Download A laser for two photon microscopy

Основы оптического
имиджинга в нейронауках
Алексей Васильевич
Семьянов
History
Santiago Ramón y Cajal
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Staining method (Golgi)
Development of precise optics
History
Electrode based techniques dominate
Extracellular 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
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
Principle of fluorescence measurment
Emission filter
STOP PASS
Emission-absorption spectrum of Fluo-4
Fluorescence measurement
Fluorescent microscope
Detector: CCD
(speed, sensitivity, resolution)
Up to 10 kHz
Light source: Mercury or Xenon Lamp
Spectrum
Stability
Filters
Charge-Coupled Devices (CCDs)
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
Charge-Coupled Devices (CCDs)
Laser scanning confocal microscopy
Detector: photomultiplier
Confocal microscope
Light source: laser
Power
Wavelength
Filters
Scanner
Principle of two photon excitation
Difference between single photon and two
photon imaging
Winfried Denk and Karel Svoboda
Neuron, Vol. 18, 351–357, March, 1997
Single photon and two photon excitation in florescent
media
Single photon and two photon excitation in florescent
media
Two-photon excitation requires IR laser
Scattering ~ (wavelength)-4
Visible light
Infrared light
IR penetrates tissue much deeper
Advantages of two photon imaging
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No out-of-focus fluorescence
Better in depth resolution
Less photobleaching of the dye
Less photodamage of the dye
Less phototoxicity for the tissue
Limitations of multiphoton imaging
1.
Two photon imaging has depth limit
out of focus light (background) > 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
Imaging laboratory
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
Two photon imaging system
RF
FL
BE
BC
AOM
(FL) femtosecond
mode-locked laser
(BC) beam condenser
(BE) beam expander
(AOM) acusto-optic
modulator
(RF) radio frequency
generator
System of mirrors and
diaphragms
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
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
pulse width in pulsing lasers
output power
beam quality
size
cost
power consumption
operating life
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
1.2
• 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
1.0
0.8
Power
• Average laser power in the spot
= 0.1 W /(2 × 10−9 cm2)
= 5 × 107 W cm−2
Instantaneous
0.6
0.4
0.2
Average
0.0
0
1
2
3
Time
4
5
Acusto-optic modulator
Acusto-optic modulator
No RF signal
0-order beam
RF signal
diffraction
Beam expander
Reversed telescope
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
l- the wavelength emitted by the laser
a0 - the beam waist radius at the laser exit
aperture
Beam expander increases a0 and allows to concentrate beam
Scanner
Focal plane
Line scan
Photomultiplier (PMT)
Photoelectron – produced
at photocathode by photon
Electrons accelerated
from 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
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
Epi and trans-fluorescence
Second harmonic generation and transmitted
fluorescence
810 nm
500 nm
Transmitted fluorescence
810 nm
405 nm
SHG
Second harmonic generation
Second harmonic generation and fluorescence imaging
Second harmonic generation and fluorescence
image of C.elegance
SHG and fluorescence images of C.elegance
Computers
Specialized computer
Scanner
PMTs
Scanning control
Image reconstruction
Computer with user interface
Computer software
Imaging laboratory
CCD
Microscope
Imaging
monitors
Electrophysiology
monitors
Manipulators
Remote controls,
keyboards
Antivibration
table
Scanners
Ext. PMTs