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Biomedical imaging Microscopy samenvatting UA
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Clear summary of the microscopy part of biomedical imaging. Fairly comprehensive.
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Biomedical Imaging Key pointsIntroduction:
o Resolution = ability to distinguish objects from one another (human eye = 0,1mm)
o Lens will increase the resolution by increasing the objects and thus the distance
between them.
o A microscope uses at least 2 lenses to increase the resolution.
Chapter 1: Microscopy Basics
Wave like properties of light
o Visible light is 700nm (infrared-ish/red) to 400nm (UV-ish/blue) in EM spectrum.
o Light is a wave; we know this thanks to the following wave-like characteristics of light:
o Light moves faster or slower depending on the density of its medium.
o The ability of a medium to bend light is given with the refraction index (n ).
o Refraction: when light passes from one medium to another it can bend. It will
bend following the Snell’s law: n1 sin α 1=n2 sin α 2
o Dispersion: The refraction doesn’t only depend on the breakings index. It also
depends on the wavelength of the light. Infrared (bigger λ ) is dispersed less
than UV-light (smaller λ ). This is the principle behind a prism.
o Reflection: If the angle in which the light reaches another medium is big
enough, it will reflect.
o Light doesn’t just bend when it encounters a difference in the breakingsindex
of a medium. Light will also bend when it goes through a small slit. This is
Diffraction. The principle of diffraction is based on interference:
o Interference between two light waves that are in phase will cause an
interference pattern with destructive (0 amplitude) and constructive (2x
amplitude) interference.
Lens theory
o 3 important rules:
o If the light from the object goes
parallel into lens. The lens will bend
it to pass the focal point.
o If the light goes from object through
the focal point, the light will bend it
to go parallel.
o If the light from object passes in the
middle of the lens, it will not bend.
o Formula’s:
o M =magnitude
o f =focal point
o d i=distance image ¿ lens
o d o =distance object ¿ lens
1 1 1 1
o = + →d i =
f d0 di 1/ f −1/ d o
, −d i
o M= (– because it’s an inverted image)
do
o With these formula’s you can calculate the
distance of the image and its magnitude.
o If the d o = f than the image will be on d i =
infinity.
To focus an image that is on infinity an
additional lens (= tube lens) is used to
focus the parallel bundles of lights to a new focal point.
A tube lens is often used in microscopy: it creates a distance between
ocular and tube lens, this gives the possibility to place ‘things’
between the objective and the tube lens, like filters for example.
o If the d o < f than the image will be on the same side of the lens as the object =
virtual image (this is the principle of a loop) (the M = a positive value) This is
the second lens (oculair) of a microscope. The eye will eventually capture the
virtual image.
Optical train of compound microscope
o 2 types of microscopes:
o Upright microscope the light comes from under the object
o Inverted microscope the light comes from above the object (better)
o Typical constellation of a compound microscope (upright):
o The light comes from light source it is bundled with condensor lens it passes
through specimen light is captured with objective lens and it forms an inverted
real image iverted image is projected on ocular that formes a virtual image The
eye captures the virtual image on retina.
o Thus, the total magnification is the sum of the magnification of objective +
magnification of ocular: M tot =M obj + M oc
o However, we often use an infinity-corrected microscope: The object is on f of the
objective lens, thus creating an infinitive image. The tube lens will bundle the parallel
light bundles and create a real image that is in front of f from ocular. Thus, the ocular
will produce a virtual image that can be captured with the eye.
o Conjugate planes = the places in light path of microscope where we have a sharp
image.
, o There are two paths that we can follow:
o Image forming path: places where the object is given with a sharp image
o Illuminating path: places where the light (filament from lamp) is given with
a sharp image.
o Image forming path: sharp images can be found on:
o Retina
o Specimen plate
o In front of ocular (= real image)
o The virtual image produced by ocular
o Light forming path: sharp light bundles can be found on:
o Lamp
o Front focal point of condenser
o Just after the objective (focal point)
o Diaphragm of the eye
o When using a microscope, you need to make sure all the conjugate planes are
sharp = Köhler illumination
o Steps Köhler illumination:
1. Sharpen image
2. Close diaphragm of condenser
3. Make sure the diaphragm of condenser is sharp (conjugate plane of front
focal plane of condensor = light forming path)
4. Center the diaphragm of condensor
5. Open the diaphgram of condenser to see the most intensity of light.
(conjugate plane of the rear focal plane of objective = light forming path,
normally you could see the filament of lamp)
Properties of optics
o Resolution limit of a microscope is 200nm.
, o Because of airy discs: every image that is created through an objective will be
‘smudged’. This limits the resolution to 200nm: When 2 airy discs overlap, and
the intensity drop of more than 25% the resolution limit is reached. The two
objects cannot be distinguished from one another.
o De smallest distance for which we can distinguish 2 points is given with Abbes
λ
Law: d=
2 n sin α
d = smallest distance for which we can distinguish 2 points
λ = wavelength light
n sin α = NA = Numerical Aperture
o Numerical Aperture with α is the half angle of captured light
through the objective. The bigger the NA, the better the
resolution will be since a broader range of light from the
specimen can be captured.
o However, with increasing NA, the portion of image
that is detected sharply is smaller.
o However, with increasing NA, the depth of field of an
image is less: we don’t see far into the tissue.
o However, with increasing NA, the distance from
specimen to objective is smaller.
o The specimen is placed in a medium with a higher diffraction index to make
sure that the diffracted light from specimen has a smaller range: this makes
NA better since n is higher (n sin α )
o Resolution does not only depend on NA. It also depends on the wavelength of the
light: λ . The bigger the wavelength, the lesser the resolution.
o Resolution also strongly depends on contrast: every object that is captured with an
objective will have smudging/blurring. This leads to a decrease in contrast. The
decrease in contrast will lead to a decrease in resolution since two close points that
are smudged, are difficult to distinguish from one another.
RESOLUTION AND ITS FACTORS ARE VERY IMPORTANT!!!
o No lens is perfect:
o Because a lens is round not every light bundle passes through same place
spherical abberations
o Because every color has a different wavelength not every lightbundle is
diffracted in the same way chromical abberations
o Because a lens has curvature image curvature
o Lenses do correct for these abberations. (given on objective)
o Apo = chromical aberration is corrected
o Plan = spherical aberration is corrected
Contrast enhancement
o Our eye can see differences in Amplitude - (intensity) and color of light (wavelength)
o It cannot see differences in phase of light (slowing down wave or diffracting wave)
o However, most specimens are phase specimens: they change the phase of light.
o Amplitude specimens change the amplitude of light.
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