1. K.U.LEUVEN Johan Hofkens Satoshi Habuchi Laboratory of Photochemistry and Spectroscopy Katholieke Universiteit Leuven - Belgium Theories and methods to study molecular interactions : microscopy

2. Overview 1. Microscope principle - Lenses and geometrical optics - Resolution of optical microscope - Image brightness 2. Application for studies of molecular interactions - Köhler illumination - Fluorescence microscopy - Confocal microscopy - Fluorescent probes - Ca 2+ measurements in cells - Nucleocytoplasmic shuttling of MAPK

3. Why doing microscopy Microscope is an instrument to produce magnified images of small objects. - Magnified image - Separate details in an image - Make details visible to eye or camera

4. Microscope Optical Components Light source Collector lens Condenser Objective Eyepiece Field Diaphragm Aperture Diaphragm Objective back focal plane Intermediate image plane

5. Lenses and Geometrical Optics

6. Refraction of light Direction change of a ray of light passing from one transparent medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface. Snell’s Law V: velocity of light in material n: refractive index material θ1θ1 θ2θ2

7. Lenses and Geometrical Optics Focal length Light from an object that is very far from the front of a lens will be brought to a focus at a fixed point behind the lens. This is known as the focal point (F) of the lens. The distance from the center of the lens to the focal plane is know as the focal length (f).

8. Lenses and Geometrical Optics Magnification of a lens Lens formulaMagnification

9. Lenses and Geometrical Optics Real image and virtual image When the distance between the object and the lens is longer than the focal length, the rays become convergent, giving the real image (inverted). When the distance between the object and the lens is shorter than the focal length, the rays can not be convergent, giving the virtual image (non inverted) which always appear upright to the observer. Real imageVirtual image

10. Lenses and Geometrical Optics Microscope conjugate field planes f obj : < 10 mm optical tube length : 160 mm f eye : few cm Example f obj : 8 mm, f eye : 25 mm object to eye distance : 250 mm

11. Lenses and Geometrical Optics Infinity-corrected optical system The region between the objective and tube lens (infinity space) provides a path of parallel light rays. Complex optical components can be placed without loosing the performance of the microscope.

12. Resolution of Optical Microscopy

13. Diffraction Light rays bend around edges – new wavefronts are generated at sharp edges

14. Resolution of Optical Microscopy Interference Constructive interferenceDestructive interference

15. Resolution of Optical Microscopy Airy pattern formation Condenser Objective Aperture Diaphragm Intermediate image plane Direct light Diffracted light Objective back focal plane

16. Resolution of Optical Microscopy Rayleigh criterion The limit at which two Airy disks can be resolved into separate entities is often called the Rayleigh criterion. Resolution R = 0.61λ / NA λ : wavelength NA : numerical aperture

17. Resolution of Optical Microscopy Numerical aperture (NA) Numerical Aperture (NA) = n(sin μ) n : refractive index of the imaging medium μ : one-half angle of the angular aperture A

18. Resolution of Optical Microscopy NA and resolution NA = 0.10NA = 0.18NA = 0.36 R = 0.61λ / NA High NA : better resolution

19. Resolution of Optical Microscopy NA and resolution Resolution and Numerical Aperture by Objective Type λ = 550 nm

20. Resolution of Optical Microscopy Wavelength and resolution R = 0.61λ / NA wavelength = 400 nmwavelength = 550 nmwavelength = 700 nm Short wavelength : better resolution

21. Resolution of Optical Microscopy Wavelength and resolution Resolution versus Wavelength NA = 0.95

22. Resolution of Optical Microscopy Axial resolution The axial range, through which an objective can be focused without any appreciable change in image sharpness, is referred as the objective depth of field. High NA : better axial resolution

23. Resolution of Optical Microscopy NA and immersion medium Low refractive indexHigh refractive index NA = n(sin μ) NA = 1.0 sin(65°) = 0.90 n = 1.00 μ = 65° NA = 1.51 sin(65°) = 1.38 n = 1.51 μ = 65° Low resolutionHigh resolution

24. Image Brightness

25. Magnification 10x NA 0.15 0.45 20x0.40 0.85 40x0.70 1.30 60x0.80 1.40 100x0.85 1.40 Transmitted light intensity 2.25 20.24 4.00 18.06 3.10 10.56 1.80 5.44 0.72 1.96

26. Köhler Illumination

27. Köhler illumination (specimen illuminating light rays) This technique is recommended by all manufactures of modern laboratory microscopes because it can produce specimen illumination that is uniformly bright and free from glare, allowing the user to realize the microscope’s full potential.

28. Köhler Illumination Köhler illumination (image-forming light rays)

29. Köhler Illumination Aperture diaphragm Aperture adjustment and proper focusing of the condenser are of critical importance in realizing the full potential of the objective. Specifically, appropriate use of the aperture diaphragm is most important in securing correct illumination, contrast, and depth of field.

30. Köhler Illumination Condenser alignment and field diaphragm opening size Correct height of the condenser is critical to quantitative microscopy and optimum photomicrography. The field diaphragm controls only the width of the bundle of light rays reaching the condenser – it does not affect the optical resolution, and the intensity of illumination. Proper adjustment of the field diaphragm is important for preventing glare. Adjust the height of the condenser Move the image of the field diaphragm to the center Open the field diaphragm until it is just beyond the field of view

31. Fluorescence Microscopy

32. Fluorescence

33. Fluorescence Microscopy Epi fluorescence and transmitted light microscopy Transmitted light microscopyEpi fluorescence microscopy

34. Fluorescence Microscopy Epi fluorescence microscopy Advantage The objective, first serving as a well corrected condenser and then as the image-forming light gatherer, is always in correct alignment relative to each of these functions. Most of the unwanted or unused excitation light reaching the specimen travels away from the objective. The area being illuminated is restricted to the area being observed The full NA of the objective is utilizable. It is possible to combine with transmitted light observation.

35. Fluorescence Microscopy Light source

36. Fluorescence Microscopy Fluorophore A good fluorophore - Large extinction coefficient ( ≈ 10 5 cm -1 M -1 ) - High fluorescence quantum yield ( > 0.8) - Large shift of the fluorescence vs. absorption, Stokes shift ( > 40 nm) - Low quantum yield of photobleaching ( < 10 -6 )

37. Fluorescence Microscopy Filters

38. Fluorescence Microscopy Detector Area detectorPoint detector

39. Fluorescence Microscopy Fluorescence images Human Cervical Adenocarcinoma Cells (HeLa Line) EGFP : Green Mito Tracker Red CMXRos : Red Peroxisomes Intracellular microtublar network Hoechst 33342 : Blue DNA in the nucleus Transformed African Green Monkey Kidney Fibroblast Cells (COS-7) Cy3 : Red Alexa Fluor 488 : Green Microtubles Cytoskeletal filamentous actin network DAPI : Blue DNA

40. Confocal Microscopy

41. Confocal principle Excitation light passes through a pinhole aperture that is situated in a conjugate plane with a scanning point on the specimen. As the laser is reflected by a dichromatic mirror and scanned across the specimen in a defined focal plane, secondary fluorescence emitted from points on the specimen (in the same focal plane) pass back through the dichromatic mirror and are focused as a confocal point at the detector pinhole aperture. The significant amount of fluorescence emission that occurs at points above and below the objective focal plane is not confocal with the pinhole (termed Out-of- Focus Light Rays). Because only a small fraction of the out-of-focus fluorescence emission is delivered through the pinhole aperture, most of this extraneous light is not detected by the photomultiplier and does not contribute to the resulting image.

42. Confocal Microscopy Resolution in confocal microscopy R lat = 0.61λ / NA Epi fluorescence microscopyConfocal fluorescence microscopy R lat = 0.43λ / NA Axial Point Spread Function Intensity Profiles

43. Confocal Microscopy Confocal microscope scanning system

44. Confocal Microscopy Confocal images

45. Application for Studies of molecular Interactions

46. Amine-Reactive ProbesThiol-Reactive Probes Fluorescent Probes General fluorescent probes target probe +

47. Fluorescent Probes Organelle-specific fluorescent probes Mitochondrion-Selective Probes

48. Fluorescent Probes Genetically encoded fluorescent probes Green fluorescent protein (GFP) targetGFP cDNA vector expression

49. Ca 2+ Measurements in Cells

50. Cameleon protein Fluorescent indicators for measuring Ca 2+ concentration. - Energy donor : ECFP ECFP EYFP CaMM13 440 nm 475 nm ECFP EYFP 440 nm 530 nm - Energy acceptor : EYFP - Linker : calmodulin (CaM) + calmodulin-binding peptide M13 (myosin light chain kinase) +4Ca 2+ -4Ca 2+

51. Ca 2+ Measurements in Cells Cameleon protein Energy transfer efficiencyCa 2+ concentration

52. Ca 2+ Measurements in Cells Ca 2+ propagation in cells HeLa cells are stimulated with histamine

53. Ca 2+ Measurements in Cells Ca 2+ propagation in cells

54. Nucleocytoplasmic Shuttling of MAPK

55. MAPK cascades The Mitogen-activated Protein Kinase (MAPK) Cascades

56. Nucleocytoplasmic Shuttling of MAPK Fused protein MAPK Dronpa MAPK-Dronpa is initially distributed throughout the cytosol and nucleus. Dronpa : GFP-like fluorescent protein COS7 cells

57. Nucleocytoplasmic Shuttling of MAPK Photoswitching of Dronpa Intense excitation at 488 nm changes Dronpa to the dim state but even weak irradiation at 400 nm restores it to the bright deprotonated form. Fluorescence could be switched on and off repeatedly.

58. Nucleocytoplasmic Shuttling of MAPK Monitoring the nuclear import and export of MAPK C ►N N ►C

59. Nucleocytoplasmic Shuttling of MAPK Monitoring the nuclear import and export of MAPK Only the nuclear accumulation of MAPK is confirmed by the normal fluorescence images. The acceleration of the bidirectional flow of MAPK across the nuclear envelope is vidualized by reversible protein highlighting.