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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 - PowerPoint PPT Presentation
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IPC Friedrich-Schiller-Universität Jena1
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
7.2 Confocal fluorescence microscopy
7. Fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena2
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-of-focus blur
As only light produced by fluorescence close to the focal plane is detected, the contrast is much better than that of wide-field microscopes.
Allows recording individual optical sections or three dimensional reconstruction of objects
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena3
The Widefield Microscope
xxyy
zz
PinholePinhole
PinholePinholePMTPMT
The Confocal Microscope
ZZ
I(Z)I(Z)
Standard Lightsource
Camera
Standard Lightsource
Camera
Lightsource Lightsource with Pinholewith Pinhole
plane spread function:
Scanning in a CLSMScanning in a CLSM
PinholePinholePMTPMT
Image of the Object:Image of the Object:
xxyy
zzLightsource Lightsource with Pinholewith Pinhole
Sample Scanning
Object scanning versus
Beam scanning
IPC Friedrich-Schiller-Universität Jena5
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 time2D or 3D imaging requires scanning over a regular raster
(i.e. a rectangular pattern of parallel scanning lines)
in the specimen: raster-scanComparison 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
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena6
Reduction of out of focus light
Resolution in confocal microscopyComparison of axial (x-z) point spread functions for widefield (left) and confocal (right) microscopy
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena7
Confocal OTF
kx,y
kz
Excite AND Detect: P(r) = PExcitation(r) PDetection(r) PSF(r) = PSFExcitation(r) PSFDetection(r)
OTF(k) = OTFExcitation(r) OTFDetection(r)
kx,y
kz
Increasing the aperture angle () enhances resolution !!
IPC Friedrich-Schiller-Universität Jena8
We have circumvented Abbe:
NAn
NANAD
emex
4sin4
)(2
1confocalmin,
IPC Friedrich-Schiller-Universität Jena9
Confocal OTFs:
WF
1 AU
0.3 AU
in-plane, in-focus OTF1.4 NA Objective
WF Limit
New Confocal Limit
Almost no transfer
IPC Friedrich-Schiller-Universität Jena10
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
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena11
Confocal laser scanning microscopy
Experimental Setup
Scanning and Descanning by same element
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Excitation
Fluorescence
Transmission Detector
IPC Friedrich-Schiller-Universität Jena12
Confocal laser scanning microscopyScan Head:
Excitation filter / Wavelength selection
Scan-System Beamsplitter Pinhole Detectors (photomultiplier)
Acousto-optic tunable filter (AOTF) for laser intensity control and wavelength selection in confocal microscopy.
Acousto Optic Tunable Filter (AOTF)
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
dichromaticbeamsplitters
excitation filter
IPC Friedrich-Schiller-Universität Jena13
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 x-direction 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.
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
dichroitic beam splitter
more detectors
variable apertures
DiffractionGrating
pinhole
IPC Friedrich-Schiller-Universität Jena14
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
Size of this maximum depends on magnification of objective and wavelength of light
Pinhole diameter needs to be adjusted on experimental parameters
< 1 Airy DiscImproved z-resolutionSignal losses
> 1 Airy DiscImproved brightnessPartial loss of confocal effect
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena15
Confocal laser scanning microscopy
Photomultiplier: As detectors photomultipliers (PMT) are used
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy •High dynamic range (Voltage can be adjusted)•Multiplication noise•Multiplicative noise•dark noise (cooling)•cosmic radiation
IPC Friedrich-Schiller-Universität Jena16
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 computerPMTs 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.
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena17
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
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
IPC Friedrich-Schiller-Universität Jena18
Widefield vs. confocal
Widefield
Confocal
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.
7. Fluorescence microscopy
7.2 Confocal fluorescence microscopy
Mouse Brain Hippocampus Smooth Muscle Sunflower Pollen Grain