Light & Flourescene Microscopy Flashcards
What is magnification the ratio of
Between image size and object size
Define resolution
Resolution is the minimum distance between two distinguishable objects in an image
How do light microscopes work
Light microscopes
Visible light is transmitted from the base through the specimen, focused by a condenser lens. The light is then magnified by the objective, an eyepiece lens which means colour can be seen because visible light is used.
Magnification can be up to …. times
1500
Resolution is up to…
significance …
Resolution is 200 nm so can show bacteria and details within nucleated cells such as nuclei, mitochondria, ribosomes and storage granules
Samples need to be prepared through …, …. and …
Explain each process
Samples need to be prepared through fixation, sectioning and staining
Fixation uses chemical cross binding to prevent decay and loss of structures. It can also be done through low temperatures.
Sectioning is cutting a sample into thin slices, so light can pass through
Staining produces contrast so structures can be distinguished
What is histochemistry
Histochemistry is the staining of cells with specific chemicals
Name 2 popular stains used for histochemistry and elanorate on them
Haematoxylin and eosin
Haematoxylin binds to nuclei and stains them dark purple because nuclei contain acids which bind to basic dyes. Structures rich in nucleic acids also stain a dark purple
Eosin binds to other structures and stains them pink, for example, basic proteins- this is seen most commonly in muscle fibres, red blood cells and epithelial cells
What is immunocytochemistry
Immunocytochemistry is the localisation of specific tissue antigens using labelled and therefore visible antibodies
Antibodies are specific and complimentary in shape to the corresponding antigen
In situ hybridisation
In situ hybridisation is the localisation of specific nucleic acid sequences
Forms hybrid DNA or RNA by the addition of a labelled molecule which is sufficiently similar to bind to the specific sequence
What are immunocytochemistry & in situ hybridisation example of - elaborate
Immunocytochemistry and in situ hybridisation are forms of fluorescence microscopy as the antibodies and complimentary nucleic acid sequences can be labelled fluorescently
Can help distinguish between similar cells in areas such as the islets of Langerhans
It can also be used on intracellular aspects with a high enough magnification, such as fluorescence in situ hybridisation or FISH
Fluorescently labelled DNA probes are annealed to an open section of DNA, if the complimentary section is present, which enables certain chromosomes to be screened for specific genes or DNA sequences.
What are the limitations of light microscopy
Light microscopy is limited by the wavelength of light, so can only show structures the same size as bigger than the wavelength of visible light
When preparing a specimen, there are often artefacts such as the dissolution of lipids, which are then lost from the specimen during fixation and embedding
What types of stains are haematoxylin & Eosin - state the significance of this
Haematoxylin is a basic dye, so binds to basophilic structures like nucleic acids
Eosin is an acidic dye, so binds to acidophilic structures like basic protein
How does confocal microscopy works
Confocal microscopy uses a laser produce a coherent beam of light this campaign, then focuses at different levels of the section, which allows a thicker sample to be viewed than a conventional laser microscope
What does GFP stand for - significance
Green fluorescent proteins (GFP) from jellyfish can be used in live in imaging of fluorescent microscopy
Different colours can also be used to show the movement of specific proteins or sequences through cells
