Slides 4

Biophotonics
Lecture 4 - The limits

of microscopy
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Last Time
Understanding diffraction
Numerical aperture and light collection
Fourier transforms in optics
Spatial frequencies
Generating arbitrary patterns at focus
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https://www.newscientist.com/article/mg19325971-600-hybrid-images-now-you-see-them/
Can you explain this in terms of spatial frequency?

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https://www.newscientist.com/article/mg19325971-600-hybrid-images-now-you-see-them/
Can you explain this in terms of spatial frequency?

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Todays Lecture
Resolution
Point Spread Function
Rayleigh Criterion
Abbe Criterion
Diffraction Limit
Seeing (a bit or a lot) smaller

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http://www.cambridgeincolour.com/tutorials/diffraction-photography.htm
At focus a circular aperture (like a lens) gives the

Airy Pattern.
Point Spread Function (PSF) is the ideal response

of an optical system to a point like illumination.
For a circular lens we have the Airy pattern

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Rayleigh Criterion
How can we resolve two objects?
Rayleigh Criterion - objects are resolved until the maximum of one

objects PSF reaches the first minimum of the second objects PSF.
Considering the Bessel function allows us to define =1.22 /D as the

angular resolution for an aperture of diameter D.
7 http://astronomy.swin.edu.au/cosmos/R/Rayleigh+Criterion
Biophotonics
Rayleigh Criterion
Well resolved


Biophotonics
Rayleigh Criterion
Well resolved Just resolved


Biophotonics
Rayleigh Criterion
Well resolved Just resolved Not resolved


Biophotonics
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Fourier theory: image made up of a range of spatial frequencies.
Imagine object made up of a series of ever finer gratings that give the
required spatial frequencies.
Imaging theory states that most of the information can be obtained if

zeroth and first order of diffraction pattern can be captured.
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Maximum angle that can be

captured:
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captured:
Constructive interference gives

path difference of m.
m =1 for first order.
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captured:

Considering this condition:
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captured:

Remembering the definition of

NA and assuming n=1:
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captured:

Remembering the definition of

NA and assuming n=1:
Abbe limit on smallest

object that can be seen!
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For a circular aperture:
Rule of thumb - minimum

resolvable size is /2
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Spatial Frequencies and The

Diffraction Limit
https://www.microscopyu.com/techniques/super-resolution/the-diffraction-barrier-in-optical-microscopy
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Depth of Field
In general we have been considering the material
resolution.
What about in the z-direction?
Depth of field is given by the distance over which

object remains in focus.
Diffraction acts in the z-direction too - also your eye

has accommodation (range of distances over
which an object remains in focus.)
Diffraction limited depth of field -
This formula can be adapted for high and low NAs!
Think also about your Gaussian Optics - what

parameter does this correspond to?
http://www.microscopyu.com/microscopy-basics/depth-of-field-and-depth-of-focus
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Biophotonics
Depth of Field
In general we have been considering the material
resolution.
What about in the z-direction?
Depth of field is given by the distance over which

object remains in focus.
Diffraction acts in the z-direction too - also your eye

has accommodation (range of distances over
which an object remains in focus.)
Diffraction limited depth of field -
This formula can be adapted for high and low NAs!
Think also about your Gaussian Optics - what

parameter does this correspond to?
http://www.microscopyu.com/microscopy-basics/depth-of-field-and-depth-of-focus
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Seeing smaller?
Use a bigger NA!
Is an NA>1 possible? YES - Change

n!!!!!
http://www.microscopyu.com/articles/formulas/formulasresolution.html
Water immersion and oil immersion
lenses give NAs up to ~1.5!
To see really small - change ! - the

electron microscope.
But electron microscopes may require

gold coatings and happen in vacuum!
- cant work with live samples
http://www.bmb.leeds.ac.uk/illingworth/6form/ 15
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Careful Design!
Sometimes you want to see large areas and
still have high resolution!
MESOLENSes allow this to happen, but they

are massive.
Low magnification but high NA gives wide

coverage at very good resolution (x4 mag,
0.5NA)
Pioneered by Brad Amos at Cambridge - now

working closely with Strathclyde.
Have a look at: https://www.theguardian.com/

science/gallery/2010/jun/30/giant-lens-
mesolens-royal-society
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Summary
Resolution
Point Spread Function
Rayleigh Criterion
Abbe Criterion
Diffraction Limit
Seeing (a bit or a lot) smaller
18

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Biophotonics

Lecture 4 - The limits

Numerical aperture and light collection

Fourier transforms in optics

Generating arbitrary patterns at focus

Can you explain this in terms of spatial frequency?

Can you explain this in terms of spatial frequency?

Point Spread Function

Seeing (a bit or a lot) smaller

At focus a circular aperture (like a lens) gives the

Point Spread Function (PSF) is the ideal response

For a circular lens we have the Airy pattern

How can we resolve two objects?

Rayleigh Criterion - objects are resolved until the maximum of one

Considering the Bessel function allows us to define =1.22 /D as the

How can we resolve two objects?

Rayleigh Criterion - objects are resolved until the maximum of one

Considering the Bessel function allows us to define =1.22 /D as the

How can we resolve two objects?

Rayleigh Criterion - objects are resolved until the maximum of one

Considering the Bessel function allows us to define =1.22 /D as the

How can we resolve two objects?

Rayleigh Criterion - objects are resolved until the maximum of one

Considering the Bessel function allows us to define =1.22 /D as the

Fourier theory: image made up of a range of spatial frequencies.

Imaging theory states that most of the information can be obtained if

Maximum angle that can be

Maximum angle that can be

Constructive interference gives

Maximum angle that can be

Constructive interference gives

Considering this condition:

Maximum angle that can be

Constructive interference gives

Considering this condition:

Remembering the definition of

Maximum angle that can be

Constructive interference gives

Considering this condition:

Remembering the definition of

Abbe limit on smallest

For a circular aperture:

Rule of thumb - minimum

Spatial Frequencies and The

What about in the z-direction?

Depth of field is given by the distance over which

Diffraction acts in the z-direction too - also your eye

Diffraction limited depth of field -

This formula can be adapted for high and low NAs!

Think also about your Gaussian Optics - what

What about in the z-direction?

Depth of field is given by the distance over which

Diffraction acts in the z-direction too - also your eye

Diffraction limited depth of field -

This formula can be adapted for high and low NAs!

Think also about your Gaussian Optics - what

Is an NA>1 possible? YES - Change

To see really small - change ! - the

But electron microscopes may require

MESOLENSes allow this to happen, but they

Low magnification but high NA gives wide

Pioneered by Brad Amos at Cambridge - now

Have a look at: https://www.theguardian.com/

Point Spread Function