Advanced light microscopy
Lecture 1
Light: 400-600nm à is already too big for certain particles; light
microscopy is not suitable then
Bright field
- Cells do not have a lot of contrast with the background
- Is suCicient if you want to count cells
There are other microscopy techniques that can increase the contrast, e.g.:
- DiCerential interference contrast microscope
- Phase contrast
- Confocal fluorescence
- Spinning disc confocal microscopy
- Super resolution
- Light sheet
What will define your choice on the type of microscope you will use?
- What is your question?
- What to consider?
o First: what question are you trying to answer?
o Second: what do you have available?
o Third: how will you analyse your images?
è Take home: you don’t always need to have the most expensive microscope available to answer your
question (in fact, it often brings problems: costs, time (samples, acquisition), qualified personnel
Light microscopy set-up
Several factors can have to do with resolution:
- DiCraction-limited resolution
- Point spread function
- Wavelengths of diCerent imaging modalities
- Factors aCecting spatial resolution
- FWHM
- Lateral vs axial spatial resolution
1
,Spatial resolution à the spatial resolution of an optical microscope is defined as the shortest distance between
two point sources of light on a specimen that can still be distinguished by the observer or camera system as
separate entities
- The distance between to items (if the center of A and the center of B are as a
certain distance from each other) determines its resolution
- At low magnification, the magnification is limiting for the resolution
The theoretical maximum resolution is proportional to wavelength
- Even with the best optics, a beam cannot be focused to a spot smaller than about half the wavelength
of light à is due to diCraction; as the wavelength increase, the minimum resolvable distance also
increases à shorter wavelength is better resolution
- So spatial resolution depends on magnification only up to a certain point
à But there is a practical limit: light microscopy is diCraction-limited (to ~200nm), as light behaves as a wave.
This is because it spreads out into a blurry pattern called an Airy disk, instead of a perfect point. This blur
spreading makes two nearby objects blur together if they are too close (more on Airy units on p. 32)
- Lower wavelength (e.g. blue light) à smaller spots; less overlap
- Larger wavelength (e.g. red light) à larger spots; more overlap à less discrimination between two
items with close distance
Resolution is not the same as detection
- Resolution: how close objects can be for you to discriminate them
- Detection: how well you can get any signal from it
Numerical aperture (NA) à says something about how much light of the sample you catch
- The higher the NA, the smaller the resolvable distance = better resolution
- Higher NA also increases the signal-to-noise ration = better detection
Refractory index (RI) à says something about the speed of light; this speed is diCerent in diCerent materials à
speed diCerence causes bending of light
- Light travels slower when it goes through an object, than it does when in a vacuum
The resolution depends on the amount of light you catch from the sample and the bending angle of the
objective
At some point when zooming in, you hit the resolution limit
Paths of light
- Absorbed
- Reflected
- Refracted; bending of light (it travels at a diCerent speed within the object); does the light
even hit the objective, or does it miss it (= out-of-focus light)
- DiCraction; spreading of light (airy disc / point-spread-function)
The speed of light in a vacuum is a constant à however in any material, it is slower than that; the actual speed
of light depends on the medium in which it travels (and material properties also change depending on the
temperature)
è Matching the refractive indexes of the glass, oil with the sample works best to not lose too much light
from the sample
2
,Refraction à how much of the path of light is bent or refracted, when entering material
- In optics, the refractive index (or refraction index) of an optical medium is a dimensionless number that
gives the indication of the light bending ability of that medium
- Refractive index (n) à n = cv
- C = the speed of light in vacuum
- V = the phase velocity of light in the medium
- With this n you can then calculate the angle of bending by using Snell’s law: 𝑛1𝑠𝑖𝑛𝜃1 = 𝑛2𝑠𝑖𝑛𝜃2
Refraction of light through a converging lens
- Parallel beamsà makes a focused beam = focal point
- Important considerations:
o Light has physical dimensions
o At small dimensions, light diCracts
o Light diCraction limit resolution
How small can a particle be and still be resolved?
- The physical size of the spot can be about half the
wavelength of light
- Above the resolution limit of particle decreases to its
size and optics are limiting
- Below the resolution of the particle appears the same, even though they are getting
smaller
è The resolution depends on the wavelength; you cannot focus smaller than half the wavelength of the
light you’re using
DiCraction limited resolution in optical microscopy
Numerical aperture (NA) = n * sin(a)
- Field of view versus depth of field (‘resolution in y-
direction)
- Low NA à most of the light is lost, and only a small
amount can be caught à you have to be in focus
- When super close to the specimen with the
objective (e.g. 100x magnification), the resolving
power is not limited by the lens, but by how much
light can enter the objective à refraction could
cause light to miss the objective à matching the
refractive index of the lens with oil minimizes the
bending of the light à goes straight into the objective
3
, Thus, the refractive index could limit the NA by limiting the angles of light that the objective can collect à
therefore you need oil to minimize this eCect
è The better you match the refraction index of everything, the better you see it all
Immersion objectives
Point spread function (PSF)
- Light behaves as a wave à diCraction forms an airy pattern
- If we image a point, the light we collect from the point source diCracts as it
passes the objective lens, resulting in a 3D interference pattern that we
call the PSF
- PSF in larger in z (axial resolution; specimen ‘depth’) than in x & y
è DiCraction and interference results in PSF
è PSF aCects the final image you get of the specimen due to convolution! You have to keep this in mind
during analysis (more on what you can do about this during analysis on p. 40)
Overview of factors that limit spatial resolution
- Magnification
- DiCraction limit
- Also scattering (e.g. dust) limits spatial resolution
- Mechanical distortions
- Imperfect optics (spheric aberrations, also in the sample!)
What is the resolution and how can you measure it? à full-width at half-maximum intensity (FWHM);
is a practical measure of resolution
- You can make a plot of the diCerences in intensity between darker and lighter spots à the
distance in the plot is representative of the resolution
- Noise can lead to over/underestimation of spatial resolution
Axial vs lateral spatial resolution à the images are in 2D whereas you are assessing a 3D structure à
the resolution refers to the X-Y resolution; the Z resolution is much lower à those structures are
imaged at the same time
Point spread function (PSF) in axial and lateral directions
4
Lecture 1
Light: 400-600nm à is already too big for certain particles; light
microscopy is not suitable then
Bright field
- Cells do not have a lot of contrast with the background
- Is suCicient if you want to count cells
There are other microscopy techniques that can increase the contrast, e.g.:
- DiCerential interference contrast microscope
- Phase contrast
- Confocal fluorescence
- Spinning disc confocal microscopy
- Super resolution
- Light sheet
What will define your choice on the type of microscope you will use?
- What is your question?
- What to consider?
o First: what question are you trying to answer?
o Second: what do you have available?
o Third: how will you analyse your images?
è Take home: you don’t always need to have the most expensive microscope available to answer your
question (in fact, it often brings problems: costs, time (samples, acquisition), qualified personnel
Light microscopy set-up
Several factors can have to do with resolution:
- DiCraction-limited resolution
- Point spread function
- Wavelengths of diCerent imaging modalities
- Factors aCecting spatial resolution
- FWHM
- Lateral vs axial spatial resolution
1
,Spatial resolution à the spatial resolution of an optical microscope is defined as the shortest distance between
two point sources of light on a specimen that can still be distinguished by the observer or camera system as
separate entities
- The distance between to items (if the center of A and the center of B are as a
certain distance from each other) determines its resolution
- At low magnification, the magnification is limiting for the resolution
The theoretical maximum resolution is proportional to wavelength
- Even with the best optics, a beam cannot be focused to a spot smaller than about half the wavelength
of light à is due to diCraction; as the wavelength increase, the minimum resolvable distance also
increases à shorter wavelength is better resolution
- So spatial resolution depends on magnification only up to a certain point
à But there is a practical limit: light microscopy is diCraction-limited (to ~200nm), as light behaves as a wave.
This is because it spreads out into a blurry pattern called an Airy disk, instead of a perfect point. This blur
spreading makes two nearby objects blur together if they are too close (more on Airy units on p. 32)
- Lower wavelength (e.g. blue light) à smaller spots; less overlap
- Larger wavelength (e.g. red light) à larger spots; more overlap à less discrimination between two
items with close distance
Resolution is not the same as detection
- Resolution: how close objects can be for you to discriminate them
- Detection: how well you can get any signal from it
Numerical aperture (NA) à says something about how much light of the sample you catch
- The higher the NA, the smaller the resolvable distance = better resolution
- Higher NA also increases the signal-to-noise ration = better detection
Refractory index (RI) à says something about the speed of light; this speed is diCerent in diCerent materials à
speed diCerence causes bending of light
- Light travels slower when it goes through an object, than it does when in a vacuum
The resolution depends on the amount of light you catch from the sample and the bending angle of the
objective
At some point when zooming in, you hit the resolution limit
Paths of light
- Absorbed
- Reflected
- Refracted; bending of light (it travels at a diCerent speed within the object); does the light
even hit the objective, or does it miss it (= out-of-focus light)
- DiCraction; spreading of light (airy disc / point-spread-function)
The speed of light in a vacuum is a constant à however in any material, it is slower than that; the actual speed
of light depends on the medium in which it travels (and material properties also change depending on the
temperature)
è Matching the refractive indexes of the glass, oil with the sample works best to not lose too much light
from the sample
2
,Refraction à how much of the path of light is bent or refracted, when entering material
- In optics, the refractive index (or refraction index) of an optical medium is a dimensionless number that
gives the indication of the light bending ability of that medium
- Refractive index (n) à n = cv
- C = the speed of light in vacuum
- V = the phase velocity of light in the medium
- With this n you can then calculate the angle of bending by using Snell’s law: 𝑛1𝑠𝑖𝑛𝜃1 = 𝑛2𝑠𝑖𝑛𝜃2
Refraction of light through a converging lens
- Parallel beamsà makes a focused beam = focal point
- Important considerations:
o Light has physical dimensions
o At small dimensions, light diCracts
o Light diCraction limit resolution
How small can a particle be and still be resolved?
- The physical size of the spot can be about half the
wavelength of light
- Above the resolution limit of particle decreases to its
size and optics are limiting
- Below the resolution of the particle appears the same, even though they are getting
smaller
è The resolution depends on the wavelength; you cannot focus smaller than half the wavelength of the
light you’re using
DiCraction limited resolution in optical microscopy
Numerical aperture (NA) = n * sin(a)
- Field of view versus depth of field (‘resolution in y-
direction)
- Low NA à most of the light is lost, and only a small
amount can be caught à you have to be in focus
- When super close to the specimen with the
objective (e.g. 100x magnification), the resolving
power is not limited by the lens, but by how much
light can enter the objective à refraction could
cause light to miss the objective à matching the
refractive index of the lens with oil minimizes the
bending of the light à goes straight into the objective
3
, Thus, the refractive index could limit the NA by limiting the angles of light that the objective can collect à
therefore you need oil to minimize this eCect
è The better you match the refraction index of everything, the better you see it all
Immersion objectives
Point spread function (PSF)
- Light behaves as a wave à diCraction forms an airy pattern
- If we image a point, the light we collect from the point source diCracts as it
passes the objective lens, resulting in a 3D interference pattern that we
call the PSF
- PSF in larger in z (axial resolution; specimen ‘depth’) than in x & y
è DiCraction and interference results in PSF
è PSF aCects the final image you get of the specimen due to convolution! You have to keep this in mind
during analysis (more on what you can do about this during analysis on p. 40)
Overview of factors that limit spatial resolution
- Magnification
- DiCraction limit
- Also scattering (e.g. dust) limits spatial resolution
- Mechanical distortions
- Imperfect optics (spheric aberrations, also in the sample!)
What is the resolution and how can you measure it? à full-width at half-maximum intensity (FWHM);
is a practical measure of resolution
- You can make a plot of the diCerences in intensity between darker and lighter spots à the
distance in the plot is representative of the resolution
- Noise can lead to over/underestimation of spatial resolution
Axial vs lateral spatial resolution à the images are in 2D whereas you are assessing a 3D structure à
the resolution refers to the X-Y resolution; the Z resolution is much lower à those structures are
imaged at the same time
Point spread function (PSF) in axial and lateral directions
4
