Transcript Lecture 2. Bright field microscopy.

Topics
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Kohler Illumination and conjugation planes
Dark field microscopy
Phase contrast
Differential interface contrast (DIC)
Conjugated Planes
Eyepiece
Condensor
Objective
Field
Diaphgram
plan
Tube Lens
Sample
plane
Image Planes
Intermediate
Image
plane
Eye
Retina
Condensor
Objective
Field
Diaphgram
plan
Tube Lens
Sample
plane
Image Planes
Intermediate
Image
plane
CCD
chip
How to avoid image formation
-- the opposite of conjugated planes
Collection
lens
Objective
Field
Diaphgram
plan
Lamp
Plane
Eyepiece
Condensor
Tube Lens
Sample
plane
Aperture
Diaphgram
plane
Back focal plane
Eye
Intermediate Retina
Image
plane
Collection
lens
Condensor
Objective
Field
Diaphgram
plan
Lamp
Plane
Tube Lens
Sample
plane
Aperture
Diaphgram
plane
Back focal plane
Eye
Retina
Retina
Objective
Sample
plane
Back focal plane
Close Aperture  Smaller Light Cone Angle  Smaller NA  Less Resolution
Darkfield Microscopy
Collection
lens
Condensor
Objective
Field
Diaphgram
plan
Lamp
Plane
Tube Lens
Sample
plane
Aperture
Diaphgram
plane
Back focal plane
Eye
Retina
Retina
Objective
Condensor
Aperture
Diaphgram
plane
Sample
plane
Back focal plane
Close Aperture  Smaller Light Cone Angle  Smaller NA  Less Resolution
Darkfield Microscopy
Objective collects
Only diffracted light
Image plane
Objective lens
Specimen
Condensor allows only
high angle rays
Stop in condenser
Source
Where is light in darkfield?
Any refractive index
change will scatter
light.
Scattering redirect
light through
objective lens.
Comparison of bright and darkfield
Brightfield Microscopy
Darkfield Microscopy
Contrast is reversed in these modalities
Key Requirement of Darkfield:
NA of Condendor > NA of Objective
High NA darkfield condenser
Visualize particles down to ~40 nm by scattering
But not resolvable
Darkfield
Brightfield,
High contrast
Closed aperture
Loss of resolution
Darkfield,
Red filter
Phase vs Amplitude
Effect of Path Length and Refractive Index
Phase shift:
Effective Pathlength:
  2 1   2  / 
  nL
Amplitude vs Phase Objects
Amplitude Objects:
Cells with stains, pigments, absorb, scatter light,
change its amplitude
Most biological objects do not absorb white light
But diffract, phase shift: phase objects
But may be hard to see with contrast by bright field
Use phase differences to visualize differences
in refractive index:
Phase contrast microscopy
Nobel Prize in 1953: Zernike
Interferometer: Convert Phase  Amplitude
Michelson interferometer
Comparison of contrast in brightfield and phase
Can see by brightfield, but phase much better
Differential Interference Contrast
Microscopy
(aka Nomarski)
Phase Contrast Microscopy:
•Visualize specimens without labeling
•Utilizes refractive index for phase differences
•Measure thicknesses
Phase Contrast Microscopy
Illumination
(zero order)
Light
diffracted by
specimen
Longer
pathway
Condenser
annulus
Condenser
specimen
Objective
lens
Phase rings
Phase contrast: Kohler-same
Conjugate planes as brightfield
Condenser annulus replaces
condenser variable diaphragm
Objective has phase ring in
Backfocal plane
S wave projects bright image of
annulus onto back aperture
(Kohler illumination, conjugate
Image planes)
Diffracted waves traverse
whole back aperture
Surround waves undeviated
Conversion of phase to amplitude
Sample retards by λ/4
Phase ring retards by λ/4
(S advanced)
S is advanced because
Of recessed (shorter) path
S,D shifted by λ/2
Combine and interfere
destructively
In image plane
S,D different places in bfp
Retarding Plate
near Back focal plane
Phase plate located near
back focal plane
Shaded to attenuate
by 70% for more contrast
Selectively manipulate either S,D wave components
Advances or retards S wave relative to D wave
With phase plate
Path Difference between S,D Waves ~ λ/4
For individual cells in tissue culture,
the optical path difference relative to fluid is relatively small:
A typical cell in monolayer culture: thickness around 5 micrometers
refractive index of approximately 1.36.
The cell is surrounded by a nutrient medium:
refractive index of 1.335
  (n2  n1 )t
Optical path difference
Gives optical path difference of 0.125 micrometer, 125 nm
Or λ/4 quarter wavelength (of green light).
Subcellular structures (organelles) generate
much smaller retardations.
PHASE CONTRAST
Phase Ring in
objective
Annulus in
condenser
in phase
¼ out
of
phase
½ out
of
phase
destructive
interference
2 forms on phase contrast:
Positive and Negative
Positive:
S is advanced
higher refractive index=
Dimmer image, P is smaller
More common
Negative:
S is retarded
higher index=
brighter image, P is larger
Cells brighter against
background
Inverse contrast between
The 2 modes
Annuli must be perfectly aligned for best contrast
Properly
aligned
Misaligned,
blurred
Differential Interface Contrast (DIC)
microscopy
Comparison of phase and DIC microscopy
DIC
Phase Contrast
Phase: path length difference: denser:darker
DIC: gradient, not quantitative
DIC is form of Kohler illumination
Without Wollastons, reduces to pol scopeCrossed polarizers
Also need strain-free objective
lenses: Like in pol scope
Selects one polarization
Wollaston splits (shears)
into perpendicular polarized
O,E rays. Located at condensor
Front focal plane (Kohler)
Shear axis is angle
between wedges
Recombine in second Wollaston
(near Back focal plane)
Need coherence for
interference in image plane
Analyzer in infinity region
Between tube lens and objective:
Crossed wrt to first polarizer:
Cancels action if no change in
Refractive index
Non-retarded light is blocked by analyzer
Differential Interference Contrast (DIC) Microscopy:
uses birefringent prisms to separate light into ordinary,
extraordinary components
Measure refractive index gradients by relative
Retardance of the two beams: no birefringence required
in specimen
Birefringent Prisms
Glan-Thompson
Wollaston
Used in DIC
From specimen
Nomarski is modified:
Small offset in wedges
Beams combine before:
Interference plane needs to
be at Back focal (diffraction)
Plane. Usually inside lens
Nomarski eases alignment
Positive Birefringence (quartz): ordinary ray faster, travel through
More of the prism, waves “catch up” at exit air interface
(regular Wollaston)
Calcite has negative birefringence: above argument is opposite
O,E Rays differentially retarded when different
Optical Path Differences Exist in specimen
OPD=   (n2  n1 )t
Bias is selective retarding of ordinary ray to increase contrast
Symmetric oil droplets
Maximum extinction
along shear axis
Sliding analyzing Wollaston changes phase relationship
Of the two beams: change linear plane or ellipticity by path length
By changing extinction across field of view: Pseudo 3d relief
Bias increases contrast, bright, dark patterns on gray background
http://micro.magnet.fsu.edu/primer/java/dic/imagecontrast/index.html
Image Formation in DIC Microscopy
Polarizer passes
One component
Exits prism
Elliptically
polarized
Larger phase change
More passed by analyzer
More contrast
No change in index:
No image in a,b
(assuming no optical path
Different paths through
Prisms compensate after second Difference in two spots)
Bias through de Senarmount compensation: quarter wave plate
Quarter wave plate, (λ/4) turns linear into circular or elliptical polarization
Either between polarizer and condenser prism
Or between objective prism and analyzer
Then rotate analyzer
Combining linear polarized light
IN PHASE
Linear Polarization
OUT OF PHASE
Elliptical Polarization
Circularly Polarized Light
• Decompose to linear polarized
light with 1/4 phase shift.
• No direction (always pass 50%
through polarizer in dependent of
polarizer orientation)
• NOT the same as unpolarized
light.
• Can be converted back to linear
polarized light with birefringent
materials (1/4 wave plate).
Combining circular polarized light
Principles of Differential Interference Contrast (DIC)
DIC turns optical path differences into amplitude contrast
DIC is based on retardance introduced by optics-not specimen
Different indices used to converts optical path,
phase gradients (refractive index) to amplitude differences
in the image
Recall phase contrast measures actual optical pathlength
DIC Not quantitative of actual refractive indices: only gradients
Not indicative of actual topography
OPD=   (n2  n1 )t
Orientation of Object relative to light polarization
can give different images, information
Perp to
shear
Parallel
to shear
Diatoms
Water flea
Gill ribs
Fish scale
Orthogonal features can be seen in each case
Thick specimens in Phase and DIC
Phase
DIC
hydrozoan
Butterfly
wing
Tapeworm
eggs
DIC does better with thicker specimens
Optical Sectioning is obtaining 3 dimensional
Stacks of images plane by plane
Volvox algae colony