1. Biophotonics lecture 16. November 2011

2. Fourier plane Point object Image f f f f  ’’ Magnification: m=1 Angles:sin(  ’)=sin(  ’=  Magnification and resolution: the Abbe limit

3. Fourier plane Point object Image f f 2f  ”” Magnification: m=2 Angles:sin(  ’)=sin(  /2   ’<  Magnification and resolution: the Abbe limit

4. Fourier plane Point object Image f f f f  ’’ Magnification: m Angles:sin(  ’)=sin(  /m Smallest distance (image):d’ = / (2 sin(  ’)) = m / (2 sin(  )) Smallest distance (sample):d = / (2 sin(  )) Magnification and resolution: the Abbe limit

5. http://biology.about.com NA=n sin  Abbe resolution limit: d= /(2NA) Magnification and resolution: the Abbe limit

6. Ewald sphere McCutchen generalised aperture 2  n/

7. Missing cone Optical Transfer Function (OTF): For incoherent microscopy techniques, e.g. fluorescence microscopy Lateral support Axial support

8. Today: -How to do optical sectioning and fill the missing cone. -How to increase the lateral support and break the Abbe-limit. -Fluorescence microscopy in general.

9. Fluorescence Microscopy advantages: -High contrast -High specificity but: -No optical sectioning in wide-field mode

10. Jablonski diagram (simplified) Energy

11. Excitation, e.g. with 488nm Vibrational relaxation Emission, e.g. with 525nm Vibrational relaxation

12. AbsorptionEmission Abbyad et al., PNAS December 18, 2007 vol. 104 no. 51 20189-20194

13. Excitation, e.g. with 488nm Emission, e.g. with 525nm Dichromatic reflector reflects blue light fluorescence Objective Sample transmits green light Tube lens Camera Blue light source High contrast:

14. Fluorochromes  Fluorescence microscopy differentiates between two kinds of fluorochromes:  Primary fluorescence (autofluorescence)  Secondary fluorescence (fluorochromation)  Fluorescence dyes  Immunofluorescence (using Antibodies)  Molecular tags (SNAP Tag,...)  Fluorescent Proteins  Applications of fluorochromes  Identification of otherwise invisible structures  Localization and identification of otherwise invisible structures  Monitoring of physiological processes  Specific detection of a protein  Using photo-physical properties of dyes (e.g. switching) for super-resolution

15. Primary fluorescence (autofluorescence)  Most samples fluoresce when excited with short-wave light  Fluorescence very often occurs for systems containing many conjugated double bonds:  e.g. chlorophyll exhibits dark red fluorescence when excited by blue or red light Moss reeds – green excitation Porphyrin ring – central unit in Chlorophyll

16. http://en.wikipedia.org/wiki/File:Chlorophyll_ab_spectra2.PNG

17.  Further examples:  Riboflavine (550nm)  NAD(P)H (460nm, 400ps)  Elastin und Collagen (305-450nm)  Retinol (500nm)  Cuticula (blue)  Lignin (> 590nm)  DNA (Ex @320nm, 390nm)  Aminoacids:  Tryptophane (348nm, 2.6ns)  Tyrosin (303nm, 3.6ns, weak)  Phenylalanine (282nm weak)  Resins, Oils Eucalyptus leaf section – UV excitation Nematode living sample – UV excitation http://en.wikipedia.org/wiki/Autofluorescence

18.  Staining (labeling) specific structures with fluorescent labels (dyes): fluorochromation  Small dye concentrations are sufficient due to high fluorescence contrast  fluorescence labels are superior than bright field dyes  Single molecule sensitivity  Fluorescence labels must selectively bind to structures or selectively accumulate in specific compartments  e.g. DAPI (= 4',6-diamidino-2-phenylindole) to label DNA (cell nuclei) exc = 358 nm em = 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 binding DAPI:

19. Secondary fluorescence (fluorochromation): Immunofluorescence V C

20.  Different groups of antibodies exist:  Polyclonal antibodies They 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 serum  Monoclonal antibodies are all identical and bind to the same epitope  Synthetic antibodies are monoclonal antibodies which are produced in-vitro i.e. via microorganism  Other systems scFv (M. Bruchez): single chain variable region anitbodies nanobodies (H. Leohardt): small (from Camelidae) and not degrated quickly inside a cell Secondary fluorescence (fluorochromation): Immunofluorescence

21. Direct immunofluorescence  For the direct or primary labeling the specific antibody for the investigated protein is labeled with the fluorochrome  The labeled antibodies are brought onto the sample and only bind specifically to the wanted protein (antigen = ligand); non bound antibodies are washed out  Detection 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 protein Secondary fluorescence (fluorochromation): Immunofluorescence

22. Indirect 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 Tubulin Antibody against tubulin generated in a rat  2. Antibody / Goat – Anti Rat fluorescently 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 sample Secondary fluorescence (fluorochromation): Immunofluorescence

23. Indirect 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 rats Secondary fluorescence (fluorochromation): Immunofluorescence

24. Dye artefacts  Bleaching:Fluorescence dye is destroyed by irradiation with light  Quenching:Fluorescence can be quenched (reduced) for large dye concentrations  Cross-Talk: Cross-Excitation:Simultaneous excitation of two dyes if their excitation wavelengths are too close to each other Bleed 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)

25. Fluorescence Microscopy Optical sectioning Missing cone

26. The confocal microscope

29. 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 Confocal fluorescence microscopy

30. 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 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 marker Confocal fluorescence microscopy

31. The 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, h illu (r-s). The point source, at r=0, will be 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 image in the pinhole plane. - Light not falling on the pinhole is blocked. Because of the de-scanning, the pinhole moves along with the scan position. - The resulting light distribution is integrated on the PMT detector, yielding the final confocal PSF:

32. The confocal PSF: illumination PSF detection PSF Detection PSF: the smaller the pinhole, the finer the detection PSF BUT: light is lost, as also IN-FOCUS light is blocked  Bad signal-to-noise

33. 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 Confocal fluorescence microscopy

34. Missing cone k x,y kzkz PSF(r) = PSF Excitation (r) PSF Detection (r) OTF(k) = OTF Excitation (r)  OTF Detection (r) k x,y kzkz  Increasing the aperture angle (  ) enhances resolution !! Missing cone has been filled !! The confocal OTF: Lateral support has been increased.

35. We have circumvented the Abbe-limit: Abbe Confocal

36. WF 1 AU 0.3 AU in-plane, in-focus OTF 1.4 NA Objective WF Limit New Confocal Limit Almost no transfer We have circumvented the Abbe-limit, BUT:

37. 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

38. Confocal laser scanning microscopy  Experimental Setup  Scanning and Descanning by same element

39. Confocal laser scanning microscopy  Scan 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) dichromatic beamsplitters excitation filter

40. 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  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 Diffraction Grating pinhole

41. 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 Disc  Improved x,y,z-resolution  Signal losses > 1 Airy Disc  Improved brightness  Partial loss of confocal effect

42. Confocal laser scanning microscopy  Photomultiplier:  As detectors photomultipliers (PMT) are used High dynamic range (Voltage can be adjusted) Multiplicative noise dark noise (cooling) cosmic radiation

43. 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.

44. 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

45. Wide-field 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