LES1: MICROSCOPY BASICS
Resolution
= the capability to spot differences between two details
- human eye: 0,1mm
- light microscope: 200nm
-> waves
WAVE LIKE PROPERTIES OF LIGHT -> photons
Refraction = visible light bends at an interface
Refractive index (n)
= the speed of light in a medium
-> n = 1 in vacuum
! refraction deceives the eye !
=> Snell’s law : n sin ⍺ (1)= n sin ⍺ (2)
Dispersion = refraction depends on the wavelength
Reflection = Above a critical angle light reflects
diffraction = when coherent light moves through a small opening, it bends
! important in microscopy !
Interference = when waves meet, amplitude changes occur
-> constructive
-> destructive
! diffraction is based on interference !
• The finer the grating, so the closer the slits are togerther, the more
spread out the maxima and minima will be
LENS THEORY
1
,OPTICAL TRAIN OF COMPOUND MICROSCOPE (light microscope)
=> to obtain a higher magnification: M = M(obj) * M(oc)
LENS1: Condenser
= the lens that captures the light and focuses it
into its focal point (not in the sample)
LENS2: Objective
= captures the light and magnifies with its convex
lens
LENS3: Oculars
= a second factor of magnification
(magnify an object that is closer to the lens than its
focal point => generate a virtual image)
LENS4: Eye-lens
= projects the image onto the retina
BUT most microscopes now position the
sample exactly on the focal point of the
objective
=> no image is generated (infinity)
EXTRA lens is added (tube lens)
=> focuses the light between the focal point
and the eyepiece
! makes more space between the lenses !
KOHLER ILLUMINATION
= the standard method for setting up a microscope’s light to get the best possible image quality
-> sharp
-> evenly lit
-> max resolution
! otherwise, the light isn’t coherent enough when passing
through the sample !
Conjugate planes - IFP
= planes of common focus, where we see the same thing
ex: our retina sees the same thing as the sample
Conjugate planes - IP
= where the lamp filament is sharp
-> should never be sharp on your eye! (divergent)
-> should be as parallel as possible on the sample
(conjugate plane has to be exaclty on the focal point)
2
,Aligning the image forming path
1. Make the Köhler hexagon as small as possible
2. Move the condenser up and down to make sure the hexagon is sharp
3. Put the hexagon in the centre
4. Open the diaphragm until it aligns with the view field
Aligning the illuminating path
=> put the condenser diaphragm at 2/3
!! the illuminating and image forming conjugate planes must each be correctly aligned within their own
system to reach Kohler illumination !!
PROPERTIES OF OPTICS
“The resolution of a microscope is defined by its objectives, BECAUSE a microscope is limited in its
resolving power”
1) “The resolution of a microscope is defined by its objectives”
Objective: collects the light coming from the sample
=> its physical properties (NA and Wavelength) determine how much detail can be separated
2) “Because a microscope is limited in its resolving power”
=> Even with more magnification, you cannot see beyond the physical optical limit (diffraction)
Airy pattern
= the reflection caused by the limited resolution
(200nm) of a microscope
D = distance between the 2 points that we can still resolve (resolution)
Lambda = wavelength
N = refractive index of the medium that we are using
Sin alfa = alfa is the angle at which the objective can catch the light
Numerical aperture (NA)
= n*sinalfa; a characteristic feature of each lens that defines how much light or diffraction patterns we
can capture (and so how good the resolution is)
Many high quality and high NA lenses rely on immersion media (WD must be very small)
=> refine n to capture more light rays for better resolution
Working distance
= distance between the lens and the sample
3
, CONTRAST ENHANCEMENT
! Contrast is directly related to resolution !
Contrast transfer function
= the level of contrast (modulation) in function of the resolution (spatial
frequency)
Lenses suffer from aberrations
Spatial aberration => plan correction
= makes sure that flat fields of the sample can be seen correctly, and won’t be rounded by the round
lenses
Chromatic aberrations => lenses
= different wavelengths break differently, and so must be corrected by lenses
ex: apo-chromatic lenses -> correct fort he 3 colours and position them at the same Z-plane
4
Resolution
= the capability to spot differences between two details
- human eye: 0,1mm
- light microscope: 200nm
-> waves
WAVE LIKE PROPERTIES OF LIGHT -> photons
Refraction = visible light bends at an interface
Refractive index (n)
= the speed of light in a medium
-> n = 1 in vacuum
! refraction deceives the eye !
=> Snell’s law : n sin ⍺ (1)= n sin ⍺ (2)
Dispersion = refraction depends on the wavelength
Reflection = Above a critical angle light reflects
diffraction = when coherent light moves through a small opening, it bends
! important in microscopy !
Interference = when waves meet, amplitude changes occur
-> constructive
-> destructive
! diffraction is based on interference !
• The finer the grating, so the closer the slits are togerther, the more
spread out the maxima and minima will be
LENS THEORY
1
,OPTICAL TRAIN OF COMPOUND MICROSCOPE (light microscope)
=> to obtain a higher magnification: M = M(obj) * M(oc)
LENS1: Condenser
= the lens that captures the light and focuses it
into its focal point (not in the sample)
LENS2: Objective
= captures the light and magnifies with its convex
lens
LENS3: Oculars
= a second factor of magnification
(magnify an object that is closer to the lens than its
focal point => generate a virtual image)
LENS4: Eye-lens
= projects the image onto the retina
BUT most microscopes now position the
sample exactly on the focal point of the
objective
=> no image is generated (infinity)
EXTRA lens is added (tube lens)
=> focuses the light between the focal point
and the eyepiece
! makes more space between the lenses !
KOHLER ILLUMINATION
= the standard method for setting up a microscope’s light to get the best possible image quality
-> sharp
-> evenly lit
-> max resolution
! otherwise, the light isn’t coherent enough when passing
through the sample !
Conjugate planes - IFP
= planes of common focus, where we see the same thing
ex: our retina sees the same thing as the sample
Conjugate planes - IP
= where the lamp filament is sharp
-> should never be sharp on your eye! (divergent)
-> should be as parallel as possible on the sample
(conjugate plane has to be exaclty on the focal point)
2
,Aligning the image forming path
1. Make the Köhler hexagon as small as possible
2. Move the condenser up and down to make sure the hexagon is sharp
3. Put the hexagon in the centre
4. Open the diaphragm until it aligns with the view field
Aligning the illuminating path
=> put the condenser diaphragm at 2/3
!! the illuminating and image forming conjugate planes must each be correctly aligned within their own
system to reach Kohler illumination !!
PROPERTIES OF OPTICS
“The resolution of a microscope is defined by its objectives, BECAUSE a microscope is limited in its
resolving power”
1) “The resolution of a microscope is defined by its objectives”
Objective: collects the light coming from the sample
=> its physical properties (NA and Wavelength) determine how much detail can be separated
2) “Because a microscope is limited in its resolving power”
=> Even with more magnification, you cannot see beyond the physical optical limit (diffraction)
Airy pattern
= the reflection caused by the limited resolution
(200nm) of a microscope
D = distance between the 2 points that we can still resolve (resolution)
Lambda = wavelength
N = refractive index of the medium that we are using
Sin alfa = alfa is the angle at which the objective can catch the light
Numerical aperture (NA)
= n*sinalfa; a characteristic feature of each lens that defines how much light or diffraction patterns we
can capture (and so how good the resolution is)
Many high quality and high NA lenses rely on immersion media (WD must be very small)
=> refine n to capture more light rays for better resolution
Working distance
= distance between the lens and the sample
3
, CONTRAST ENHANCEMENT
! Contrast is directly related to resolution !
Contrast transfer function
= the level of contrast (modulation) in function of the resolution (spatial
frequency)
Lenses suffer from aberrations
Spatial aberration => plan correction
= makes sure that flat fields of the sample can be seen correctly, and won’t be rounded by the round
lenses
Chromatic aberrations => lenses
= different wavelengths break differently, and so must be corrected by lenses
ex: apo-chromatic lenses -> correct fort he 3 colours and position them at the same Z-plane
4
