History

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History

• Santiago Ramón y Cajal
• Staining method (Golgi)
• Development of precise optics

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History
Electrode based techniques dominateExtracellular
electrodes, patch clamp, sharp
electrode Calcium indicators developed The
principle of confocal imaging was patented by
Marvin Minsky in 1961- most of the excitation
outside of focus-information cut by
pinhole Two-photon excitation concept first
described by Maria Göppert-Mayer in 1931.
Two-photon microscopy was pioneered by Winfried
Denk in the lab of Watt W. Webb at Cornell
University in 1990- all light is taken no
pinhole
Winfried Denk
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History
Second harmonic generation - photons
interacting with a nonlinear material are
effectively "combined" to form new photons with
twice the energy, and therefore twice the
frequency and half the wavelength of the initial
photons P. A. Franken, A. E. Hill, C. W. Peters,
and G. Weinreich at the University of Michigan,
in 1961 In neuroscience used first in 2004
WW.Webb
real-time optical recording of neuronal
action potentials using SHG Sacconi L, Dombeck
DA, Webb WW. PNAS 2006
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Principle of fluorescence measurment
Emission-absorption spectrum of Fluo-4
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Fluorescence measurement
Detector CCD (speed, sensitivity,
resolution) Up to 10 kHz Light source Mercury
or Xenon Lamp Spectrum Stability Filters
Fluorescent microscope
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Charge-Coupled Devices (CCDs)
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Charge-Coupled Devices (CCDs)
CCD - photon detector, a thin silicon wafer
divided into a geometrically regular array of
thousands or millions of light-sensitive regions
Pixel - picture element
metal oxide semiconductor (MOS) capacitor
operated as a photodiode and storage device
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Charge-Coupled Devices (CCDs)
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Laser scanning confocal microscopy
Detector photomultiplier Light source
laser Power Wavelength Filters Scanner
Confocal microscope
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Principle of two photon excitation
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Difference between single photon and two photon
imaging
Winfried Denk and Karel Svoboda Neuron, Vol. 18,
351357, March, 1997
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Single photon and two photon excitation in
florescent media
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Single photon and two photon excitation in
florescent media
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Two-photon excitation requires IR laser
IR penetrates tissue much deeper
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Advantages of two photon imaging

• No out-of-focus fluorescence
• Better in depth resolution
• Less photobleaching of the dye
• Less photodamage of the dye
• Less phototoxicity for the tissue

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Limitations of multiphoton imaging

1. Two photon imaging has depth limit out of focus
   light (background) gt 1000 mm Theer, Hasan, Denk.
   Opt Lett. 2003
2. Scanner frame rate is relatively slow compare to
   open field imaging
3. light with wavelength over 1400 nm may be
   significantly absorbed by the water in living
   tissue limits multiphoton excitation
4. IR lasers are expensive

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Imaging laboratory
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Two photon imaging system
(FL) femtosecond mode-locked laser (BE) beam
expander (GM) pair of galvanometer scanning
mirrors (SL) scan-lens intermediate optics (DM)
dichroic mirror (OBJ) objective lens (PMT)
photomultiplier detector (HAL) computer
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Two photon imaging system
(FL) femtosecond mode-locked laser (BC) beam
condenser (BE) beam expander (AOM) acusto-optic
modulator (RF) radio frequency generator System
of mirrors and diaphragms
RF
FL
BE
BC
AOM
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Laser as a light source
Light Amplification by the Stimulated Emission of
Radiation
Constructed on different principles wavelength
(tunable) 1P in IR 2P in in visible spectrum
Technical considerations pulse width in pulsing
lasers output power beam quality size cost power
consumption operating life
A laser for two photon microscopy tuning range
690 to over 1050 nanometers pulse widths 100
femtoseconds Pulse frequency 80 MHz average power
2 W
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Why a pulsed laser?

• Average laser power at the specimen 100 mW,
  focused on a diffraction-limited spot
• Area of the spot 2 10-9 cm2
• Average laser power in the spot 0.1 W /(2
  10-9 cm2) 5 107 W cm-2
• Laser is on for 100 femtoseconds every 10
  nanoseconds therefore, the pulse duration to
  gap duration ratio 10-5
• Instantaneous power when laser is on 5 1012
  W cm-2

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Acusto-optic modulator
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Acusto-optic modulator
No RF signal
0-order beam
RF signal
diffraction
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Beam expander

• The radius of the spot at the focus
  (aberration-free microscope objective, at
  distance z)
• a(z) lf/pa0
• where f - focal length of the lens
• - the wavelength emitted by the laser
• a0 - the beam waist radius at the laser exit
  aperture

Reversed telescope
Beam expander increases a0 and allows to
concentrate beam
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Scanner
Focal plane Line scan
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Photomultiplier (PMT)
Photoelectron produced at photocathode by
photon Electrons acceleratedfrom one dynode to
another (voltage drop)
Quantum efficiency
Quantum efficiency - of photons which will
produce photoelectron (depends on thickness of
photocathode) 30 is good quantum efficiency
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Parameters of PMT
Gain depends on the number of dynodes and
voltage Dark current (thermal emissions of
electrons from the photocathode, leakage current
between dynodes, stray high-energy
radiation) Spectral sensitivity depends on the
chemical composition of the photocathode
gallium-arsenide elements from 300 to 800 nm
not uniformly sensitive
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Epi and trans-fluorescence
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Second harmonic generation and transmitted
fluorescence
810 nm
810 nm
405 nm
500 nm
SHG
Transmitted fluorescence
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Second harmonic generation
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Second harmonic generation and fluorescence
imaging
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Second harmonic generation and fluorescence image
of C.elegance
SHG and fluorescence images of C.elegance
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Computers
Specialized computer
Computer with user interface
Scanner
PMTs
Scanning control Image reconstruction
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Computer software
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Imaging laboratory
CCD
Scanners Ext. PMTs
Microscope
Electrophysiologymonitors
Imagingmonitors
Manipulators
Remote controls, keyboards
Antivibrationtable