Exploring Microscopy: From History to Modern Techniques

1. Detection systems

2. Course outline

3. Scale

4. Scale: 100 μm

5. Optical microscopy

6. Life under a microscope Watch out! A cover slide!

7. History of microscopy

8. History of microscopy

9. History of microscopy 1665 1673 1880 History of microscopy 1720

10. Today’s microscopy

11. Bright-field microscopy

12. Microscope resolution • Also called resolving power • Ability of a lens to separate or distinguish small objects that are close together • Light microscope has a resolution of 0.2 micrometer • wavelength of light used is major factor in resolution shorter wavelength  greater resolution

13. Bright-field microscopy • produces a dark image against a brighter background • Cannot resolve structures smaller than about 0.2 micrometer • Inexpensive and easy to use • Used to observe specimens and microbes but does not resolve very small specimens, such as viruses

14. Bright-field microscopy • has several objective lenses (3 to 4) • Scanning objective lens 4X • Low power objective lens 10X • High power objective lens 40X • Oil immersion objective lens 100X • total magnification • product of the magnifications of the ocular lens and the objective lens • Most oculars magnify specimen by a factor of 10

15. Microscope objectives

16. Microscope objectives

17. Working distance

18. Oil immersion objectives

19. Bright-field image of Amoeba proteus

20. Darki-field microscopy • Uses a special condenser with an opaque disc that blocks light from entering the objective lens • Light reflected by specimen enters the objective lens • produces a bright image of the object against a dark background • used to observe living, unstained preparations

21. Dark-field image of Amoeba proteus

22. Microscope image

23. Fluorescence microscopy

24. Excitation sources • Lamps • Xenon • Xenon/Mercury • Lasers • Argon Ion (Ar) 353-361, 488, 514 nm • Violet 405 405 nm • Helium Neon (He-Ne) 543 nm, 633 nm • Helium Cadmium (He-Cd) 325 - 441 nm • Krypton-Argon (Kr-Ar) 488, 568, 647 nm

25. Arc lamp excitation spectra Xe Lamp   Irradiance at 0.5 m (mW m-2 nm-1)  Hg Lamp     

26. Fluorescent microscope Arc Lamp EPI-Illumination Excitation Diaphragm Excitation Filter Ocular Dichroic Filter Objective Emission Filter

27. Standard band pass filters 630 nm band pass filter white light source transmitted light 620 -640 nm light

28. Standard long pass filters 520 nm long pass filter white light source transmitted light >520 nm light

29. Standard short pass filters 575 nm short pass filter white light source transmitted light <575 nm light

30. Fluorescence • Chromophores are components of molecules which absorb light • E.g. from protein most fluorescence results from the indole ring of tryptophan residue • They are generally aromatic rings

31. Jablonski diagram absorption internal conversion fluorescence intersystem crossing S1 T0 -hν +hν internal conversion S0 radiationless transition transition involving emission/absorption of photon

32. Simplified Jablonski diagram S’1 S1 hvex hvem Energy S0

33. Fluorescence • The longer the wavelength the lower the energy • The shorter the wavelength the higher the energy e.g. UV light from sun causes the sunburn not the red visible light

34. Some fluorophores Common Laser Lines 350 457 488 514 610 632 300 nm 400 nm 500 nm 600 nm 700 nm PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid

35. Stokes shift Change in the energy between the lowest energy peak of absorbance and the highest energy of emission Stokes Shift is 25 nm Fluorescein molecule 520 nm 495 nm Fluorescence Intensity Wavelength

36. Excitation saturation • The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime τf) • Optical saturation occurs when the rate of excitation exceeds the reciprocal of τf • In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in 1 second requires a dwell time per pixel of 2 x 10-6 sec. • Molecules that remain in the excitation beam for extended periods have higher probability of interstate crossings and thus phosphorescence • Usually, increasing dye concentration can be the most effective means of increasing signal when energy is not the limiting factor (i.e. laser based confocal systems) Material Source: Pawley: Handbook of Confocal Microscopy

37. Photo-bleaching • Defined as the irreversible destruction of an excited fluorophore • Methods for countering photo-bleaching • Scan for shorter times • Use high magnification, high NA objective • Use wide emission filters • Reduce excitation intensity • Use “antifade” reagents (not compatible with viable cells)

38. Quenching Not a chemical process • Dynamic quenching Collisional process usually controlled by mutual diffusion • Typical quenchers oxygen Aliphatic and aromatic amines (IK, NO2, CHCl3) • Static Quenching Formation of ground state complex between the fluorophores and quencher with a non-fluorescent complex (temperature dependent – if you have higher quencher ground state complex is less likely and therefore less quenching

39. Excitation and emission peaks % Max Excitation at 488568 647 nm Fluorophore EXpeak EMpeak FITC 496 518 87 0 0 Bodipy 503 511 58 1 1 Tetra-M-Rho 554 576 10 61 0 L-Rhodamine 572 590 5 92 0 Texas Red 592 610 3 45 1 CY5 649 666 1 11 98 Material Source: Pawley: Handbook of Confocal Microscopy

41. Probes for proteins Probe Excitation Emission FITC 488 525 PE 488 575 APC 630 650 PerCP™ 488 680 Cascade Blue 360 450 Coumerin-phalloidin 350 450 Texas Red™ 610 630 Tetramethylrhodamine-amines 550 575 CY3 (indotrimethinecyanines) 540 575 CY5 (indopentamethinecyanines) 640 670

42. Probes for nucleotides Hoechst 33342 (AT rich) (uv) 346 460 DAPI (uv) 359 461 POPO-1 434 456 YOYO-1 491 509 Acridine Orange (RNA) 460 650 Acridine Orange (DNA) 502 536 Thiazole Orange (vis) 509 525 TOTO-1 514 533 Ethidium Bromide 526 604 PI (uv/vis) 536 620 7-Aminoactinomycin D (7AAD) 555 655

43. GFP GFP - Green Fluorescent • GFP is from the chemiluminescent jellyfish Aequorea victoria • excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm • contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser- Tyr-Gly at positions 65-67 of the primary sequence • Major application is as a reporter gene for assay of promoter activity • requires no added substrates

44. Multiple emissions • Many possibilities for using multiple probes with a single excitation • Multiple excitation lines are possible • Combination of multiple excitation lines or probes that have same excitation and quite different emissions • e.g. Calcein AM and Ethidium(ex 488 nm) • emissions 530 nm and 617 nm

45. Energy transfer Non radiative energy transfer – a quantum mechanical process of resonance between transition dipoles • Effective between 10-100 Å only • Emission and excitation spectrum must significantly overlap • Donor transfers non-radiatively to the acceptor • PE-Texas Red™ • Carboxyfluorescein-Sulforhodamine B

46. Fluorescence resonance energy tranfer FRET Molecule 1 Molecule 2 Fluorescence Fluorescence DONOR ACCEPTOR Intensity Absorbance Absorbance Wavelength

47. Confocal microscopy

48. Confocal microscopy • confocal scanning laser microscope • laser beam used to illuminate spots on specimen • computer compiles images created from each point to generate a 3-dimensional image

49. Benefits of confocal microscopy Reduced blurring of the image from light scattering Increased effective resolution Improved signal to noise ratio Clear examination of thick specimens Z-axis scanning Depth perception in Z-sectioned images Magnification can be adjusted electronically

50. The different microscopes Fluorescent Microscope Confocal Microscope Arc Lamp Laser Excitation Pinhole Excitation Diaphragm Excitation Filter Excitation Filter Ocular PMT Objective Objective Emission Filter Emission Filter Emission Pinhole