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Optical Microscopy. Lecture 1. Concepts we will discuss in this lecture: . Natures of light Mechanism of Optical Imaging system The Use of Lenses and the Problem of Lenses Spatial Resolution. Some Properties of Light. Both lasers and white light sources used in microscopy. Laser.

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Presentation Transcript
slide2

Concepts we will discuss in this lecture:

  • Natures of light
  • Mechanism of Optical Imaging system
  • The Use of Lenses and the Problem of Lenses
  • Spatial Resolution
slide3

Some Properties of Light

Both lasers and white light sources used in microscopy

Laser

White light

Chromatic

Polarization

Phase

Direction

slide4

Monochromatic vs white light

450 nm

600 nm

White light contains all, or most, of the colors of the visible spectrum.

Lasers are Monochromatic

(very narrow frequency distribution)

Both white light, lasers used in microscopy techniques

slide5

Polarization of Light

Plane where electric field vector lies, E=Eºcos(ωt)

Perpendicular to direction of propagation

s= horizontal

p= vertical

Vertical

for propagation

Parallel to floor

Circular polarization:

H,V (s,p) 90 degrees

out of phase

horizontal

elliptical polarization: less than 90 out of phase

This nature used extensively

In microscopy: pol microscopy, DIC, SHG

slide6

Particle (Quantized) Behavior

  • Light interacting with matter: absorption, reflection
  • photon smallest unit- energy corresponds to frequency ()
  • h=6x10-34 J*s Planks constant
  • ~10-19 J for visible light (=600 nm)
  • best for describing absorption, emission of light
  • Best for describing how detectors work (photomultipliers, Diodes)
slide7

Wave Behavior

Constructive, destructive interference

0, 180 degrees Limiting cases for complete constructive, Destructive interference, respectively

Underlies image formation in almost all forms of microscopy:

phase, DIC, polarization,

Some advanced forms of confocal

slide8

Representations of Light

Absorption,

lasers

Interference,

Image formation

Good for modeling

Light propagation:

Ray Tracing

Not real form

Wave, particle duality physically important

Some phenomenon described by both

slide11

Converging (focusing) Lens

  • The parallel rays converge at the second focal point F‘.
  • The first focal point is at the front. All rays originated at
  • This point become parallel to the axis after the lens.
slide12

Diverging (defocusing) Lens

Focal length is negative

To an eye on the right-hand side, these diverging rays will

Appear to be coming from the point F’: the second focal point.

slide13

medium

index

air (STP)

1.00029

water (20° C)

1.33

crown glass

1.52

flint glass

1.65

Snell’s Law

where q1 is the angle of incidence, q2 is the angle of refraction

slide15

Single-lens Imaging system

Real image: if rays intersect and unite in image plane

and can be projected onto some surface in image plane

Two-lens Imaging system

Virtual Image: if rays diverge, but backwards extensions

converge and intersect behind specimen

slide16

A slightly more complicated imaging system aka old microscope

Eye is part of optical

system of microscope

slide17

Infinity Corrected Microscopes: last 15 Years

Infinity optics allows insertion of

Filters, analyzers without changing tube length, or final image

Infinity=parallel

slide19

Thin lens formula 

Basic Formulae in air

Object plane

Image plane

Lensmakers equation 

some conventions
Some Conventions
  • S is distance from the object; S’ is distance from the image
  • Sign conventions: m = positive for inverted image; negative for upright
  • Sign conventions: f = positive for converging lens; negative for diverging lens
slide25

Inverted vs Upright Geometries

  • Upright:
  • Move stage for focusing (unless fixed stage)
  • Optical path is simpler
  • Easier for immersion (long working distance)
  • Inverted:
  • Move objective for focusing
  • Better access for live cells in culture
  • Electrophysiology
  • Harder for oil, water immersion.
slide31

How to Calculate?

Sellmeier Equations

All but quartz

Quartz

These values are tabulated (e.g. CVI Laser, Melles Griot)

slide36

Spherical Aberration could also be caused by the use of the cover glass-slip.

A correction collar might be found on the objective to set the thickness of the glass-slip.

If no correction collar can be found, the objective is corrected for a 0.17 mm glass-slip.

what s important for a microscope
What’s Important for a Microscope?
  • Contrast is necessary to detect detail from background

light from an object must either be different in intensity or color (= wavelength) from the background light:

Both used in light and fluorescence microscopy

  • Resolution fundamentally limited by diffraction

diffraction occurs at the objective lens aperture

slide43

l

n sinq

Numerical Aperture (N.A.)

q

specimen

Objective

lens

Image plane

From diffraction theory

d

N.A. = n sinq

Minimum spot

NA= radius/focal length

~250 nm in visible

Abbe` Limit

Resolution only determined by NA and wavelength

slide44

Electromagnetic Spectrum

Visible region used for

Light microscopy small

Part of EM spectrum

Resolution limit :λ/2

~200 nm:

Visible good for

Live specimens:

Cells, organelles

Ideal for micron sized

structures

EM, X-ray cannot

do live imaging

slide45

Consider microscope object as simple grating

Spacing of Grating and Diffraction Pattern

S=3 microns

S=12 microns

Inverse relationship (transform) of

object spacing (or size) and diffraction pattern

slide46

Double-slit Experiment

Condition for

Constructive interference:

a sinθ = nλ

n = 0, 1, 2,  3 …

Afterfocusing:

d = f λ / a

slide49

d1

Tube

Lens

Fringe spacing in the image:

d2 = f’ λ / d1 = f’ λ a / f λ = M a

Requires at least one of the first order diffraction spot in order to form the image.

slide50

Diffracted Spots in back focal plane

  • No specimen diffraction: no image
  • Specimen diffraction: no collection, no image
  • 0th and first order diffraction
  • 0th and first and second order diffraction
  • better resolution

Abbe showed need for central and diffracted spot

slide52

Visualizing objects below the diffraction limit

Subresolution beads

Appear same size

60 nm

800 nm

slide53

Diffraction from self-luminous spot: delta function source

Impossible to remove

interference rings:

Separated exactly by

n

Absence of light between

Rings is due to

destructive interference

Light from each point of the object is spread out in the microscope because light diffracts at the edges of the lens

Central spot is 0th order diffraction or Airy disk

Contains 84% of power

slide54

Aperture size, Interference, and Resolution

Con inter at P’

Destr at P’’

Full aperture

Interference in

image plane

P’-P’’ distance

Smaller for full

aperture

Reduced

aperture

Always fill

Lens aperture

For highest

resolution

Maxima larger, max, min further apart:

Covers more cone cells

or camera pixels:

less resolution

resolution
RESOLUTION

The resolution of a microscope is the shortest distance two points can be separated and still be observed as 2 points.

Not resolved

just resolved

Well resolved

MORE IMPORTANT THAN MAGNIFICATION !!

slide56

High NA

Low NA

Limits on NA and Resolution?

Air: NA= 0.95 fora =70 degrees

Immersion increase n:

NA= 1.4a =67 degrees (oil) n~1.5

1.2 (water) n=1.33

Higher index materials for greater resolution?

Some exist: methyl iodide, smelly, toxic

Also need higher index coverslips, slides

slide57

Useful Magnification

Useful Magnification (total) = 500 to 1000 • NA (Objective)

More mag does not help, and

decreases image quality through artifacts, diffraction

Limit comes from rod separation in the eye

slide58

Depth of Field:

Axial resolving power

Defined only by NA2

Small Depth of Field at high NA

Focusing critical at high NA

slide59

Gromit captured at f/22 (left) and at f/4 (right).

f = image distance / effective diameter of the lens