Lecture 5

Diffraction & interference in image formation

Abbe’s theory

- Interference between 0th- and higher-order diffracted rays in the image

plane generates image contrast and determines the limit of spatial
resolution
Lecture 5

Diffraction & interference in image formation

Defining diffraction and interference

- diffraction: spreading of light that occurs when a beam of light
interacts with an object
- Light reaches a geometric shadow by diffraction

- A point source of light in the microscope: diffracted at the

margins of the objective lens, causing the image of the point to
look like a spot. Diffraction image of a copper mesh grid

- Image of a point source of light in the microscope: a

diffraction pattern of a point source

- Specimen absorbs or diffracts light. Otherwise, invisible!

- Two sources of diffraction in the microscope:

Specimen & the aperture of the obj.

- diffraction: scattering & diverging light

- Interference: recombination & summation of two or

more superimposed waves. Process of creating the
real image of an object in the focal plane in the Θ: inversely proportional to the size of the particles
microscope
Lecture 5

Diffraction & interference in image formation

Defining diffraction and interference

- Interference: constructive & destructive

- Diffraction & interference: redistribution of light

Lecture 5

Diffraction & interference in image formation

The diffraction image of a point source of light

- Image of a point source: a diffraction pattern created by the
action of Interference: in the image plane
- 0th-order diffraction spot, 1st-, 2nd-, …. Rings: Airy disk.

- Airy disk: result of diffraction by the aperture of the lens

-size of the diffraction disk radius (camera, telescope)
d=1.22 λ(f/D) f: focal length, D: lens diameter, f/D: f-number
% for detail: see Fraunhofer diffraction pattern

-size of the diffraction disk radius (microscope)

d=1.22 λ/2NA NA: numerical aperture
 Determines diffraction-limit & resolution limit
Lecture 5

Diffraction & interference in image formation

Constancy of optical path length between the object & the image
- The number of vibrations & the transit time from an object
point to the image point: constant, independent of optical path.

- Optical path length (OPL) = nt (n: refractive index, t: thickness of the object)

-Number of vibrations = nt/λ (homogenous medium)

% note!! frequency = constant  λ and c of light decrease during transit through the
lens, c (medium) = c/n
-Number of vibrations passing two different media= n1t1/λ1+ n2t2/λ2
Lecture 5

Diffraction & interference in image formation

Effect of aperture angle on diffraction spot size

- Reducing the aperture angle  decrease spatial resolution
Lecture 5

Diffraction & interference in image formation

Diffraction grating & D

-Grating equation
mλ=d sinθ
-Θ increases when, d small, λ large.
Lecture 5

Diffraction & interference in image formation

Abbe’s theory for image formation in the microscope

- Interference between 0th- and higher-order diffracted rays in the image

plane generates image contrast and determines the limit of spatial
resolution

- At least two orders of diffraction must be captured

- Collecting larger the number of diffraction orders  the sharper and

better resolved image

- Diffraction pattern focused on the back

focal plane of objective

- Image formation in the image plane due to

the interference of undeviated (0th) and
deviated (higher-order) components

- Diffraction + objective (collecting diffracted

rays) = image
Lecture 5

Diffraction & interference in image formation

Abbe’s theory for image formation in the microscope

-At least two orders of diffraction must be captured

-Multiple orders of diffracted light collected  sharper image
-Diffraction pattern & image in the image plane  inverse transforms of
each other
Lecture 5

Diffraction & interference in image formation

Preservation of coherence

- Partially coherent light is required for image formation (phase contrast,

DIC, etc)
Lecture 6

Diffraction & spatial resolution

Numerical Aperture

- Lenses that can capture light over a wide angle give better resolution
than lenses collecting light over a narrower angle.
- NA = nsinθ, air, n=1. Oil, n=1.515

- Why oil immersion?

- Dry lens limit: angle 41 degree, beyond that are lost by TIR.
Lecture 6

Diffraction & spatial resolution

Spatial Resolution

- Resolving power of the microscope (self-luminous: fluorescence Micro., dark-field Micro.,

d=0.61 λ/NA d: Airy disk radius, NA: numerical aperture

- Resolving power of the microscope (bright-field, when the condenser NA < obj. NA)

d=1.22 λ/(condenser NA+obj NA)

- Rayleigh criterion: two adjacent objects are resolved, when

the central diffraction spot (Airy disk) of one point coincides with the first minimum of
the other point.
Lecture 6

Diffraction & spatial resolution

Spatial Resolution

- Increasing the aperture angle increases resolving power of the microscope

- cf) reducing the intensity of lamp  not by closing diaphragm, but the voltage supply

Condenser aperture open & close

- If d = 0.5 um, objects smaller than d appear as 0.5 um objects in the image.
Lecture 6

Diffraction & spatial resolution

Depth of field

- Depth of field: the thickness of the optical section along the z-axis within which objects
in the specimen are in focus

For diffraction limited optics, Z=nλ/NA2

For x-y, d=0.61 λ/NA

- The method to measure the DOF of a objective.

-Using diffraction grating,
-putting obliquely on the slide using a cover slip, etc.
-Measuring the width, w, of the focused zone (clearly grating image)
-Z=nwtan(a)
Lecture 6

Diffraction & spatial resolution

Optimizing the microscope image: spatial resolution vs contrast

-(left), maximal spatial resolution: condenser front aperture = the back aperture of Obj

- poor image by (1) polychromatic illumination: monochromatic illumination increases

resolution by factor of 2
(2) scattered light, less coherent light

-(right), condenser front aperture = 70% of the back aperture of Obj

contrast up, but spatial resolution down
-Reducing the amount of stray light
-Increase coherent light