Основы оптического Основы оптического имиджинга в нейронаукахимиджинга в нейронауках

Алексей Васильевич Алексей Васильевич СемьяновСемьянов

HistoryHistory

Santiago Ramón y Cajal

• Staining method (Golgi) • Development of precise optics

History 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

HistoryHistory

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 measurmentPrinciple of fluorescence measurment

Emission filter

STOP PASS

Emission-absorption spectrum ofEmission-absorption spectrum of Fluo-4 Fluo-4

Fluorescence measurementFluorescence 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)

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)Charge-Coupled Devices (CCDs)

Laser scanning confocal microscopyLaser scanning confocal microscopy

Confocal microscope

Detector: photomultiplier

Light source: laser

Power

Wavelength

Filters

Scanner

Principle of two photon excitationPrinciple of two photon excitation

Difference between single photon and two Difference between single photon and two photon imagingphoton imaging

Winfried Denk and Karel Svoboda

Neuron, Vol. 18, 351–357, March, 1997

Single photon and two photon excitation in florescent Single photon and two photon excitation in florescent mediamedia

Single photon and two photon excitation in florescent Single photon and two photon excitation in florescent mediamedia

Two-photon excitation requires IR laserTwo-photon excitation requires IR laser

Scattering ~ (wavelength)4

Visible light

Infrared light

IR penetrates tissue much deeper

Advantages of two photon imagingAdvantages 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

LimitationsLimitations of multiphoton imaging of multiphoton imaging

1. Two photon imaging has depth limit out of focus light (background) > 1000 m 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 laboratoryImaging laboratory

Two photon imaging systemTwo 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 systemTwo 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

FL

BC

AOM

RF

BE

Laser as a light sourceLaser as a light source

Constructed on different principles

wavelength (tunable) 1P in IR 2P in in visible spectrum Technical considerations

pulse width in pulsing lasersoutput powerbeam qualitysizecostpower consumptionoperating life

A laser for two photon microscopy:tuning range 690 to over 1050 nanometerspulse widths ~ 100 femtosecondsPulse frequency 80 MHzaverage power 2 W

Light Amplification by the Stimulated Emission of Radiation

Time

0 1 2 3 4 5

Pow

er

0.0

0.2

0.4

0.6

0.8

1.0

1.2Instantaneous

Average

Why a pulsed laser?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

Acusto-optic modulatorAcusto-optic modulator

Acusto-optic modulatorAcusto-optic modulator

No RF signal

RF signal

0-order beam

diffraction

Beam expanderBeam expander

The radius of the spot at the focus (aberration-free microscope objective, at distance z):

a(z) = f/a0

where f - focal length of the lensthe wavelength emitted by the laser

a0 - the beam waist radius at the laser exit aperture

Beam expander increases a0 and allows to concentrate beam

Reversed telescope

ScannerScanner

Focal plane

Line scan

Photomultiplier (PMT)Photomultiplier (PMT)

Quantum efficiency - % of photons which will produce photoelectron (depends on thickness of photocathode)

30% is good quantum efficiency

Photoelectron – produced

at photocathode by photon

Electrons acceleratedfrom one dynode to another

(voltage drop)

Quantum efficiency

Parameters of PMTParameters 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-fluorescenceEpi and trans-fluorescence

Second harmonic generation and transmitted Second harmonic generation and transmitted fluorescencefluorescence

810 nm

405 nm

SHG

810 nm

500 nm

Transmitted fluorescence

Second harmonic generationSecond harmonic generation

Second harmonic generation and fluorescence imagingSecond harmonic generation and fluorescence imaging

Second harmonic generation and fluorescence Second harmonic generation and fluorescence image of C.eleganceimage of C.elegance

SHG and fluorescence images of C.elegance

ComputersComputers

Scanner

PMTs

Specialized computer Computer with user interface

Scanning control

Image reconstruction

Computer softwareComputer software

Imaging laboratoryImaging laboratory

Imagingmonitors

Electrophysiologymonitors

Remote controls,

keyboards

Antivibrationtable

Manipulators

Microscope

CCD

Scanners

Ext. PMTs