Transcript Confocal laser scanning microscopy - Friedrich-Schiller

7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Reduction of out of focus light
Excitation light excites fluorescence more or less within the whole sample
Out of focus fluorescence light is not imaged sharply
Out of focus fluorescence reduces especially for thick samples the image quality
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Reduction of out of focus light
A confocal microscope uses focused
laser illumination and a
pinhole in an optically conjugate plane
in front of the detector to eliminate out-offocus blur
As only light produced by fluorescence
close to the focal plane is detected, the
contrast is much better than that of widefield microscopes.
Allows recording individual
optical sections or
three dimensional reconstruction of objects
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TheWidefield
ConfocalMicroscope
Microscope
The
plane spread function:
I(Z)
Camera
Pinhole
PMT
Pinhole
Z
y
z
x
Standard
Lightsource
Lightsource
with Pinhole
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Scanning in a CLSM
Image of the Object:
PMT
Pinhole
Object scanning
versus
Beam scanning
y
z
Lightsource
with Pinhole
Sample Scanning
x
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Reduction of out of focus light
In contrast to widefiled fluorescence microscopy where
the whole sample is illuminated in confocal microscopy
only one point in the sample is illuminated at a time
2D or 3D imaging requires scanning over a regular raster
(i.e. a rectangular pattern of parallel scanning lines)
in the specimen: raster-scan
Comparison widefiled vs. confocal
Linewise scanned image
Cell in its meta-/ana-phase.
Plasma membrane is stained with a red
fluorescing antibody while the
spindle apparatus is labeled with a green
fluorescent marker
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7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Reduction of out of focus light
Resolution in confocal microscopy
Comparison of axial (x-z) point spread functions for widefield (left) and confocal (right)
microscopy
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Confocal OTF
Excite AND Detect: P(r) = PExcitation(r) PDetection(r)
PSF(r) = PSFExcitation(r) PSFDetection(r)
OTF(k) = OTFExcitation(r)  OTFDetection(r)
kx,y
kz
kx,y
a
kz
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Increasing the aperture angle (a) enhances resolution !!
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We have
circumvented Abbe:
Dmin, confocal

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
4n sin a
1

NA NA
2(

)

ex

em
4 NA
IPC Friedrich-Schiller-Universität Jena

Confocal OTFs:
in-plane, in-focus OTF
1.4 NA Objective
WF Limit
1 AU
WF
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0.3 AU Almost no transfer
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New Confocal Limit
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
 In confocal laser scanning microscopy laser light is focused to a small point at the
focal plane of the specimen and moved / scanned by a computer controlled scanning
mirror in the X-Y direction at the focal plane.
 The fluorescent emission is sent through a pinhole
and recorded by a photon multiplier tube (PMT)
 An image is assembled with the help of a computer
 Advantages:
 Good axial out-of focus suppression
 Quantification of fluorescence intensity
 Simultaneous recording of different dyes in different channels
 Disadvantages:
 High costs (why?)
 Artifacts due to coherence of laser and laser fluctuations
 High amount of photo-bleaching
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7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
Experimental Setup
Fluorescence
 Scanning and Descanning
by same element
Excitation
Transmission
Detector
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7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
 Scan Head:
 Excitation filter /
Wavelength selection
 Scan-System
 Beamsplitter
 Pinhole
 Detectors (photomultiplier)
dichromatic
beamsplitters
excitation
filter
Acousto Optic Tunable Filter (AOTF)
Acousto-optic tunable filter (AOTF) for laser intensity
control and wavelength selection in confocal microscopy.
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7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
Scan System:
 Mirror system is used to scan laser
beam line by line over the sample
 Mirror system consists of two rotating
mirrors; one for scanning the laser in xdirection and the other for movement in
the y-direction
(almost parfocal, f-lens, 4-Galvo idea)
Beam separation
 In confocal microscopy several
wavelength bands can be detected in
parallel. Beam splitting is performed by
dichroitic mirrors + filters,
prisms, diffraction gratings + apertures.
Diffraction
Grating
variable apertures
pinhole
more detectors
dichroitic beam splitter
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7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
Pinhole:
 Pinhole in the optically conjugate sample plane in front
of the detector to eliminate out-of-focus blur
can be adjusted continuously in its size
 Pinhole size determines how much out-of-focus light is
eliminated and how much light reaches the detector
 The smaller the pinhole the better the axial resolution
the smaller the brightness
 Pinhole diameter = 1 Airy disc:
Pinhole diameter corresponds to diameter of dark ring
< 1 Airy Disc
 Improved z-resolution
 Signal losses
 Size of this maximum depends on magnification of
objective and wavelength of light
 Pinhole diameter needs to be adjusted on experimental
parameters
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> 1 Airy Disc
Improved brightness
Partial loss of confocal effect
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
 Photomultiplier:
 As detectors photomultipliers (PMT) are used
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•High dynamic range
(Voltage can be adjusted)
•Multiplication noise
•Multiplicative noise
•dark noise (cooling)
•cosmic radiation
IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
Photomultiplier:
 PMT collects and amplifies incoming photons / electrons and reacts quickly
and sensitive on incoming lights
 PMTs do not generate an image!
Image is generated by a computer
PMTs amplify brightness i.e. intensity of incoming light
 PMTs see black and white!
Wavelength of incoming light is irrelevant for PMTs
 In order to measure different wavelengths the light must be filtered and
distributed onto several detectors. Every single detector displays the
intensity of the selected wavelength area.
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7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Confocal laser scanning microscopy
Modern detectors:
 GAsP PMTs, high efficiency
 avalanche photo diodes (APDs),
extremely efficient, small area, low maximum rate
 APD arrays (expensive)
 APD/PMT Hybrid detectors
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7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Widefield vs. confocal
Widefield
Mouse Brain Hippocampus
Smooth Muscle
Confocal
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Sunflower Pollen Grain
Comparison of widefield (upper row)
and laser scanning confocal
fluorescence microscopy images
(lower row).
(a) and (b) Mouse brain hippocampus
thick section treated with primary
antibodies to glial fibrillary acidic
protein (GFAP; red), neurofilaments
H (green), and counterstained with
Hoechst 33342 (blue) to highlight
nuclei.
(c) and (d) Thick section of rat
smooth muscle stained with
phalloidin conjugated to Alexa Fluor
568 (targeting actin; red), wheat germ
agglutinin conjugated to Oregon
Green 488 (glycoproteins; green),
and counterstained with DRAQ5
(nuclei; blue).
(e) and (f) Sunflower pollen grain
tetrad autofluorescence.
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