Biomedical imaging
Microscopy:
Biochemistry: tells you what and how much = how much genes and which are active ,…
Microscopy: tells you when and where = show the tissue and cell dynamics + structures in toto
bv visualising individual cells moving and forming structures in embryology non-invasively
Evolution of microscopy:
Robert Hooke: invented a compound microscope → saw the openings where cells were in cork
First compound microscope: works with a combination of 2 lenses → can exceed the resolution limit
of 1 lens
Diagnostic relevance: microscopes used for detection of rogue cells by searching cells with an off phenotype
Microscopy data: data size and complexity have vastly increased → automated to get the statistically
relevant data
The eye:
Anatomy:
Lens: held in a cavity and shielded by the cornea + iris (diaphragm)
Light: passes through the iris (tuned to allow light to pass if it’s light/dark) and through
the vitreum on the retina
Retina: contains photosensitive cells of rods and cones
Signaling: signal from rods and cones passed to nerves which assemble in 1 optic nerve
Limit: eyes have a resolution limit of 0,1mm -> anything smaller will appear as a point
➢ Use lenses to magnify things under 0,1mm
Microscopy basics:
Properties of light:
Electromagnetic spectrum: visible light is a fraction of the spectrum between 400-700nm
➢ > 770nm: infrared , radiowaves , microwaves
➢ < 390nm: ionizing waves (ultraviolet , X-rays , gamma rays , …)
Wavelength: distance between the maxima of a wave
➢ Wave energy: the shorter wavelength , the higher energy -> can damage cells
Amplitude: hight of the peaks
Phase: where the peak starts
Polarisation: direction of the wave
➔ Properties of the wave are defined by the medium in wich the wave travels
, Refractive index 𝒏: speed of light in vacuum / speed of light in medium
➢ The slower light goes through the medium the higher the refractive
index will be bv higher index in water
𝑛1 sin 𝛼2
Snell’s law: 𝑛2
= sin 𝛼1
Light bending: when light passes from a lower to a bigger 𝑛 the light will be bent towards the normal
Normal = line placed 90° on the surfaces
Dispersion:
Dispersion: the amount of refraction of a wave depends on the wavelength
➢ Red light is less sensitive to refraction than blue light because it has a higher wavelength
➢ White light will be dispersed into it’s component because they refract differently
Reflection:
Reflection: at the critical angle light will reflect and not refract
➢ Reflection under the same angle it came in with 𝜃𝑖 = 𝜃𝑟
Diffraction:
Diffraction: light will bend and spread out when passing through a narrow opening
Bending: the amount the light will be bent depends on the opening of the
slit in relation to the wavelength
➢ The smaller the slit the stronger light will be bent
Diffraction pattern:
Central spot: a strongly lit central spot
→ central light is not bent so it will reach the screen first
Darker spot: the bent light layers cancel eachother out = destructive interference
= minima
Lighter spot: the bent light layers amplify eachother = constructive interference
= maxima
…
Interference:
Interference: occurs when 2 waves meet and converts phase into amplitude changes
Constructive interference:
Double amplitude: 2 waves with the same phase will amplify eachother and make a
wave double in amplitude
Destructive interference:
No amplitude: 2 waves are shifted by 0,5𝜆 will cancel eachother out because the
maxima of 1 wave correlates with the minima of the other so you get no wave
Diffraction = based on interference
Single slit: the bent light will start interfering and form a diffraction pattern
Double slit: light will interfere and form an interference pattern
Maxima: 𝑑 sin 𝜃 = 𝑚𝜆 -> depends on the angle 𝜃 , distance between slits 𝑑 , wavelength 𝜆
,Lens theory:
Perfect convex lens:
• Optical axis: axis through the centre of the lens
• Focal point: light through the lens is focussed in this point in an
inverse pattern
• Focal distance: distance of the focal point from the lens
Convex lens rules:
Parallel rays converge at the focal point:
Rays from the focal point exit the plane in parallel:
Rays passing through the centre are unaffected:
Calculate magnification:
1 1 1
Lens maker equation: 𝑑0
+𝑑 =𝑓
𝑖
𝑑 ℎ𝑖
Magnification: 𝑀 = − 𝑑𝑖 = ℎ0
0
1
𝒅𝟎 > 𝟐𝒇 -> 𝑀 = − 2 : the object is inverted (-) and has become smaller (1/2) =minification
𝒅𝟎 = 𝟐𝒇 -> 𝑀 = −1 : the object is inverted (-) and is the same size (1)
𝒇 < 𝒅𝟎 < 𝟐𝒇 -> 𝑀 = −2 : the object is inverted (-) and has become bigger (2)
𝒅𝟎 = 𝒇 -> 𝑀 = ∞ : there is no object because the lights through the lens move parallel
➢ Focus this object with another lens
𝒅𝟎 < 𝒇 -> 𝑀 = +2 : there is a virtual image on the same side of the lens and not inverted (+)
➢ Visible with a magnifier (ocular)
Compound microscope:
Lamp
Field diaphragm
Condenser: condenses light on the sample
Sample
Objective: magnifying lens
Eyepieces: magnifying lens + eyes are the detectors and also have a lens
Camera film plane: lens projects the light on the camera screen
, Upright compound microscope: lenses are above the sample + light shines from underneath
= looking at cells from above
Inverted compound microscope: lenses are below the sample + light shines from above
= looking at cells from below
Compound microscope = 2 lens magnifier:
• Objective
• Occular
3. Ocular: generates a virtual image on the same side of
the lens and this will be projected on the retina (f > obj)
→ object of ocular is the image of objective
2. Condenser: focusses light
1. Objective: generates a real inverted image on the
other side of the lens (2f > obj > f)
Total magnification: 𝑀𝑡𝑜𝑡 = 𝑀𝑜𝑏𝑗 ∗ 𝑀𝑜𝑐
Infinity optics: object positioned at the focal plane of the
objective so the image is at infinity
→ more space to put extra lenses to enhance the contrast
Conjugate planes:
= planes where the focus is the same
Image-forming path: conjugate planes where the image is in focus
Retina: image focussed in the eye
Intermediate plane: location where the objective focussed the image
Object / specimen plane
Field stop diaphragm: light focussed from the collector lens
Illuminating-path: conjugate planes where the light filament is in focus
→ only see light if it’s in focus with the image + image needs to be illuminated continuously
Iris: diaphragm of the eye
Rear focal plane of objective
Front focal plane of condenser
Lamp
= Köhler illumination
Microscopy:
Biochemistry: tells you what and how much = how much genes and which are active ,…
Microscopy: tells you when and where = show the tissue and cell dynamics + structures in toto
bv visualising individual cells moving and forming structures in embryology non-invasively
Evolution of microscopy:
Robert Hooke: invented a compound microscope → saw the openings where cells were in cork
First compound microscope: works with a combination of 2 lenses → can exceed the resolution limit
of 1 lens
Diagnostic relevance: microscopes used for detection of rogue cells by searching cells with an off phenotype
Microscopy data: data size and complexity have vastly increased → automated to get the statistically
relevant data
The eye:
Anatomy:
Lens: held in a cavity and shielded by the cornea + iris (diaphragm)
Light: passes through the iris (tuned to allow light to pass if it’s light/dark) and through
the vitreum on the retina
Retina: contains photosensitive cells of rods and cones
Signaling: signal from rods and cones passed to nerves which assemble in 1 optic nerve
Limit: eyes have a resolution limit of 0,1mm -> anything smaller will appear as a point
➢ Use lenses to magnify things under 0,1mm
Microscopy basics:
Properties of light:
Electromagnetic spectrum: visible light is a fraction of the spectrum between 400-700nm
➢ > 770nm: infrared , radiowaves , microwaves
➢ < 390nm: ionizing waves (ultraviolet , X-rays , gamma rays , …)
Wavelength: distance between the maxima of a wave
➢ Wave energy: the shorter wavelength , the higher energy -> can damage cells
Amplitude: hight of the peaks
Phase: where the peak starts
Polarisation: direction of the wave
➔ Properties of the wave are defined by the medium in wich the wave travels
, Refractive index 𝒏: speed of light in vacuum / speed of light in medium
➢ The slower light goes through the medium the higher the refractive
index will be bv higher index in water
𝑛1 sin 𝛼2
Snell’s law: 𝑛2
= sin 𝛼1
Light bending: when light passes from a lower to a bigger 𝑛 the light will be bent towards the normal
Normal = line placed 90° on the surfaces
Dispersion:
Dispersion: the amount of refraction of a wave depends on the wavelength
➢ Red light is less sensitive to refraction than blue light because it has a higher wavelength
➢ White light will be dispersed into it’s component because they refract differently
Reflection:
Reflection: at the critical angle light will reflect and not refract
➢ Reflection under the same angle it came in with 𝜃𝑖 = 𝜃𝑟
Diffraction:
Diffraction: light will bend and spread out when passing through a narrow opening
Bending: the amount the light will be bent depends on the opening of the
slit in relation to the wavelength
➢ The smaller the slit the stronger light will be bent
Diffraction pattern:
Central spot: a strongly lit central spot
→ central light is not bent so it will reach the screen first
Darker spot: the bent light layers cancel eachother out = destructive interference
= minima
Lighter spot: the bent light layers amplify eachother = constructive interference
= maxima
…
Interference:
Interference: occurs when 2 waves meet and converts phase into amplitude changes
Constructive interference:
Double amplitude: 2 waves with the same phase will amplify eachother and make a
wave double in amplitude
Destructive interference:
No amplitude: 2 waves are shifted by 0,5𝜆 will cancel eachother out because the
maxima of 1 wave correlates with the minima of the other so you get no wave
Diffraction = based on interference
Single slit: the bent light will start interfering and form a diffraction pattern
Double slit: light will interfere and form an interference pattern
Maxima: 𝑑 sin 𝜃 = 𝑚𝜆 -> depends on the angle 𝜃 , distance between slits 𝑑 , wavelength 𝜆
,Lens theory:
Perfect convex lens:
• Optical axis: axis through the centre of the lens
• Focal point: light through the lens is focussed in this point in an
inverse pattern
• Focal distance: distance of the focal point from the lens
Convex lens rules:
Parallel rays converge at the focal point:
Rays from the focal point exit the plane in parallel:
Rays passing through the centre are unaffected:
Calculate magnification:
1 1 1
Lens maker equation: 𝑑0
+𝑑 =𝑓
𝑖
𝑑 ℎ𝑖
Magnification: 𝑀 = − 𝑑𝑖 = ℎ0
0
1
𝒅𝟎 > 𝟐𝒇 -> 𝑀 = − 2 : the object is inverted (-) and has become smaller (1/2) =minification
𝒅𝟎 = 𝟐𝒇 -> 𝑀 = −1 : the object is inverted (-) and is the same size (1)
𝒇 < 𝒅𝟎 < 𝟐𝒇 -> 𝑀 = −2 : the object is inverted (-) and has become bigger (2)
𝒅𝟎 = 𝒇 -> 𝑀 = ∞ : there is no object because the lights through the lens move parallel
➢ Focus this object with another lens
𝒅𝟎 < 𝒇 -> 𝑀 = +2 : there is a virtual image on the same side of the lens and not inverted (+)
➢ Visible with a magnifier (ocular)
Compound microscope:
Lamp
Field diaphragm
Condenser: condenses light on the sample
Sample
Objective: magnifying lens
Eyepieces: magnifying lens + eyes are the detectors and also have a lens
Camera film plane: lens projects the light on the camera screen
, Upright compound microscope: lenses are above the sample + light shines from underneath
= looking at cells from above
Inverted compound microscope: lenses are below the sample + light shines from above
= looking at cells from below
Compound microscope = 2 lens magnifier:
• Objective
• Occular
3. Ocular: generates a virtual image on the same side of
the lens and this will be projected on the retina (f > obj)
→ object of ocular is the image of objective
2. Condenser: focusses light
1. Objective: generates a real inverted image on the
other side of the lens (2f > obj > f)
Total magnification: 𝑀𝑡𝑜𝑡 = 𝑀𝑜𝑏𝑗 ∗ 𝑀𝑜𝑐
Infinity optics: object positioned at the focal plane of the
objective so the image is at infinity
→ more space to put extra lenses to enhance the contrast
Conjugate planes:
= planes where the focus is the same
Image-forming path: conjugate planes where the image is in focus
Retina: image focussed in the eye
Intermediate plane: location where the objective focussed the image
Object / specimen plane
Field stop diaphragm: light focussed from the collector lens
Illuminating-path: conjugate planes where the light filament is in focus
→ only see light if it’s in focus with the image + image needs to be illuminated continuously
Iris: diaphragm of the eye
Rear focal plane of objective
Front focal plane of condenser
Lamp
= Köhler illumination
