1. IPC Friedrich-Schiller-Universität Jena 1 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

2. IPC Friedrich-Schiller-Universität Jena 2 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

3. IPC Friedrich-Schiller-Universität Jena 3 The Widefield Microscope xyz Pinhole Pinhole PMT The Confocal Microscope ZI(Z) Standard Lightsource Camera Standard Lightsource Camera Lightsource with Pinhole plane spread function:

4. Scanning in a CLSM PinholePMT Image of the Object: xyz Lightsource with Pinhole Sample Scanning Object scanning versus Beam scanning

5. IPC Friedrich-Schiller-Universität Jena 5 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 7. Fluorescence microscopy 7.2 Confocal fluorescence microscopy

6. IPC Friedrich-Schiller-Universität Jena 6 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 7. Fluorescence microscopy 7.2 Confocal fluorescence microscopy

7. IPC Friedrich-Schiller-Universität Jena 7 Confocal OTF k x,y kzkz Excite AND Detect: P(r) = P Excitation (r) P Detection (r) 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 !!

8. IPC Friedrich-Schiller-Universität Jena 8 We have circumvented Abbe:

9. IPC Friedrich-Schiller-Universität Jena 9 Confocal OTFs:  WF 1 AU 0.3 AU in-plane, in-focus OTF 1.4 NA Objective WF Limit New Confocal Limit Almost no transfer

10. IPC Friedrich-Schiller-Universität Jena 10 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

11. IPC Friedrich-Schiller-Universität Jena 11 Confocal laser scanning microscopy  Experimental Setup  Scanning and Descanning by same element 7. Fluorescence microscopy 7.2 Confocal fluorescence microscopy Excitation Fluorescence Transmission Detector

12. IPC Friedrich-Schiller-Universität Jena 12 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) 7. Fluorescence microscopy 7.2 Confocal fluorescence microscopy dichromatic beamsplitters excitation filter

13. IPC Friedrich-Schiller-Universität Jena 13 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 Diffraction Grating pinhole

14. IPC Friedrich-Schiller-Universität Jena 14 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 z-resolution  Signal losses > 1 Airy Disc  Improved brightness  Partial loss of confocal effect 7. Fluorescence microscopy 7.2 Confocal fluorescence microscopy

15. IPC Friedrich-Schiller-Universität Jena 15 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

16. IPC Friedrich-Schiller-Universität Jena 16 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. 7. Fluorescence microscopy 7.2 Confocal fluorescence microscopy

17. IPC Friedrich-Schiller-Universität Jena 17 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

18. IPC Friedrich-Schiller-Universität Jena 18 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