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2.1 What was the difference in the microscopy studies of Hooke, published in 1665, and Leeuwenhoek, published in 1678?
Hooke was the first to identify cells (name derived from the Latin Cella) in cork, whereas van Leeuwenhoek identified and examined bacteria, protozoa and spermatozoa.
2.2 List the five important components of a light microscope and briefly outline the role of each.
A light microscope must contain the following important components:
- A light source – illuminate the specimen;
- A condenser – containing the diaphragm which controls the amount of light illuminating the specimen;
- A stage – holds the specimen in place above the light source;
- An objective lens – has a short focal length which falls on the specimen;
- An ocular lens – provides additional magnification and allows specimen to be focused for visualisation.
2.3 Explain the term resolving power.
The resolving power is the smallest distance between two points on a specimen to allow those points to be seen as separate. The greatest resolving power is seen where points very close together can be identified as separate.
2.4 Define the term Stokes shift.
The Stokes shift describes the longer wavelength of light which is produced following excitation of a fluorochrome. As excitation energy is lost, the wavelength of emitted light increases.
2.5 Briefly compare and contrast transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
TEM is an important technique for investigating the structure of cells. It has high resolving power and permits a very detailed and accurate representation of the cell to be produced. SEM does not penetrate the cell membrane and as such provides a method of scanning the surface of a specimen to provide surface detail.
In both cases, the electron beam is focused by electromagnetic lenses within a vacuum.
2.6 What does forward scatter and side scatter represent in terms of cell structure and identity?
Forward scatter and side scatter can be measured using light scattering technologies. Forward scatter represents the size of the cell and side scatter represents internal complexity.
2.7 Explain how hydrodynamic focusing can facilitate cell enumeration.
Hydrodynamic focusing involves the delivery of cells in single file to a detector. Cells are injected into the middle of a fast-moving sheath flow. The central cells of interest move more slowly than the sheath flow and are organised into single file for counting.
2.8 Describe the work of Kohler and Milstein, and explain how this is applied to flow cytometry.
Kohler and Milstein developed hybridoma technology. By fusing specific antibody- producing B-cells with myeloma cells (derived from antibody-producing B-lineage cancers) it was possible to produce large quantities of antibodies that would recognise only one epitope. Depending on the specific epitope used to stimulate the B cells, it is possible to produce monoclonal antibodies to a broad range of antigens – these are monoclonal antibodies. This manufacturing process produces antibodies of known specificity that can be labelled with particular fluorochromes and used to detect specific antigens expressed on or within target cells.
2.9 How many autosomes are present in a somatic cell?
There are 44 autosomes and two sex chromosomes in a somatic cell.
2.10 Explain the difference between heterochromatin and euchromatin and how these are demonstrated in G-banding and R-banding.
Euchromatin is transcriptionally active whereas heterochromatin is inactive. As such, euchromatin has an open structure and heterochromatin structure is closed. Euchromatin is represented by light bands in G-banding and heterochromatin is shown by dark bands. The opposite is true for R-banding, hence its name.
2.11 Explain the role of Thermophilus aquaticus in PCR.
Thermus aquaticus (Taq) is a thermostable polymerase used in PCR reactions to amplify DNA sequences through the assembly of nucleotides.
2.12 Define the term restriction fragment length polymorphism.
Describes heterogeneity in gene structure leading to polymorphism. These polymorphisms are identified according to recognition sequences which can be cleaved by specific restriction enzymes.
2.13 Differentiate between reporter probes and SYBR green I.
Reporter probes contain recognised specific sequences and two additional important elements. The 5’ end expresses a fluorochrome and the 3’ end a quencher. The DNA polymerase digests the reporter probe in the 5’ to 3’ direction, first releasing the fluorescent probe. This inhibits quenching leading to fluorescence which can be detected. The quenching tag is then released.
SYBR green I binds to double-stranded DNA in a non-sequence-specific manner. When bound, fluorescence is generated.
2.14 What is isolated from target cells to measure gene expression?
Rather than looking at the protein products of genes to assess gene expression, messenger RNA (mRNA) is isolated for further investigation.