BIOMEDICAL IMAGING
HOOFDSTUK 1: MICROSCOPY BASICS
WAVE LIKE PROPERTIES OF LIGHT
- Light has a dual property: particle and wave light is just radiation
- Anything with a short wavelength and thus a higher energy is ionizing radiation
- Visible light: 400-700 nm
- Blue is lower in wavelength and red higher
- Slows down in media other than vacuum
- Refraction: when light moves from one medium in another the light will
bend because of the change in refractive index
speed of light ∈vacuum
o Refractive index n =
speed ∈medium
o It will slow down in a thicker
o Snell’s law: n1sinα1 = n2sinα2 the angle will be smaller when the refractive index becomes
bigger
o How higher the refractive index how more it will be bend + the direction of bending will
be towards the normal
- Refraction is dependent on the wavelength dispersion =
wavelength dependent refraction = every wavelength will be bend
a little different f.a. prisma
o Depending on medium and angle the light pass or no
longer pass
o If u reach a critical angle light will reflect and not refract: angle = angle incidence
- Diffraction: property of light to bend when it reaches and opening = coherent light spreads out when
passing a narrow slit
o The smaller the opening, the stronger the light will be bend
o It changes the way light is perceived and how it is projected on a flat plan important for
microscopy
o It generates a characteristic pattern diffraction pattern made by the different speeds of
Important formular: d sin θ =
mλ
the different parts + the interaction, pos of neg
o Interference: when 2 waves meet
o Constructive or destructive interference
,LENS THEORY
1
2
- A lens focusses light in the focal point at a certain distance f from the lens
o Rule 1: parallel rays converge at the focal point
o Rule 2: rays from the focal point plane exit in parallel
o Rule 3: rays that pass though the centre are unaffected
- Calculating the magnification 3
OPTICAL TRAIN OF COMPOUND MICROSCOPE
Inverted microscope: like a normal upright microscope but the lenses are inverted, they are below the sample
hole.
Condenser: condenses the light onto the sample and thus focusses the light on it and then the light diverses
again and gets captured by the lens detector, commonly the eye
THE COMPOUND MICROSCOPE IS A TWO-LENS MAGNIFIER
The first lens that really makes the magnification is
the objective.
The lens works as a slide projector, it makes a real
inverted image on the other side of the lens the
object needs to be position at least closer than 2x
focal distance, else it will not magnify
The real image is formed outside of the objective and
then captured by the second lens, the ocular
,generate a virtual image: acts as magnifier that gives an image on the same side so we can see it because it is
projected on our retina
A virtual image is made if the object is
positioned closer than the focal distance
The compound lens system achieves higher magnification.
We can see the virtual image because our eye has a lens itself and
that projects the image on our retina
We can add different structural components in between the lenses
for different properties but we then need more space in-between the lenses. But the lenses are put in an exact
measured distance for the optimal view infinity optics: the object is positioned exactly at the focal plane of
the objective infinity image if we don’t position any lenses in between then the image would just
continue because of the parallel rays we can put anything in between without effecting the focus after
we put in everything we want we put a tube lens which focusses the light before the focal plane of the eye
piece
Everything needs to be well aligned.
Within a microscope slide box there are several positions
where the focus is the same = conjugate planes
In image forming path: 4 planes
Intermediate image plane = where the objective focusses
the image = 3th conjugate plane
4th = before the sample = above the collector lens = where
we control how much of the field is illuminated = where
we position the diaphragm
In illuminating path:
Conjugate planes where the amp filament is in focus
we don’t want to see that there can not be a conjugate
plane at the position of the sample light should there
move parallel light should be focussed in front of the
focal plane of the condenser = 1st conjugate plane
PROPERTIES OF OPTICS (HEEL BELANGRIJK DEEL)
, A microscope has a resolution limit because of diffraction.
When passing the lens aperture, light diffracts
The smaller the distance between the patterns, de more
the light will get bend larger diffraction pattern + less
resolution
The moment the gaps become wider, there are more
maxima the objective can detect.
Diffraction takes place but that isn’t a problem because
if we can capture everything, we can make the image.
But if we lose some maxima, we can’t regenerate the
complete image anymore. This is because the objective
can only capture a part of the light lose resolution
f imposes a
resolution limit.
Resolution limit= limit where u have a difficulty win spotting maxima
België (BE)between the 2 we can no longer resolve the object
Rayleigh limit = 25% in intensity drop
λ
Optical resolution limit: d =
2n∗sin α
n*sin α = numerical aperture NA
Small d = good resolution
NA = the opening of the lens = the angle the objecti<ve can make, how much light it can capture or how
strongly it can focus the light. It also defines the resolution.
The resolution is only defined by 2 things: the wavelength and the NA, not the magnification
Working distance= distance u have between
the sample and the lens
Is connected to the NA: the larger the opening
the closer u have to be to ur sample.
With a high NA u have a small
depth off field.
Most lenses work in dry environment but ur samples are often
embedded in a oily solution refractive index differences some high class lenses are made
so u can use immersion media: u can put a drop of the solution on the lens minimize
refractive index differences + capture more lightrays higher resolution
HOOFDSTUK 1: MICROSCOPY BASICS
WAVE LIKE PROPERTIES OF LIGHT
- Light has a dual property: particle and wave light is just radiation
- Anything with a short wavelength and thus a higher energy is ionizing radiation
- Visible light: 400-700 nm
- Blue is lower in wavelength and red higher
- Slows down in media other than vacuum
- Refraction: when light moves from one medium in another the light will
bend because of the change in refractive index
speed of light ∈vacuum
o Refractive index n =
speed ∈medium
o It will slow down in a thicker
o Snell’s law: n1sinα1 = n2sinα2 the angle will be smaller when the refractive index becomes
bigger
o How higher the refractive index how more it will be bend + the direction of bending will
be towards the normal
- Refraction is dependent on the wavelength dispersion =
wavelength dependent refraction = every wavelength will be bend
a little different f.a. prisma
o Depending on medium and angle the light pass or no
longer pass
o If u reach a critical angle light will reflect and not refract: angle = angle incidence
- Diffraction: property of light to bend when it reaches and opening = coherent light spreads out when
passing a narrow slit
o The smaller the opening, the stronger the light will be bend
o It changes the way light is perceived and how it is projected on a flat plan important for
microscopy
o It generates a characteristic pattern diffraction pattern made by the different speeds of
Important formular: d sin θ =
mλ
the different parts + the interaction, pos of neg
o Interference: when 2 waves meet
o Constructive or destructive interference
,LENS THEORY
1
2
- A lens focusses light in the focal point at a certain distance f from the lens
o Rule 1: parallel rays converge at the focal point
o Rule 2: rays from the focal point plane exit in parallel
o Rule 3: rays that pass though the centre are unaffected
- Calculating the magnification 3
OPTICAL TRAIN OF COMPOUND MICROSCOPE
Inverted microscope: like a normal upright microscope but the lenses are inverted, they are below the sample
hole.
Condenser: condenses the light onto the sample and thus focusses the light on it and then the light diverses
again and gets captured by the lens detector, commonly the eye
THE COMPOUND MICROSCOPE IS A TWO-LENS MAGNIFIER
The first lens that really makes the magnification is
the objective.
The lens works as a slide projector, it makes a real
inverted image on the other side of the lens the
object needs to be position at least closer than 2x
focal distance, else it will not magnify
The real image is formed outside of the objective and
then captured by the second lens, the ocular
,generate a virtual image: acts as magnifier that gives an image on the same side so we can see it because it is
projected on our retina
A virtual image is made if the object is
positioned closer than the focal distance
The compound lens system achieves higher magnification.
We can see the virtual image because our eye has a lens itself and
that projects the image on our retina
We can add different structural components in between the lenses
for different properties but we then need more space in-between the lenses. But the lenses are put in an exact
measured distance for the optimal view infinity optics: the object is positioned exactly at the focal plane of
the objective infinity image if we don’t position any lenses in between then the image would just
continue because of the parallel rays we can put anything in between without effecting the focus after
we put in everything we want we put a tube lens which focusses the light before the focal plane of the eye
piece
Everything needs to be well aligned.
Within a microscope slide box there are several positions
where the focus is the same = conjugate planes
In image forming path: 4 planes
Intermediate image plane = where the objective focusses
the image = 3th conjugate plane
4th = before the sample = above the collector lens = where
we control how much of the field is illuminated = where
we position the diaphragm
In illuminating path:
Conjugate planes where the amp filament is in focus
we don’t want to see that there can not be a conjugate
plane at the position of the sample light should there
move parallel light should be focussed in front of the
focal plane of the condenser = 1st conjugate plane
PROPERTIES OF OPTICS (HEEL BELANGRIJK DEEL)
, A microscope has a resolution limit because of diffraction.
When passing the lens aperture, light diffracts
The smaller the distance between the patterns, de more
the light will get bend larger diffraction pattern + less
resolution
The moment the gaps become wider, there are more
maxima the objective can detect.
Diffraction takes place but that isn’t a problem because
if we can capture everything, we can make the image.
But if we lose some maxima, we can’t regenerate the
complete image anymore. This is because the objective
can only capture a part of the light lose resolution
f imposes a
resolution limit.
Resolution limit= limit where u have a difficulty win spotting maxima
België (BE)between the 2 we can no longer resolve the object
Rayleigh limit = 25% in intensity drop
λ
Optical resolution limit: d =
2n∗sin α
n*sin α = numerical aperture NA
Small d = good resolution
NA = the opening of the lens = the angle the objecti<ve can make, how much light it can capture or how
strongly it can focus the light. It also defines the resolution.
The resolution is only defined by 2 things: the wavelength and the NA, not the magnification
Working distance= distance u have between
the sample and the lens
Is connected to the NA: the larger the opening
the closer u have to be to ur sample.
With a high NA u have a small
depth off field.
Most lenses work in dry environment but ur samples are often
embedded in a oily solution refractive index differences some high class lenses are made
so u can use immersion media: u can put a drop of the solution on the lens minimize
refractive index differences + capture more lightrays higher resolution
