Fluorescence Microscopy and Bioimage Processing Flashcards
equation for magnification
v/u=magnification
v = image distance
u = object distance
*the closer an object is to the lens, the higher the magnification
equation for total magnification
magnification eyepiece x magnification objective = total magnification
what is an inverted microscope?
- the object being viewed is upside down
- the observer looks up into the microscope from below
- used for observing samples suspended in liquid
what is an upright microscope?
- object being viewed is in an upright position
- observer looks down into microscope from above
- used for observing samples on a slide
what does the eye piece do/consist of?
ocular and tube,
magnifies the primary image produced by the objective
what is the role of the condenser?
aligns light rays into straight path. Adjust it for the objective
what is spherical aberration?
a phenomenon in which the image can be blurry because the light rays are not focused on the same point.
what is chromatic aberration?
a phenomenon where the image is sharp, but different colours show.
definition of numerical aperture
The numerical aperture (NA) of a microscope objective is a measure of its ability to gather light and
resolve fine specimen detail at a fixed object distance
calculate numerical aperture
Numerical Aperture (NA) = n x (sin m(mew))
m = angle of one-half the angular aperture (A)
n = Refractive Index of imaging medium
relationship between numerical aperture and resolution?
The higher the total numerical aperture, the better the resolution.
refraction
bending of light, occurs as light passes from one medium into another medium with a
different refractive index.
refractive index
a dimensionless number that gives the indication of the light bending ability of that medium.
resolution
measure of the microscopes ability to distinguished the smallest distance between two points
on a specimen as separate entities.
resolution calculation
R = λ/(2 x NA) (where λ is the wavelength and NA is the numerical aperture)
what is fluorescence?
Fluorescence is the property of some atoms/molecules to absorb light (the excitation: Ex) of short
wavelength and emitting (Em) light of longer wavelength.
what is the Stokes shift?
The distance between the excitation and emission peaks is known as the Stokes shift.
give an advantage to fluorescence microscopy
collect images in more than one colour
what is confocal microscopy?
Confocal microscopy is an advanced light microscopy method which utilises a ‘pinhole’ to eliminate
out of focus light.
what are the stages of image processing?
1) image acquisition, 2) image processing and 3) image analysis.
what filters are used in image processing?
deconvolution, Gaussian blur, subtract background
what is the purpose of filters?
improve the effectiveness of image analysis
what is deconvolution?
corrects the systematic error of blur (loss of contrast in smaller features) and reconstructs true image.
what is Gaussian blur?
image is convoluted with a Gaussian function for smoothing to reduce the image noise.
what is subtract background?
Removes backgrounds from images. A local background value is determined for every pixel by averaging over a very large area.
what is thresholding?
pixels are divided into foreground (bright structures) and background (dark structures).
what is segmentation?
- Image segmentation is the process of partitioning a digital image into multiple segments.
- reduces the complexity of the image and makes subsequent analysing easier
- erode and dilate
