A Brief History of Light Microscopy And How It Transformed Biomedical Research

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1 A Brief History of Light Microscopy And How It Transformed Biomedical Research Suewei Lin Office: Interdisciplinary Research Building 8A08 TEL:

2 Microscope = To View Small

3 Antony van Leeuwenhoek ( )

4 Robert Hook ( )

5 How simple lens microscope works

6 Compound Microscope

7 Light as a probe of matter!="# $=%"=%!/#

8 Light interacts with matter

9 Aberrations of a simple lens

10 Objective lens designs Cheap Red-blue corrected Less expensive color-corrected Bright good resolution Very expensive Highly color-corrected Bright High resolution

$Refractive$ 1.0 Water: 1.

12 Numerical aperture (NA) resolving power = #/2NA Refractive index (RI) Air: 1.0 Water: 1.3 Glycerol: 1.47 Glass: 1.5 Oil: 1.52

13 Bright field Phase contrast Differential interference Stained

14 An unstained brain

15 Camillo Golgi ( )

16 Santiago Ramón y Cajal ( )

17 Neuron theory

18 Nobel laureates in chemistry 2008

19 Aequorea victoria Osamu Purified Cloned & seq Douglas Prasher Robert Mutated Martin Expressed Improved

20 The power of differential labelling

22 Seeing signaling pathway & protein-protein interaction Seeing cell-cell interaction Fluorescence Resonance Energy Transfer Seeing neural activity Seeing protein modification

23 Observing protein-protein interaction with FRET

24 Observing functioning synapses

25 Physical basis of fluorescence upward arrow: absorption downward arrow: fluorescence emission wavy lines: heat

26 Absorption and emission spectra of fluorescein

27 Light source

28 Filters can be used to isolated specific wavelength

29 Filter modules

30 The operation of filter cubes

31 Light-emitting diode (LED)

32 A four-color LED setup

33 The thickness problem

34 Confocal laser scanning microscopy

35 Pinhole is the main mechanism for optical sectioning in confocal microscopy

36 Confocal laser scanning microscopy

37 Scanning control mechanism

38 Confocal vs. Wildefield Microscopy

39 Effect of confocal parameters on image quality

40 3D reconstruction of a fly brain

41 Fluorescence recovery after photobleaching (FRAP)

42 Increasing speed by spinning disks

43 Increasing speed by spinning disks

44 Eric Betzig Light sheet Microscopy

45 Whole brain activity imaging

46 Two-photon excitation Maria Göppert-Mayer ( )

47 Two photon vs confocal

48 Localised excitation

49 Localized excitation

50 Advantages and disadvantages of two-photon microscopy Near-infrared radiation penetrates tissues better: good for imaging thick specimens. The single-spot excitation causes less photodamage overall. Good for inducing photochemical reactions only on the focal plane: e.g. photoactivation of fluorescence proteins. Lower resolution compared to confocal microscopy

51 A specific subset of dopaminergic neurons responds to water

52 Less photon toxicity is the key Holtmaat & Svoboda 2009

53 Marching from high-resolution to super-resolution

54 Ernst Abbe ( ) Theoretical resolution limit for light microscopy & = #/2NA

55 Light microscopes only allow us to see a small portion of the world

56 Airy disc formation

57 Two airy discs

58 Image of a single GFP protein

60 Stefan Hell The RESOLFT concept REversible Saturable Optical Fluorescence Transitions

61 STED microscopy STimulated Emission Depletion microscopy

62 STED depletion lasers

63 STED Microscopy

64 PALM/STORM microscopy Eric Betzig PALM: photo activated localization microscopy STORM: stochastic optical reconstruction microscopy

65 PALM/STORM microscopy

Current microscopy limit Electron microscopy Light microscopy Superresolution microscopy Unaided eye Human height Length of some nerve and muscle cells Chicken egg Frog egg Human egg

66 Current microscopy limit Electron microscopy Light microscopy Superresolution microscopy Unaided eye Human height Length of some nerve and muscle cells Chicken egg Frog egg Human egg Nucleus Most plant and animal cells Most bacteria Mitochondrion Smallest bacteria Viruses Ribosomes Proteins Lipids Small molecules Atoms 10 m 1 m 0.1 m 1 cm 1 mm 100!m 10!m 1!m 100 nm 10 nm 1 nm

Cell Structure and Function

Cell Structure and Function Dead White Men Who Discovered (and were made of) Cells: Anton Van Leeuwenhoek Robert Hooke Where the Magic Happened Schleiden Cell Theory All plants are made of cells Schwann