Fourier-transformation & Optics

Biophotonics lecture16. November 2011FourierplanePoint objectImageffffaaMagnification: m=1Angles:sin(a)=sin(a) a=aMagnification and resolution: the Abbe limitFourierplanePoint objectImageff2f2faaMagnification: m=2Angles:sin(a)=sin(a) /2 a 590nm)DNA (Ex @320nm, 390nm)Aminoacids:Tryptophane (348nm, 2.6ns)Tyrosin (303nm, 3.6ns, weak)Phenylalanine (282nm weak)Resins, OilsEucalyptus leaf section UV excitation

Nematode living sample UV excitationhttp://en.wikipedia.org/wiki/AutofluorescenceStaining (labeling) specific structures with fluorescent labels (dyes): fluorochromationSmall dye concentrations are sufficient due to high fluorescence contrast fluorescence labels are superior than bright field dyesSingle molecule sensitivityFluorescence labels must selectively bind to structures or selectively accumulate in specific compartmentse.g. DAPI (= 4',6-diamidino-2-phenylindole) to label DNA (cell nuclei)

lexc = 358 nmlem = 461 nm

Fluorescence image of Endothelium cells. Microtubili are labeld in green, while actin filaments are labeled red. DNA within cell nuclei are stained with DAPI.Secondary fluorescence (fluorochromation)some dyes unquench upon bindingDAPI:Immunofluorescence (antibody staining) is the labeling of specific proteins with an antibody which is visualized by a dyeAntibodies are proteins which are used by the immune sys-tem to identify and neutralize foreign substances (antigens)Antibodies are made of two large heavy chains (~440 amino acids) and two small light chains(~220 amino acids) C-region is similar for all antibodies, while the V-region is extremely variable and forms the specific binding sitefor the antigen i.e. every antibody can recognize andbind two antigensSpecificity: antibody-antigen reaction. The part of the antigen (protein) recognized by an antibody is called an epitope. Highly specific interaction, called induced fit, allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organismSecondary fluorescence (fluorochromation): Immunofluorescence

VC1919Different groups of antibodies exist:Polyclonal antibodiesThey are a mixture of antibodies secreted against a specific antigen, each recognizing a different epitope i.e. bind to different areas of the protein. The protein (e.g. tubulin) for which a special antibody should be generated is injected into a suitable mammal (mostly rats, mice or goats).Antibodies against the protein are produced by the mammalian immune response and can be isolated from the blood serumMonoclonal antibodiesare all identical and bind to the same epitope Synthetic antibodiesare monoclonal antibodies which are produced in-vitro i.e. via microorganismOther systemsscFv (M. Bruchez): single chain variable region anitbodiesnanobodies (H. Leohardt): small (from Camelidae) and not degrated quickly inside a cellSecondary fluorescence (fluorochromation): Immunofluorescence2020Direct immunofluorescenceFor the direct or primary labeling the specific antibody for the investigated protein is labeled with the fluorochromeThe labeled antibodies are brought onto the sample and only bind specifically to the wanted protein (antigen = ligand); non bound antibodies are washed outDetection of the bound antibodies via the attached fluorochrome Localization of the wanted protein

An interphase female human fibroblast cell. Arrow points to the corresponding X chromosome (right). Labeling of a DNA-associated histone proteinSecondary fluorescence (fluorochromation): Immunofluorescence2121Indirect immunofluorescence Two sets of antibodies; Primary antibody detects antigen A subsequent, secondary (indirect), dye-coupled antibody recognizes the primary antibody. Signal amplification (several secondaries bind to one primary)Color palette separating staining from target

Example:1. Antibody / Rat Anti TubulinAntibody against tubulin generated in a rat2. Antibody / Goat Anti Ratfluorescently labeled antibody against all rat antibodies (generated in a goat)Negative test: Primary antibody is left out in order to test if the fluorescence labeled secondary antibody binds unspecifically to the sampleSecondary fluorescence (fluorochromation): Immunofluorescence2222Indirect immunofluorescence

GPCR transfected HEK cells:Double staining:For an identification of single cells the dye Hoechst 33342 was employed (cell nuclei: blue);Cell bound primary mouse anti-GPCR antibodies were detected by secondary goat anti-mouse Ig(H+L) antibodies labeled with Alexa Fluor 488 (GPCR-protein: green).The fluorescence labeled secondary antibodies can be employed for all antibodies produced within one animal e.g. goat serum against rats reacts with all primary antibodies produced in ratsSecondary fluorescence (fluorochromation): Immunofluorescence2323Der Begriff G-Protein-gekoppelter Rezeptor (kurz GPCR, fr englisch G protein-coupled receptor) wird in der Biologie fr Rezeptoren in der Zellmembran verwendet, die Signale ber GTP-bindende Proteine (kurz G-Proteine) in das Zellinnere weiterleiten (Signaltransduktion). G-Protein-gekoppelte Rezeptoren sind fr die Verarbeitung von Licht-, Geruchs- und einer Vielzahl von Geschmacksreizen verantwortlich. Sie spielen eine entscheidende Rolle bei Entzndungsprozessen, der gezielten Zellbewegung (Chemotaxis), dem Transport von Stoffen durch die Zellmembran (Endozytose und Exozytose) sowie beim Zellwachstum und bei der Zelldifferenzierung.

Dye artefactsBleaching:Fluorescence dye is destroyed by irradiation with lightQuenching:Fluorescence can be quenched (reduced) for large dye concentrationsCross-Talk:Cross-Excitation:Simultaneous excitation of two dyes if their excitation wavelengths are too close to each otherBleed through:In case the emission spectra overlap too much both dyes will be detected but to different amounts

Can be compensated by calibration and an inverse matrix technique"Spectral unmixing"Secondary fluorescence (fluorochromation)2424Fluorescence Microscopy

Optical sectioning

Missing cone

The confocal microscope

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 objectsConfocal 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 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

Line-wise 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 markerConfocal fluorescence microscopyThe confocal PSF:

Beam scan scan position: s- For each scan position s the illumination beam is moved to this position s,i.e. we illuminate with the shifted excitation PSF, hillu(r-s). The point source, at r=0, willbe illuminated with and emit fluorescence with an intensity proportional to- The sample is a point source, fixed at position 0. - This emission light is imaged with the PSF h(r) of the optical system and forms an imagein the pinhole plane.- Light not falling on the pinhole is blocked. Because of the de-scanning, the pinhole movesalong with the scan position.- The resulting light distribution is integrated on the PMT detector, yielding the finalconfocal PSF:The confocal PSF:

illuminationPSFdetectionPSFDetection PSF: the smaller the pinhole, the finer the detection PSFBUT: light is lost, as also IN-FOCUS light is blocked Bad signal-to-noiseReduction of out of focus light Resolution in confocal microscopyComparison of axial (x-z) point spread functions for widefield (left) and confocal (right) microscopy

Confocal fluorescence microscopyMissing conekx,ykzPSF(r) = PSFExcitation(r) PSFDetection(r) OTF(k) = OTFExcitation(r) OTFDetection(r) kx,ykzaIncreasing the aperture angle (a) enhances resolution !!Missing conehas been filled !!The confocal OTF:

Lateral support hasbeen increased.We have circumvented the Abbe-limit:

AbbeConfocal

WF1 AU0.3 AUin-plane, in-focus OTF1.4 NA ObjectiveWF LimitNew Confocal LimitAlmost no transferWe have circumvented the Abbe-limit, BUT:

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 suppressionQuantification of fluorescence intensitySimultaneous recording of different dyes in different channels Disadvantages:High costs (why?)Artifacts due to coherence of laser and laser fluctuationsHigh amount of photo-bleachingConfocal laser scanning microscopyExperimental Setup

Scanning and Descanning by same element

Confocal laser scanning microscopyScan Head:Excitation filter / Wavelength selectionScan-SystemBeamsplitterPinholeDetectors (photomultiplier)

Acousto-optic tunable filter (AOTF) for laser intensity control and wavelength selection in confocal microscopy.Acousto Optic Tunable Filter (AOTF)

dichromaticbeamsplittersexcitation filterConfocal laser scanning microscopyScan System:Mirror system is used to scan laser beam line by line over the sampleMirror system consists of two rotating mirrors; one for scanning the laser in x-direction and the other for movement in the y-direction

Beam separationIn confocal microscopy several wavelength bands can be detected in parallel. Beam splitting is performed by dichroitic mirrors + filters,prisms, diffraction gratings + apertures.

dichroitic beam splittermore detectorsvariable aperturesDiffractionGratingpinholeConfocal laser scanning microscopyPinhole:Pinhole in the optically conjugate sample plane in front of the detector to eliminate out-of-focus blur can be adjusted continuously in its sizePinhole size determines how much out-of-focus light is eliminated and how much light reaches the detectorThe smaller the pinhole the better the axial resolution the smaller the brightnessPinhole diameter = 1 Airy disc:Pinhole diameter corresponds to diameter of dark ringSize of this maximum depends on magnification of objective and wavelength of lightPinhole diameter needs to be adjusted on experimental parameters

< 1 Airy DiscImproved x,y,z-resolutionSignal losses> 1 Airy DiscImproved brightnessPartial loss of confocal effectConfocal laser scanning microscopyPhotomultiplier:As detectors photomultipliers (PMT) are used

High dynamic range (Voltage can be adjusted)Multiplicative noisedark noise (cooling)cosmic radiationConfocal laser scanning microscopyPhotomultiplier:PMT collects and amplifies incoming photons / electrons and reacts quickly and sensitive on incoming lightsPMTs do not generate an image!Image is generated by a computerPMTs amplify brightness i.e. intensity of incoming lightPMTs 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.Confocal laser scanning microscopyModern detectors:GAsP PMTs, high efficiencyavalanche photo diodes (APDs), extremely efficient, small area, low maximum rateAPD arrays (expensive)APD/PMT Hybrid detectors

Wide-field vs. confocal

WidefieldConfocal

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 HippocampusSmooth Muscle Sunflower Pollen Grain