Microscopy

Question 1

What is Light Microscopy?

Answer 1

Instrument for visualising fine detail (microscope) using an ordinary light beam.

Question 2

What is Bright-Field Microscopy?

Answer 2

A stained preparation examined with the use of ordinary light that passes through the sample. The detail and quality of the image depends on the resolving power which determines the images clarity and richness of detail.
A condenser focuses the light onto the object that is to be studied. The objective lens then enlarges and projects the image towards the observer. The objective lens can give different magnifications of x4, x10 and x40. The two eyepieces or ocular lenses magnifies this image another x10 and projects it to the viewer.

Question 3

Why is the specimen stained in bright light microscopy?

Answer 3

To provide contrast between the image and the image background.

Question 4

What is Resolving Power?

Answer 4

The smallest distance between two particles which can be seen as separate objects.

Question 5

What is the maximum resolving power of a light microscope?

Answer 5

0.2 Micrometres

Question 6

What is the maximum magnification of a light microscope?

Answer 6

1500x

Question 7

What is Phase-Contrast Microscopy?

Answer 7

Allows for samples to be examined without the need for staining them. The tissue section is usually transparent and colourless. However, cellular detail is difficult to see because all parts of the specimen have roughly similar optical density.

Question 8

How does Phase-Contrast Microscopy work?

Answer 8

1. A condenser annulus modifies the light beam.
2. When the sample in inserted, some parts of the light is scattered which like the direct light is also refocused at the detector.
3. To distinguish between the direct light and scattered light by sample, a phase plate in inserted.
4. The scattered light passes through the thicker part of the plate compared to the direct light, which causes a shift in phase for the scattered light.
5. When the scattered light interferes with the direct light, this creates phase contrast.
6. It is due to the intensity differences that allows for the transparent specimen to be seen.

https://www.youtube.com/watch?v=vr4tYUnaHNQ

Question 9

What is a Fluorescent microscope?

Answer 9

An optical microscope which uses fluorescent samples.
1. It consists of magnifying lenses, a dichroic mirror and filters.
2. The first filter will select the light which will excite the fluorophores in the sample.
3. The light illuminates the fluorescent sample.
When the fluorophores in the sample are illuminated with the proper wavelength, they emit a fluorescent light of a different colour and wavelength
4. The second filter and dichroic mirror select only the emitted light. Therefore the microscope only detects the fluorescent part of the sample.
5. Fluorescent microscope is used to detect the presence and localisation of very small amounts of molecules in biological samples.

https://www.youtube.com/watch?v=BYwHLhgP1qI

Question 10

What are fluorophores?

Answer 10

A fluorescent chemical compound that can re-emit light upon light excitation.

Question 11

What is a Confocal Microscope?

Answer 11

A confocal microscope is a special type of fluorescent microscope. Two aperture pinholes are positioned in the confocal position. Light passes through the first pinhole which targets the light on a small part of the sample. If flurophores are present on this point of the sample, light is emitted which is then filtered by the filter and dichroic mirror. The second pinhole positioned on the focal plane selects only the light from the targeted area, therefore only collecting the targeted light.

The surface can be scanned by either moving the sample or the light beam. This can give a 2D reconstruct of the image from one give height.

One can then move vertically to obtain images from different heights which can be used to obtain a 3D reconstruct in proper softwares.

Confocal Microscopy is used to detect fluorescent molecules in 3D with good spacial resolution.

https://www.youtube.com/watch?v=BYwHLhgP1qI

Question 12

How do you calculate the maximum magnification?

Answer 12

Obtained by multiplying the magnification power of the objective and ocular lenses.

Question 13

What is electron microscopy?

Answer 13

Electron microscopy is microscopy based on the interaction of cell components with beams of electrons. The beams have a shorter wavelength than that of light and give a 1000x increase in resolution There are two types: Transmission Electron microscopy and Scanning electron microscopy. The electron microscope in general has a resolution on 3 nanometers and a magnification of up to 400,000.

Question 14

What is freezing fixation?

Answer 14

Another quick preparation method is freezing the cell with liquid nitrogen. This however, does not deactivate the degradative enzymes but just prevents them from activating. Once the cell has melted the enzymes will once again be activated.

Question 15

What is Transmission Electron Microscopy?

Answer 15

Transmission electron microscopy is an imaging system using electron beams instead of light to produce an image of around 3nm with the magnification of 400,000 times. Usually used to examine very thin resin embedded tissue structure (40-90nm) at magnifications of up to 120,000 times.

The TE microscope column is in a vacuum and beams of electrons are emitted from the top by a cathode that travels to an anode at an accelerating voltage of between 60kV to 120kV. The electrons pass through a hole in the anode which are focused electromagnetically by electron circular coils similar to the effect of optical lenses on light. Some electrons interact with the specimen causing for the electrons to be absorbed or scattered, while some electrons pass through the specimen. The electrons that reach the objective lens forms an image which is then magnified on a fluorescent screen or a charged coupled device (CCD) monitor and camera.

In the specimen image, the areas where the beam was absorbed or deflected, the image (electron dense) is appeared dark and areas where the electrons pass through the specimen (electron lucent) appear bright. This is why TEM produces a black, white and grey image.

Question 16

What is Scanning electron microscopy?

Answer 16

SEM is similar to TEM however, the beam does not pass through the specimen. Instead the specimen is coated in a very thin metal compound (often gold) reflecting the electrons beam and scanning the specimen. The reflected electrons are captured by the detecter which produces signals to present a black and white image. The image is easy to interpret as it is illuminated in 3d with shadowed detail.

Question 17

What is Autoradiography?

Answer 17

The method of localising newly synthesised macromolecules in a cell or tissue section.

Radioactively labelled metabolites (nucleotides, amino acids, sugar) are incorporated into specific macromolecules (DNA, RNA, Proteins, Glycoproteins, Polysaccharides) which emit weak radiation only from the region where the molecules are located. Slides with radio-labelled cells or tissue sections are coated in a dark room with photographic emulsions in which silver bromide crystals act as micro-detectors for radiation. After adequate time in lightproof boxes, the slides are developed photographically. The silver bromide crystals reduced by the radiation produce small black grains of metallic silver which under light microscopes and TEM, indicate the location go the radio labelled macromolecules in the tissue.

Question 18

What is cell culture?

Answer 18

Allows for the direct observation of cellular behaviour with phase contact microscope, and experiments that are impossible to be done intact with the body can be done in vitro.

Question 19

What does primary cell cultures mean?

Answer 19

Cells and tissues are grown in complex solution whose compositions are known (amino acids, salts, vitamins). Serum or other growth factors are added. Cells to be cultured are dispersed mechanically or enzymatically from the tissue or organ and with sterile procedures are placed in a clean dish. This preparation is called primary cell cultures.

Question 20

How do some cells maintain in vitro for long periods of time?

Answer 20

Some cells maintain in vitro for long periods of time as they become immortalised due to changes in the cell (a process called transformation) and constitute a permanent cell line.

Question 21

How can macromolecules be identified and localised with tagged compounds?

Answer 21

The tagged compound attaches to a specific macromolecule of interest. The compound is also tagged with a label so that it is visible to light in light or electron microscopy. The common used labels are fluorescent compounds, radioactive atoms that can be localised with autoradiography or enzymes such as peroxidase which can be detected in enzyme histochemical procedures.

Examples of the molecules that tag with others:

Phalloidin: Interacts with the actin proteins of microfilaments
Protein A: Interacts with the Fc region of antibodies. Can therefore be used to localise naturally occurring or applied antibodies bound to cell structures.
Lectins: Binds to carbohydrates so can be used to localise specific sugars or sequences of sugar residues.

Question 22

What is immunohistochemistry?

Answer 22

A method of localising many specific proteins through the binding of antibodies to the protein. A tissue section which is believed to have the protein of interest is incubated in a solution containing antibodies (monoclonal or polyclonal) which will bind specifically to the protein of interest. After a rinse the antibodies can be seen bound to the protein. The antibody is commonly tagged with compounds to allow the antibody to be seen bound through either a light microscope or an electron microscope. Common bounding tags are fluorescent compounds, peroxidase or alkaline phosphatase for histochemical detecting or gold particles dense in electrons to be examined using TEM.

Question 23

What does monoclonal and polyclonal mean?

Answer 23

Monoclonal means antibodies that can only bind to one antigen. Polyclonal means antibodies that can bind to several antigens.

Question 24

What is direct and indirect immunohistochemistry/immunocytochemistry?

Answer 24

Direct IHC is when the antibody with the tagged compound binds directly to the protein of interest (primary antibody). Indirect means that one antibody binds directly to the protein of interest and another antibody with the tagging compound binds to the first antibody (second antibody is called the secondary antibody)

Question 25

What is hybridization?

Answer 25

Hybridization implies the binding of two single stands of nucleic acids whose strands are complementary.

Question 26

What is In Situ Hybridisation?

Answer 26

The process of determining a particular sequence of DNA, such as a gene or part of a gene, identifying the cells in which specific mRNA is contained, and determining the localisation of a specific gene in a chromosome. First the DNA or RNA must be denatured through heat or other agents. A strand that is complementary to the sequence of nucleotides which are of interest is modified to contain a nucleotide tag which can be radioactive isotopes to be localised through autoradiography or tagged with a small compound that can be detected through immunocytochemistry. This modified strand is called the probe. It attaches to the sequence of interest and after a wash, the hybridised probe becomes visible through its label.

Question 27

What is enzyme histochemistry?

Answer 27

The method of localising enzymes through enzymatic activity. A tissue that is believed to contain the enzyme of interest is immersed in a solution which contains its substrate. The enzyme is allowed to act on the substrate. The section is then put in contact with a marker compound which reacts to the product of the enzymatic action. The final product from the marker, which must be insoluble and visible to electron microscopes and light microscopes, precipitates at where the enzymatic activity happened revealing the location of the enzyme if present.

Examples of enzymes that can be detected using histochemistry are: Phosphatase, Dehydrogenates and Peroxidase.

Question 28

What is polarising microscopy?

Answer 28

Polarising microscopy allows the recognition of stained or unstained structures containing highly organised subunits. When light passes through a polarising filter, it exits vibrating only in one direction. If a second filter is placed in the microscope above the first filter with its main axis perpendicular to the first, no light can pass through. However, if the tissue containing highly oriented macromolecules is placed in between the 2 structures, due to their repetitive structures, the light emerging from the polariser rotates and appears as bright structures against a dark background. The ability to rotate the direction of vibration of the light is called birefringence and is a future in crystalline substances or substances with highly oriented molecules such as cellulose, collagen, microtubules and actin filaments.