Fluorescence Microscopy and Bioimage Processing Flashcards
Total Magnification =
Magnification of Eyepiece x Magnification of Objective
What is the difference between an inverted and an upright microscope?
- Inverted = objective system is below the sample - used to visualise cells etc. in a petri dish, no need to disturb the environment
- Upright = often used for embedded samples - ie you have taken a sample - objective is above the sample
List the omponents of a light microscope
- Eyepiece - occular and tube
- Revolving nosepiece
- Objective
- Stage
- Stage clip
- Condenser
- Light source
- Coarse and fine focus
- are
- base
Define Refraction
Bending of light occurs as light passes from one medium to another with a different refractive index
Define Refractive index
A dimensionless number that gives the indication of the light bending ability of that medium
- Light bends through water as water has a different density to air
Air - 1.0003
Water - 1.33
Glycerin - 1.47
Immersion Oil - 1.515
Glass - 1.52
Define Numerical Aperature
NA of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed objective distance
NA = n x (sinu)
u= angle of one half of the angular aperature
n = Refractive index of imaging medium
The higher the total na the better the resolution
Define Resolution
R of optical microscopy is physically limited. The R of microscope objective is defined as the smallest distance between two points on a specimen that can still be distinguished as 2 separate entities
What is fluorescence?
- The property of some atoms/molecules to absorb light of short wavelength and emitting light of a longer wavelength
- The distance between the excitation and emission peaks is known as the Stokes shift
Name the 2 fluorescence labelling strategies
- Antibodies - can be direct or indirect
- Gene transfer - Formation/Binding a small - molecule fluorophore to the gene
Name the components of the fluorescnence microscope that is different to a light microscope
- Filter turret
- UV shield
- filters
- Collecter lens
- Microscope control circuit board- Tungstem Halogen lamphouse
- Epi-fluorescence lamphouse
- Digital camera systems
Define confocal microscopy
Is an advanced light microscopy method which utilises a pinhole to eliminate out of focus light
Define images and pixels
- Digital images are composed of picture elementes - Pixels
- Each pixel has a numeric value
- 8-bit = unsigned integer - 2^8 = 256 different pixel values
- Digital images are a matrix of numbers = information
- Images that look the same can contain different pixel values
Define RGB images
- In general, each colour is represented using 3 8 bit unsigned integers
- The number of bits used to represent each pixel in RGB space is called the pixel depth
- Each integer value defines how much of each primary colour should be mixed together to create the final colour
- not very good at quantitative analysis
What does deconvolution filter do?
- Corrects the systemic error of blue (loss of contrast in smaller features) and reconstructs the true image
What does Gaussian blue filter do?
Image is convoluted with a Gaussian function for smoothing to reduce the image noise
What does the subtract background filter do?
Removes backgrounds from images. A local background vaue determined for every pixel by averaging over a very large area.
Describe thresholding
A technique for dividing an image into foreground and background pixels
Describe segmentation
The process of partitioning a digital image into multiple segments
- Segmentation helps to reduce the complexity of the image and make subsequent processing easier
- Label each pixel in an image so that pixels with the same label have similar visual characteristics
- Commonly used to find objects and boundaries and quantify numbers, sizes, intensity and density
