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ADVANCED
ADVANCED METHODS
METHODS FOR
FOR
CONFOCAL
CONFOCAL MICROSCOPY
MICROSCOPY III
III
Jean-Yves Chatton / Yannick Krempp / Sept. 2006
Workshop outline
•
•
Simultaneous fluorescence and transmitted
images (DIC, Nomarski) on a confocal
microscope
3D and confocal microscopy
Interference of electromagnetic waves
A fraction of the light is deviated when it encounters certain parts of the specimen. This deviated
light (i.e. DIFFRACTED) becomes 1/2 wavelength out of phase with respect to the non-diffraced
light (1/4 for non-absorbing objects)
Image formation (transmission microscopy)
Intermediate image
The image produced by an objective is
congugated with the specimen, i.e. each
point is geometrically related to a
corresponding point in the specimen
Back focal plane
Objective
Æ Each point in the specimen is
represented by a correponding point in
Specimen
the image
Condenser
Light source
Principles of phase contrast
Resulting
wave
Undeviated
light
Resulting
wave
Wave accelerated
by λ/4
Diffracted wave
Diffracted wave
λ/4 out-of-phase
Differential interference contrast microscopy
(DIC, Nomarski)
Georges Nomarski (1919 – 1997)
A Polish born physicist and optics theoretician, who
adopted France as his home after World War II.
Nomarski is credited with numerous inventions and
patents, including a major contribution to the wellknown differential interference contrast (DIC)
microscopy technique.
Contrast technique that enhances the gradient of
optical path in the specimen (i.e. gradient of thickness
and refractive index)
Bright field
Human buccal epithelial cells, Obj. 100X
DIC contrast
Differential interference contrast microscopy
(DIC, Nomarski)
William Hyde Wollaston (1766 – 1828)
One of the most influent scientist of his time. Although
trained as a physician, he studied and made advances in
several fields as chemistry, physics, botany,
crystallography, optics, astronomy, and mineralogy.
He is at the origin of the major invention of the so-called
Wollaston prism, that is fundamentally important to
interferometry and differential interference (DIC) contrast
microscopy.
Principles of differential interference contrast
Analyzer
Wollaston
prism
Wollaston
prism
Polarizer
From: Inoué et Spring, "Video Microscopy",
Plenum Press, 1997
Shading and relief effect in DIC microscopy
Shear effect
Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson
Comparison phase contrast and DIC microscopy
Phase contrast
DIC contrast
• Phase contrast yields image intensity values
as a function of specimen optical path length
magnitude, with very dense regions (those
having large path lengths) appearing darker
than the background
• Optical path length gradients (thickness,
refractive index), i.e. the rate of change in the
direction of wavefront shear) are primarily
responsible for introducing contrast into
specimen images.
• Specimen features that have relatively low
thickness, or a refractive index less than the
surrounding medium, are rendered much
lighter when superimposed on the medium
gray background.
• Steep gradients in path length generate
excellent contrast, and images display a
pseudo three-dimensional relief shading that
is characteristic of the DIC technique.
• Regions having very shallow optical path
slopes, such as those observed in extended,
flat specimens, produce insignificant contrast
and often appear in the image at the same
intensity level as the background.
Comparison phase contrast and DIC microscopy
DIC
Phase
Buccal Epithelium
Section of rodent
renal tissue
Obelia (marine
polypus)
Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson
Halos in phase contrast and DIC microscopy
Erythrocytes
HeLa cells
Algae, Zygnema
Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson
Depth of field in phase contrast and DIC microscopy
Phase
DIC
Obelia (polypoid)
Butterfly scales
Eggs of canine
cucumber
tapeworm
Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson
Comparison phase contrast and DIC microscopy
Characteristics
Phase Contrast
DIC
Image Brightness
(Brightfield = 100 Percent)
1.3 Percent
0.36 - 2.3 Percent
Epi-Fluorescence Light Loss
(Brightfield = 0 Percent)
28 Percent
73 Percent
Lateral Resolution
Condenser
Annulus
Restricted
Superior
Axial Resolution
(Depth Discrimination)
Poor
Superior
Illuminating Aperture
10 Percent of
Objective NA
Variable
Phase Shift
Detection Limit
< λ/100
< λ/100
Utility at High Phase Shifts
Not Useful
Useful
Azimuthal Effects
No
Yes
Halos and Shade-Off
Yes
No
Stained Specimens
Not Useful
Useful
Birefringent Specimens
Useful
Not Useful
Birefringent Specimen
Containers
Yes
No
Brightfield Image
Deterioration
Slight
None
Cost
Moderate
High
Köhler illumination
• Production of a magnified image of the
lamp filament that is focused at the level
of the aperture plane of the condensor.
• Opening or closing this diaphragm
controls the angle of the light cone that
reaches the specimen, which is
determining the image resolution along
with the numerical aperture of the
objective (i.e. the global numerical
aperture of the optical system).
• Since the light source is not focused at
the level of the specimen, the light is
diffuse and speads evenly at the level of
the speciment and does not suffer from
deteriorations due to dust and glass
defects in the condensor.
Conventional DIC Observation
DIC observation in confocal mode
Laser
(polarized light)
This DIC image is for free as it is formed using
the light that has not been absorbed by the
fluorescence specimen
Photomultiplier
Setting up the microscope for DIC observation
2
1
3
Transmitted light detector
Simultaneous fluorescence and DIC acquisition
Cortical neurons
With GFP expressing cell
Simultaneous fluorescence and DIC acquisition
Living brain slice
(rat hippocampus)
Sulforhodamine 101
(astrocyte stain)
Next...
ADVANCED METHODS FOR
CONFOCAL MICROSCOPY III
Arnaud Paradis 26 Septembre 2006
Adapted by Yannick KREMPP
Part I Optical sectioning series acquisition (z-stack)
- Standard acquisition (XYZ scan)
- Z-line (scan xz)
Part II 3D views, capabilities with the LSM Software
-Gallery
-3D Depth Code
-3D Projections xyz
-3D Animations
Part III Zeiss LSM Software vs Imaris
Z-STACK ACQUISITION
Standard Z-Stack acquisition
HOW TO SET UP THESE PARAMETERS ?
THERE IS NO RULE.
AS ALWAYS IT IS A TRADE-OFF
BETWEEN SPEED AND QUALITY
Z-STACK ACQUISITION
Standard Z-Stack acquisition
SOME CLUES…
If SPEED is what you need (fragile samples, you’re in a hurry, etc…):
-Decrease the number of slices (or increase the size of the interval) with « keep slices »
enabled, WITH A CONSTANT SLICE THICKNESS
+SPEED
THE TRADE-OFF:
You have now gaps in your stack.
-Increase the slice thickness (or decrease the slice number) with « keep interval » enabled.
+SPEED
THE TRADE-OFF:
The pinhole opens more, the Z
resolution is poor.
Z-STACK ACQUISITION
Standard Z-Stack acquisition
SOME CLUES…
If QUALITY is what you need (fixed samples, you want the cover of Nature magazine,
etc…):
Try to fullfill the Nyquist-Shannon criterium (signal sampling theorem).
+QUALITY
YOU NEED AT LEAST AN OVERLAY OF
50% OF 2 CONSECUTIVE IMAGE PLANES
THE TRADE-OFF
You need plenty of time, and a very
resistant dye regarding bleaching
50% overlay
Z-STACK ACQUISITION
Standard Z-Stack acquisition
THE EASY WAY
NB : In the case of multiple channel acquisitions, you have to verify that you have
the same optical slice thickness for the different channels. Change the pinhole size
if necessary.
Z-STACK ACQUISITION
Standard Z-Stack acquisition
Part I Optical sectioning series acquisition (z-stack)
- Standard acquisition (XYZ scan)
- Z-line (scan xz)
Part II 3D views, capabilities with the LSM Software
-Gallery
-3D Depth Code
-3D Projections xyz
-3D Animations
Part III Zeiss LSM Software vs Imaris
Z-STACK ACQUISITION
Z-Line
A Z-Line acquistion allows you to scan a line (x) at
the focal plane along the z axis.
As for the conventional XYZ stack you can change
the number of focal planes in the interval.
Z-STACK ACQUISITION
Z-Line – drawing the line
X
y
Z-STACK ACQUISITION
Z-Line – The result
X
Higher limit
Z
current line.
Lower limit
Z-STACK ACQUISITION
Z-Line – Intensity profile
Part I Optical sectioning series acquisition (z-stack)
- Standard acquisition (XYZ scan)
- Z-line (scan xz)
Part II 3D views, capabilities with the LSM Software
-Gallery
-3D Depth Code
-3D Projections xyz
-3D Animations
Part III Zeiss LSM Software vs Imaris
Z-STACK VIEWING
Gallery
Part I Optical sectioning series acquisition (z-stack)
- Standard acquisition (XYZ scan)
- Z-line (scan xz)
Part II 3D views, capabilities with the LSM Software
-Gallery
-3D Depth Code
-3D Projections xyz
-3D Animations
Part III Zeiss LSM Software vs Imaris
Z-STACK VIEWING
Depth Coding
Part I Optical sectioning series acquisition (z-stack)
- Standard acquisition (XYZ scan)
- Z-line (scan xz)
Part II 3D views, capabilities with the LSM Software
-Gallery
-3D Depth Code
-3D Projections xyz
-3D Animations
Part III Zeiss LSM Software vs Imaris
Z-STACK VIEWING
Projections – Orthogonal projections
Z-STACK VIEWING
Projections – Cut view
Z-STACK VIEWING
Projections - MIP
MIP
Maximum Intensity Projection
Z-STACK VIEWING
Projections - Transparency
Transparency tweaking
Part I Optical sectioning series acquisition (z-stack)
- Standard acquisition (XYZ scan)
- Z-line (scan xz)
Part II 3D views, capabilities with the LSM Software
-Gallery
-3D Depth Code
-3D Projections xyz
-3D Animations
Part III Zeiss LSM Software vs Imaris
Z-STACK VIEWING
Projections – Rotating the projection
Rotation
around
the Y axis
Part I Optical sectioning series acquisition (z-stack)
- Standard acquisition (XYZ scan)
- Z-line (scan xz)
Part II 3D views, capabilities with the LSM Software
-Gallery
-3D Depth Code
-3D Projections xyz
-3D Animations
Part III Zeiss LSM Software vs Imaris
IMARIS
Section mode
Z
X
Y
Y
X
Z
Z-stack of mouse kidney in Section display with MIP mode
IMARIS
Section mode
dx
dy
Extended view
IMARIS
Section mode
Extended view – Blend rendering
IMARIS
Surpass mode (Real-time 3D)
Blend
IMARIS
Surpass mode (Real-time 3D)
MIP
IMARIS
Surpass mode (Real-time 3D)
MIP+ Isosurface + Grid
IMARIS
Surpass mode (Real-time 3D)
Isosurface only - Low threshold
Isosurface only - High threshold
IMARIS
Surpass mode (Real-time 3D)
Isosurface – Low details
Isosurface – High details
IMARIS
Animation mode – movie recording