A Brief History of Light Microscopy And How It Transformed Biomedical Research Suewei Lin Office: Interdisciplinary Research Building 8A08 Email: sueweilin@gate.sinica.edu.tw TEL: 2789-9315
Microscope = To View Small
Antony van Leeuwenhoek (1632-1723)
Robert Hook (1635-1703)
How simple lens microscope works
Compound Microscope
Light as a probe of matter!="# $=%"=%!/#
Light interacts with matter
Aberrations of a simple lens
Objective lens designs Cheap Red-blue corrected Less expensive color-corrected Bright good resolution Very expensive Highly color-corrected Bright High resolution
Numerical aperture (NA) resolving power = #/2NA Refractive index (RI) Air: 1.0 Water: 1.3 Glycerol: 1.47 Glass: 1.5 Oil: 1.52
Bright field Phase contrast Differential interference Stained
An unstained brain
Camillo Golgi (1843-1926)
Santiago Ramón y Cajal (1879-1930)
Neuron theory
Nobel laureates in chemistry 2008
Aequorea victoria Osamu Purified Cloned & seq Douglas Prasher Robert Mutated Martin Expressed Improved
The power of differential labelling
Seeing signaling pathway & protein-protein interaction Seeing cell-cell interaction Fluorescence Resonance Energy Transfer Seeing neural activity Seeing protein modification
Observing protein-protein interaction with FRET
Observing functioning synapses
Physical basis of fluorescence upward arrow: absorption downward arrow: fluorescence emission wavy lines: heat
Absorption and emission spectra of fluorescein
Light source
Filters can be used to isolated specific wavelength
Filter modules
The operation of filter cubes
Light-emitting diode (LED)
A four-color LED setup
The thickness problem
Confocal laser scanning microscopy
Pinhole is the main mechanism for optical sectioning in confocal microscopy
Confocal laser scanning microscopy
Scanning control mechanism
Confocal vs. Wildefield Microscopy
Effect of confocal parameters on image quality
3D reconstruction of a fly brain
Fluorescence recovery after photobleaching (FRAP)
Increasing speed by spinning disks
Increasing speed by spinning disks
Eric Betzig Light sheet Microscopy
Whole brain activity imaging
Two-photon excitation Maria Göppert-Mayer (1906-1972)
Two photon vs confocal
Localised excitation
Localized excitation
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
A specific subset of dopaminergic neurons responds to water
Less photon toxicity is the key Holtmaat & Svoboda 2009
Marching from high-resolution to super-resolution
Ernst Abbe (1840-1905) Theoretical resolution limit for light microscopy & = #/2NA
Light microscopes only allow us to see a small portion of the world
Airy disc formation
Two airy discs
Image of a single GFP protein
Stefan Hell The RESOLFT concept REversible Saturable Optical Fluorescence Transitions
STED microscopy STimulated Emission Depletion microscopy
STED depletion lasers
STED Microscopy
PALM/STORM microscopy Eric Betzig PALM: photo activated localization microscopy STORM: stochastic optical reconstruction microscopy
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 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