Download Phase Contrast

Basic Microscopy
– An Overview –
October 2005
Protistology Course
MBL, Woods Hole, MA
Agenda
Brief History of the Microscope
What‘s a microscope?
Definition of Magnification
Conventional Viewing Distance
Leeuwenhoek > Compound > Stereo
Microscope
The “Telescope”, a simple detour
How to make the specimen visible – Contrast!
Definition of Contrast
Techniques:







Brightfield
Phase
Darkfield
Pol
DIC (Differential Interference Contrast)
Fluorescence
Optical Sectioning – an expansion of Fluorescence
Setting up the Microscope (Lab)
Koehler Illumination
Resolution & Empty Magnification
What is “Magnification”?
MB ~ 2x MA
A
B
Objects appear to the eye at different magnifications, depending on their
distance from the eye. Accommodation (lens) will make it possible.
Conventional Viewing Distance
?
250 mm
1x
“Magnification” 1x
1x
f = 250 mm
1x
Magnification via Single Lens
1x
f = 250 mm
Magnifying Glass (Loupe)
M 
5x
Example:
f=50mm
250mm
f Lens
The “simple” microscope
Leeuwenhoek
Microscope
The current -corrected
Compound Microscope
Eyepiece
Tube lens
(Zeiss: f=164.5mm)
Objective
M
250mm
fObjective

fTube
250mm
M 
fTube
fObjective


MCompound Microscope  MObjective 
250mm
fEyepiece
250mm
fEyepiece
MEyepiece
Q: What happens if we take the objective away?
Eyepiece
∞
Tube lens
∞
Objective
M

(Zeiss: f=164.5mm)
fTube
250mm
M Telescope 
250mm
fEyepiece

f Tube
f Eyepiece
Answer: We have created a “Telescope”
AxioImager
Upright Research Microscope
Axiovert 200
Inverted Research Microscope
The basic light microscope types
Upright microscope
.
Inverted microscope
Illumination via Transmitted Light
The specimen must be transparent !
Upright microscope
.
Inverted microscope
Illumination via “Reflected” (Incident) Light
Eg. Fluorescence, Opaque Samples
Upright microscope
.
Inverted microscope
Mixed Illumination
Upright microscope
.
Inverted microscope
Other Ways to Illuminate
Reflectors
Ring Lights
Fiber Optics
LED’s
Etc.
“Couldn’t one build a
microscope for both
eyes, and thereby
generate spatial
images?”
Question addressed to
Ernst Abbe in 1896
by Horatio S. Greenough
1897 – the first Stereo Microscope in
the world, built by Zeiss, according to
the “Greenough” principle
1896: Drawing by Horatio S. Greenough
Greenough Type
Introduced first
by Zeiss - 1897
Telescope Type
Introduced first
by Zeiss - 1946
Greenough Stereo Microscopes
SteMi DV4
Greenough Stereo Microscopes
SteMi 2000
(2000-C, 2000-CS)
Research Stereo Microscopes
SteREO Discovery V12
SteREO Lumar V12
How to make the specimen visible –
CONTRAST!
Agenda
Definition of Contrast
Techniques:







Brightfield
Phase
Darkfield
Pol
DIC (Differential Interference Contrast)
Fluorescence
Optical Sectioning – an expansion of Fluorescence
0 Units
50 Units
100 Units
C ONTRAST
50 Units
50
50 – 100 / 50 + 100 =
Brightness of Specimen - Brightness of Background
Brightness of Specimen  Brightness of Background
50
-0.33
Common Illumination Techniques
•
•
•
•
•
•
Brightfield
Darkfield
Phase Contrast
Polarized Light
DIC (Differential Interference Contrast)
Fluorescence (and related techniques)
Brightfield
• For naturally absorbing or stained samples
• True Color Representation
• Proper Technique for Measurements
•Spectral
•Dimensional
Paramecium bursaria
Condenser diaphragm open
Condenser Diaphragm almost closed
Paramecium bursaria
Different Staining Techniques
Indian Ink Staining
Feulgen Staining
Silver Staining
Phase Contrast
(Frits Zernike 1934)
- “Halo” effect > Reduced resolution
+ No staining necessary
+ Good Depth of Field
+ Easy alignment
+ Orientation independent
+ Repeatable setup
+ Works with plastic dishes
Required Components
for Phase Contrast:
1.
Objective with built-in
Phase Annulus
2. Condenser or Slider
with Centerable Phase
Ring for illumination
(Ph0, 1, 2 or 3)
Required Adjustment:
Superimpose Phase Ring of
condenser over (dark)
phase plate of objective
(after Koehler Illumination)
•Illumination bypasses
Specimen > no phase shift
•Illumination passes
through thin part of
Specimen > small phase
retardation
•Illumination passes
through thick part of
Specimen > larger phase
retardation
Phase Shifts:
Cells have higher n than water. Light moves slower in
higher n, consequently resulting in a phase retardation
Phase shift depends on n and on thickness of specimen
detail
4. Non-diffracted and diffracted light are
focused via tube lens into intermediate
image and interfere with each other; ¼+¼=
½ wave shift causes destructive interference
i.e. Specimen detail appears dark 
Tube Lens
Objective
Specimen
Condenser
3. Affected rays from specimen, expressed by
the higher diffraction orders, do not pass
through phase ring of objective
>¼ wave retarded 
2. Objective Phase Ring
a) attenuates the non-diffracted 0th Order
b) shifts it ¼ wave forward 
1.
Illumination from Condenser Phase Ring
(“0” Order) > meets phase ring  of
objective
Paramecium bursaria
Phase
Contrast
Rhipidodendron
Phase
Contrast
Cochliopodium
Phase
Contrast
Lyngbya Bacteria
Phase
Contrast
Darkfield
No staining necessary
Detection of sub-resolution details possible
Excellent, reversed contrast
Central Darkfield via “hollow cone”
Oblique Darkfield via Illumination from the side
Not useful for Measurements (sizes exaggerated)
Low NA
Objective
Required conditions for
Darkfield:
Illumination Aperture must be
larger than objective aperture
I.e. direct light must bypass
observer
High NA
Objective
Iris Diaphragm
Paramecium bursaria
Darkfield
Polarized Light
Polarized Light
Specimen is placed between 2 crossed
polarizers.
Only light produced by birefringent
particles (e.g. crystals) or coming from the
edges of particles (“edge birefringence”) is
visible.
Looks sometimes like Darkfield
Orientation-specific (linear Pol)
Linear / circular Polarized Light
Birefringent Material
Background
Brightfield
Polarized Light
Color of
sample and background
modified by wave plate
Pol + Red I
Polarized Light
Polarizer 2
(Analyzer)
When Polarizers are
crossed, only items that
rotate the plane of
polarization reach the
detector.
Wave plate adds color
Specimen
Polarizer 1
Required / Recommended
Components:
• Polarizer (fixed or rotatable)
• Analyzer (fixed or rotatable)
• Strain-free Condenser and Objective
• Rotating, centerable Stage
• Wave plate and/or Compensator
• Crossline Eyepiece
DIC
(Differential Interference Contrast
after Nomarski)
High Contrast and high resolution
Control of condenser aperture for optimum contrast
Changes GRADIENTS into brightness differences
3-D Image appearance
Color DIC by adding a wave plate
Best contrast / resolution via different DIC sliders
Orientation-specific > orient fine details
perpendicular to DIC prism
DIC
Observing local differences in phase retardation
9 Image
8 Tube lens
7 Analyzer (7a with Wave Plate)
6 Wollaston Prism behind objective
5 Objective
4 Specimen
3 Condenser with receptacle for prisms
2 Wollaston Prism before condenser
1 Polarizer
Wollaston Prism
Birefringence (Different
refractive index for different
polarization orientations)
Polarized beam, under 45˚ to prism,
gets split into “ordinary” and
“extraordinary” beam
Required Components for DIC:
• Nosepiece with DIC receptacles
• Polarizer (or Sénarmont Polarizer)
• Low Strain Condenser and Objective*
• DIC Prisms for Condenser
(# I or II or III)
• Appropriate DIC Slider for each objective
• Analyzer (or Sénarmont Analyzer)
• *Not needed for New Plas-DIC (up to 40x)
Paramecium bursaria
DIC
Interference
Fluorescence
• Easy to set up > Objective = Condenser
• Highly specific technique, wide selection of
markers
• Detection and Identification of Proteins,
Bacteria, Viruses
• Basics for
–
–
–
–
–
Special Techniques eg. TIRF, FRET, FRAP etc.
3-D imaging
Deconvolution
Structured Illumination
Confocal Techniques
Epi - Fluorescence
Observation port
Excitation Filter
FL
Light
Source
Example: Specimen containing
green fluorescing Fluorochrome
Emission Filter
Dichromatic Mirror
Epi - Fluorescence
Filter Sets
Example
Curve for a typical GFP filter set
Epi - Fluorescence
(Specimen containing green
fluorescing Fluorochrome)
Observation port
Excitation Filter
FL
Light
Source
Specimen containing green
fluorescing Fluorochrome
Emission Filter
Dichromatic Mirror
Paramecium bursaria
Fluorescence
How to improve Fluorescence
Imaging in a major way:
•Optical Sectioning
Optical sectioning – increased
contrast and sharpness
Overview of
Optical sectioning Methods
1.
–
–
Confocal and Multi-photon
Laser Scanning Microscopy
Pinhole prevents out-of-focus light getting to the
sensor(s) (PMT - Photomultiplier) (30 – 70 µm)
Multi Photon does not require pinhole (90 – 500 µm)
2. Spinning disk systems
–
–
A large number of pinholes (used for excitation
and emission) is used to prevent out-of-focus light
getting to the camera
E.g. Perkin Elmer, Solamere ( up to 30 µm)
3. Structured Illumination
–
–
Moving grid represents the reference for in-focus
information
Zeiss Apotome (10-50 µm)
Overview of
Optical sectioning Methods
- cont‘d -
4. Total Internal Reflection Fluorescence
(TIRF)
–
–
High NA Objective projects beam at angle
which exceeds critical angle.
Area touching cover slip (evanescent field)
is typically smaller than 200 nm
5. Deconvolution
–
–
Point-Spread function (PSF) information is
used to calculate light back to its origin
Post processing of an image stack
Limited Depth of Field
With Standard Microcopy
 Amber fossil
(Chironomide)
 Thickness app.
300 µm
 Conventional
incident light
Optical Sectioning +
Extended Focus Software
 Amber fossil
(Chironomide)
 Thickness app.
300 µm
 Conventional
incident light
 3D reconstruction