Flourescence Flashcards
What is Fluorescence?
What is Fluorescence Microscopy?
What are the common light sources in Fluorescence Microscopy?
◦ Fluorescence is when a substance absorbs light, then emits it as light of longer wavelength
◦ Fluorescence Microscopy is when UV lights are emitted (short wavelength) then the substance emits it back as visible light
◦ Mercury or Halogen lamps are common light source
◦ Thioflavin T, Thioflavin S, Auramine-rhodamine, FITC, DAPI, Texas Red
What wavelengths are regular microscopes viewed in?
How are Fluorescent microscopes used?
Regular microscopes view in the wavelengths of 400-700 nm, and use a light bulb for visualization.
- Fluorescent microscopes use a more intense light source, while the substance then emits a lower energy light with a longer wavelength
What is the light source filtered through?
What is the excitation filters function?
What is the function of the second filter? What kind of light does it allow to pass?
Where is the light then observed?
Light source is filtered through an excitation filter
The excitation filter only allows the selected wavelength to pass to the sample
*Next, the light is split with a second filter that only allows the emitted light from the specimen to pass
*This light is then viewed under the microscope
What histological structure demonstrates Autofluorescence?
Why would Autofluorescence be a problem?
◦ Some naturally fluorescing components include collagen
◦ Photobleaching, it limits time.
Fluorescence
◦ Primary antibody
◦ Secondary Antibody (different animal) linked with fluorescent dye
◦ Binds primary antibody and fluoresces
Fluorescence
◦ Primary antibody
◦ Secondary Antibody (different animal) linked with fluorescent dye
◦ Binds primary antibody and fluoresces
What is the excited range for FITC?
What is the emitted range for FITC?
FITC: lime green dots, black background
Excited 493-495
Emitted 519-525
What is the excited range for DAPI?
What is the emitted range for DAPI?
DAPI: blue strawberries with green leaves
Excited 377-458
Emitted 420-489
Describe the Direct Method (Staining Method).
What is the antibody labeled with?
Direct Method: Labeled antibody of known specificity is used to identify antigens in the patient’s tissue.
◦ Antibody is labeled with fluorescent dye (FITC) or labeled with an enzyme for subsequent reaction with a chromogen.
Describe the Indirect Method (Staining Method).
What is another name for this method?
◦ Indirect Method: Uses two antibodies. First antibody is an unlabeled defined antibody and the second labeled antibody is used to localize the first. Also known as “two-step” indirect method.
Describe the Indirect Method 3-Step (Staining Method)
◦ Indirect Method Three-Step. Same as “Two-Step” but with third antibody.
(Immunofluorescence)
What is Fluorochrome?
What can be observed when fluorochrome is attached or conjugated to an antibody?
- Fluorochrome is a dye that absorbs light and then emits its own light at a longer wavelength (called fluorescence).
- When attached or conjugated to an antibody, sites of reaction between antigen and antibody can be observed.
What are the most common fluorochromes used in Immunofluorescence?
- Most commonly used fluorochromes in IF is FITC and rhodamine (Texas Red).
What is Auramine rhodamine?
What does it stain in AFB cell walls?
Auramine rhodamine is usually used as a marker for what?
Auramine rhodamine:
◦ Auramine O and Rhodamine B
◦ Auromine O stains mycolic acid in AFB cell walls, along with Rhodamine B
◦ Usually used as a marker for the bacteria as Ziehl Neelsen method is much more specific
