ipc friedrich-schiller-universität jena 1 reduction of out of focus light excitation light excites...

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



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




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



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





Resolution in confocal microscopyComparison of axial (x-z) point spread functions for widefield (left) and confocal (right) microscopy




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 !!


We have circumvented Abbe:

NAn

NANAD

emex

4sin4

)(2

1confocalmin,


Confocal OTFs:

WF

1 AU

0.3 AU

in-plane, in-focus OTF1.4 NA Objective

WF Limit

New Confocal Limit

Almost no transfer


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





Experimental Setup

Scanning and Descanning by same element



Excitation

Fluorescence

Transmission Detector


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)



dichromaticbeamsplitters

excitation filter



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.



dichroitic beam splitter

more detectors

variable apertures

DiffractionGrating

pinhole



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





Photomultiplier: As detectors photomultipliers (PMT) are used


7.2 Confocal fluorescence microscopy •High dynamic range (Voltage can be adjusted)•Multiplication noise•Multiplicative noise•dark noise (cooling)•cosmic radiation



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.





Modern detectors:

GAsP PMTs, high efficiency

avalanche photo diodes (APDs), extremely efficient, small area, low maximum rate

APD arrays (expensive)

APD/PMT Hybrid detectors




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.



Mouse Brain Hippocampus Smooth Muscle Sunflower Pollen Grain

ipc friedrich-schiller-universität jena 1 reduction of out of focus light excitation light excites...

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