Summary Biomedical Imaging PH
3Ba BMW
2025-2026
Summary Microscopy Part
Ch1: Microscopy basics
1. Wave-like properties of light
Visible light is a fraction of the electromagnetic spectrum
• Light duality: light can be seen as a particle (photon) AND as a wave
• Visible light is but a mini fraction of the EM spectrum
o Range from 400nm to 700nm
o UV-ish (400nm) to green (500nm) to red light (700nm)
• Properties of light:
o Wavelength = distance between 2 maxima of a wave
▪ Short wavelength = high energy
o Frequency = number of waves per second
o Amplitude = hight of a peak
o Phase = where the peak starts
Visible light slows down in media other than vacuum
• Speed of light in vacuum = 3.00E8 m/s
• In other media light will slow down = REFRACTION
o Every medium that slows down the speed of light
has a certain refractive index
▪ Refractive index = speed of light in vacuum / speed in medium
▪ Higher the index = slower the light will propagate
• When light doesn’t come in straight but at an angle, it will brakes and
will bend
o When light breaks from thin medium to dens medium
(low n to higher n)
➔ it will bend TOWARDS the normal
o This is Snell’s law: n1sina1 = n2sina2
• Refraction deceives the eye (object isn’t where you see it)
Refraction depends on the wavelength = DISPERSION
• DISPERSION = recreation of white light will result in refraction in
its individual components (colours)
o Colour will deviate inversely proportional related with their
wavelength
(blue will bend more than red)
• Above a critical angle, light reflects
o More dens medium → more REFLECTION
Coherent light spreads out when passing a slit = DIFFRACTION
• DIFFRACTION = light will bend out when passing a small opening
Page 1 of 63
,Summary Biomedical Imaging PH
3Ba BMW
2025-2026
o Smaller opening = stronger deviation of light
o At the opening we see a spherical wave
• Diffraction only takes place when the slit is smaller
than the wavelength
• Diffraction generates a characteristic pattern
consisting of a strong light focal spot alternated by
darker spots
o Intuitive: the central part is un-deviated and will reach the surface quicker, then
you get different layers that can interact with each other
INTERFERENCE happens when waves meet
• INTERFERENCE coverts phase into amplitude changes
• when 2 waves interact, they can create 2 possible diffraction patters:
o Amplify each other’s signal = CONSTRUCTIVE
INTERFERENCE
▪ complete constructive interference =
amplitude x 2
o Destruct each other’s signal = DESTRUCTIVE
INTERFERENCE
▪ Phase difference = half of wavelength
Diffraction is based on interference
• Diffraction pattern = result of the interference of the deviated light
• Maxima is related to:
o Angle
o Distance between the slits
o Wavelength
➔ d*sinθ = m*λ
2. Lens theory
A perfect convex lens
• A perfect convex lens has a focal point = point where light
gets focussed
• Light coming from the focal point passing through the lens,
exit parallel
Rules lens theory
Parallel rays converge at the Rays from the focal plane exit in Rays that pass through the
focal point parallel centre are unaffected
A convex lens magnifies
• Based on 3 lines we can find out where an object is in space
o Parallel line with the optical axis will de deviate through the focal point
Page 2 of 63
,Summary Biomedical Imaging PH
3Ba BMW
2025-2026
o 2nd line through the focal point will exit parallel with the optical axis
o 3rd line passing through the centre is unaffected
• Based on this drawing we can visualize where the object is in space
• Formulas:
1 1 1
o Lens maker equation : 𝑑0 + 𝑑𝑖 = 𝑓
𝑑𝑖 ℎ𝑖
o Magnification: 𝑀 = − =
𝑑0 ℎ0
Different situations
d0 f Image distance di Magnification Image
d0 > 2f 1/3f + 1di = 1f di = 3/2f M = -1/2 Smaller, real, inverted
d0 = 2f 1/di = 1/2f di = 2f M = -1 Same size, real, inverted
d0 between f and 2f di = 3f M = -2 Bigger, real, inverted
d0 = f di = 0 M = infinity No image
d0 <f di = f M = +2 Bigger, virtual, upright
Commentary
• minus sign before di means the image is in front of the lens
• minus sign before magnification means the image is inverted
• Magnification lower than 1 means image is smaller
• “no image” is used in microscopes → second lens needed for focus
3. Optical train of compound microscope
Anatomy of the compound microscope
• We know different microscopes
o Inverted microscope → objective underneath the sample
o Upright microscope → objective on top of the sample
• In general a microscope consist of:
o Lightsource (lamp)
o Condenser (to focus light)
o Objective (for magnification)
o Ocular (eyepiece) (for extra magnification to create virtual image to the
eye)
• 2 step process: Mtot = Mobjective * Mocular
The compound microscope is a two-lens magnifier
• The objective magnifies the specimen
o A real inverted image is formed on the other side of the lens when the
specimen is at least 2f away from the lens
o In the end it will be a real image because it is projected on to the retina
• We use 2 lenses: objective for initial magnification and the ocular for further
magnification
o Result: virtual image that gets projected through the lens of the eye on
to the retina
Page 3 of 63
, Summary Biomedical Imaging PH
3Ba BMW
2025-2026
Finite vs. infinity optics
• Finite:
o The real image is closer to the lens than the focal
distance ➔ virtual image formed behind the
ocular
• Infinity:
o Problem: if we want higher contrast, there needs
to be space between the lenses BUT every
deviation can lead to no imaging
o Solution: specimen placed at focal point ➔ no image BUT
image formed at infinity
▪ More time to focus image
▪ At some point you need to focus ➔ tube lens will
focus light
The concept of conjugate planes
• Quality of the microscope is depend on its setting
o This can be adjusted via conjugate planes
• Conjugate planes = location where the image is focussed/sharp
o Place where specimen is
o Retina
o Intermediate image (formed by objective and used by
ocular to create virtual image)
o Field stop diaphragm (determines how much light goes
through)
• ALSO illuminating path has conjugate planes (where lamp filament is sharp)
o We don’t want to see this because we want the specimen to be illuminated
homogenic ➔ NO CONJUGATE PLANE ON RETINA
▪ Lamp filament
▪ Front focal plane of condenser
▪ Rear focal plane of objective
▪ Iris diaphragm of the eye
4. Properties of optics (always on exam!!!)
Resolution limit
• Resolution limit is measure of the ability of the objective to distinguish two points in the
image (eye = 0,1mm)
o No lens is perfect ➔ we don’t see points but a
diffraction pattern
o You get an airy disk that’s surrounded by concentric
rings = AIRY PATTERN
Page 4 of 63
3Ba BMW
2025-2026
Summary Microscopy Part
Ch1: Microscopy basics
1. Wave-like properties of light
Visible light is a fraction of the electromagnetic spectrum
• Light duality: light can be seen as a particle (photon) AND as a wave
• Visible light is but a mini fraction of the EM spectrum
o Range from 400nm to 700nm
o UV-ish (400nm) to green (500nm) to red light (700nm)
• Properties of light:
o Wavelength = distance between 2 maxima of a wave
▪ Short wavelength = high energy
o Frequency = number of waves per second
o Amplitude = hight of a peak
o Phase = where the peak starts
Visible light slows down in media other than vacuum
• Speed of light in vacuum = 3.00E8 m/s
• In other media light will slow down = REFRACTION
o Every medium that slows down the speed of light
has a certain refractive index
▪ Refractive index = speed of light in vacuum / speed in medium
▪ Higher the index = slower the light will propagate
• When light doesn’t come in straight but at an angle, it will brakes and
will bend
o When light breaks from thin medium to dens medium
(low n to higher n)
➔ it will bend TOWARDS the normal
o This is Snell’s law: n1sina1 = n2sina2
• Refraction deceives the eye (object isn’t where you see it)
Refraction depends on the wavelength = DISPERSION
• DISPERSION = recreation of white light will result in refraction in
its individual components (colours)
o Colour will deviate inversely proportional related with their
wavelength
(blue will bend more than red)
• Above a critical angle, light reflects
o More dens medium → more REFLECTION
Coherent light spreads out when passing a slit = DIFFRACTION
• DIFFRACTION = light will bend out when passing a small opening
Page 1 of 63
,Summary Biomedical Imaging PH
3Ba BMW
2025-2026
o Smaller opening = stronger deviation of light
o At the opening we see a spherical wave
• Diffraction only takes place when the slit is smaller
than the wavelength
• Diffraction generates a characteristic pattern
consisting of a strong light focal spot alternated by
darker spots
o Intuitive: the central part is un-deviated and will reach the surface quicker, then
you get different layers that can interact with each other
INTERFERENCE happens when waves meet
• INTERFERENCE coverts phase into amplitude changes
• when 2 waves interact, they can create 2 possible diffraction patters:
o Amplify each other’s signal = CONSTRUCTIVE
INTERFERENCE
▪ complete constructive interference =
amplitude x 2
o Destruct each other’s signal = DESTRUCTIVE
INTERFERENCE
▪ Phase difference = half of wavelength
Diffraction is based on interference
• Diffraction pattern = result of the interference of the deviated light
• Maxima is related to:
o Angle
o Distance between the slits
o Wavelength
➔ d*sinθ = m*λ
2. Lens theory
A perfect convex lens
• A perfect convex lens has a focal point = point where light
gets focussed
• Light coming from the focal point passing through the lens,
exit parallel
Rules lens theory
Parallel rays converge at the Rays from the focal plane exit in Rays that pass through the
focal point parallel centre are unaffected
A convex lens magnifies
• Based on 3 lines we can find out where an object is in space
o Parallel line with the optical axis will de deviate through the focal point
Page 2 of 63
,Summary Biomedical Imaging PH
3Ba BMW
2025-2026
o 2nd line through the focal point will exit parallel with the optical axis
o 3rd line passing through the centre is unaffected
• Based on this drawing we can visualize where the object is in space
• Formulas:
1 1 1
o Lens maker equation : 𝑑0 + 𝑑𝑖 = 𝑓
𝑑𝑖 ℎ𝑖
o Magnification: 𝑀 = − =
𝑑0 ℎ0
Different situations
d0 f Image distance di Magnification Image
d0 > 2f 1/3f + 1di = 1f di = 3/2f M = -1/2 Smaller, real, inverted
d0 = 2f 1/di = 1/2f di = 2f M = -1 Same size, real, inverted
d0 between f and 2f di = 3f M = -2 Bigger, real, inverted
d0 = f di = 0 M = infinity No image
d0 <f di = f M = +2 Bigger, virtual, upright
Commentary
• minus sign before di means the image is in front of the lens
• minus sign before magnification means the image is inverted
• Magnification lower than 1 means image is smaller
• “no image” is used in microscopes → second lens needed for focus
3. Optical train of compound microscope
Anatomy of the compound microscope
• We know different microscopes
o Inverted microscope → objective underneath the sample
o Upright microscope → objective on top of the sample
• In general a microscope consist of:
o Lightsource (lamp)
o Condenser (to focus light)
o Objective (for magnification)
o Ocular (eyepiece) (for extra magnification to create virtual image to the
eye)
• 2 step process: Mtot = Mobjective * Mocular
The compound microscope is a two-lens magnifier
• The objective magnifies the specimen
o A real inverted image is formed on the other side of the lens when the
specimen is at least 2f away from the lens
o In the end it will be a real image because it is projected on to the retina
• We use 2 lenses: objective for initial magnification and the ocular for further
magnification
o Result: virtual image that gets projected through the lens of the eye on
to the retina
Page 3 of 63
, Summary Biomedical Imaging PH
3Ba BMW
2025-2026
Finite vs. infinity optics
• Finite:
o The real image is closer to the lens than the focal
distance ➔ virtual image formed behind the
ocular
• Infinity:
o Problem: if we want higher contrast, there needs
to be space between the lenses BUT every
deviation can lead to no imaging
o Solution: specimen placed at focal point ➔ no image BUT
image formed at infinity
▪ More time to focus image
▪ At some point you need to focus ➔ tube lens will
focus light
The concept of conjugate planes
• Quality of the microscope is depend on its setting
o This can be adjusted via conjugate planes
• Conjugate planes = location where the image is focussed/sharp
o Place where specimen is
o Retina
o Intermediate image (formed by objective and used by
ocular to create virtual image)
o Field stop diaphragm (determines how much light goes
through)
• ALSO illuminating path has conjugate planes (where lamp filament is sharp)
o We don’t want to see this because we want the specimen to be illuminated
homogenic ➔ NO CONJUGATE PLANE ON RETINA
▪ Lamp filament
▪ Front focal plane of condenser
▪ Rear focal plane of objective
▪ Iris diaphragm of the eye
4. Properties of optics (always on exam!!!)
Resolution limit
• Resolution limit is measure of the ability of the objective to distinguish two points in the
image (eye = 0,1mm)
o No lens is perfect ➔ we don’t see points but a
diffraction pattern
o You get an airy disk that’s surrounded by concentric
rings = AIRY PATTERN
Page 4 of 63
