprehensive definition below. How has your definition changed?


Summarise the key factors that determine optical resolution in microscopy





Explain why the highest power objective on your microscope has a much closer working distance
than the low-power lens.


Now, using your sense of scale, give an example of the smallest biological object that can be resolved
with the 40x objective.


Using the formula provided, calculate the resolution of the 40x objective on your microscope?











What form of microscopy can go beyond the limitations of light microscopy?


Summarise why H&E is a useful stain for interpreting this example of breast cancer histopathology.


Can you think of an example of a question that would require specific labelling in living cells to get
an answer?


Can we expect to obtain better resolution with a fluorescence microscope than a microscope that uses
transmitted light? Why?

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