Light and Fluorescence Microscopy Flashcards
Resolution definition
The smallest distance between two structures at which they can be seen as two separate entities. Determines the quality of the image, its clarity and richness of detail.
Resolution of a light microscope
approximately 0.2 micro-metres
What can be viewed?
Nuclei, mitochondria. ribosome, storage granules and bacteria
Three steps to prepare specimens for light microscopy
Fixation, sectioning and staining
Fixation definition
pieces of organ are placed as soon as possible after removal from the body into solutions of stabilising and cross-linking fixatives.
Why are tissues cut into fine fragments?
Fixative must diffuse fully into tissue
What fixative is used in light microscopy?
37% formaldehyde
Function of fixation
Preserve tissue structure and prevent degradation by enzymes released from cells or microorganisms
Sectioning definition
Cutting the tissue into thin specimen
What occurs before sectioning?
The fixed tissue is embedded in a material that imparts firm consistency, such as paraffin.
Stages of sectioning
Embedded, fixed tissue is placed into a microtome.
Paraffin sections cut between 3-10 micrometers in thickness. Specimen then placed on a slide.
Staining definition
Adding a dye to differentiate between different materials in the cells
What dye is often used?
Hematoxylin and eosin H&E
Basophilic vs acidophilic
Hematoxylin binds to basophilic organelles with a net negative charge
Eosin binds to acidophilic organelles, with a net positive charge
What colour is hematoxylin and what does it bind?
Purple- DNA, RNA, ribosomes, GAGs and cartilage matrix
What colour is eosin and what does it bind?
Pink- other cytoplasmic structures and collagen. Most proteins stain pink, especially cytoplasm of muscle, red blood cells and epithelial cells
Immunohistochemistry definition
Based on specific reactions between an antibody and antigen labeled with visible markers, often fluorescent compounds. Then viewed using light microscopy.
Two types of immunohistochemistry explained
Direct- labelled antibody binds to protein of interest
Indirect- Sequential application of two antibodies and washing steps. Antibody added, then washed, another antibody added with tag, then washed, enzyme added that binds with tag
Hybridisation definition
Implies the specific binding between two single strands of nucleic acid, which occurs under appropriate conditions if the strands are complementary.
In situ hybridisation definition
Nucleic acid sequences in solution applied directly to prepared tissues and cells, where they diffuse into the nuclei and bind to RNA/DNA.
What form of nucleic acid is used?
Probes
Fluorescence microscopy explained
Specimen illuminated with a light at a specific wavelength, which is absorbed by fluorophores, causing them to emit light at a longer wavelength (at a different colour than absorbed light)
