Основы оптического имиджинга в нейронауках

1. Основы оптического имиджинга в нейронауках Алексей Васильевич Семьянов

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

3. History Electrode based techniques dominateExtracellular electrodes, patch clamp, sharp electrode Calcium indicators developed The principle of confocal imagingwas 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 microscopywas pioneered by Winfried Denk in the lab of Watt W. Webb at Cornell University in 1990- all light is taken: no pinhole Winfried Denk

4. 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

5. Emission filter STOP PASS Principle of fluorescence measurment Emission-absorption spectrum of Fluo-4

6. Fluorescence measurement Detector: CCD (speed, sensitivity, resolution) Up to 10 kHz Light source: Mercury or Xenon Lamp Spectrum Stability Filters Fluorescent microscope

7. Charge-Coupled Devices (CCDs)

8. 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

9. Charge-Coupled Devices (CCDs)

10. Laser scanning confocal microscopy Detector: photomultiplier Light source: laser Power Wavelength Filters Scanner Confocal microscope

11. Principle of two photon excitation

12. Difference between single photon and two photon imaging Winfried Denk and Karel Svoboda Neuron, Vol. 18, 351–357, March, 1997

13. Single photon and two photon excitation in florescent media

14. Single photon and two photon excitation in florescent media

15. Scattering ~ (wavelength)-4 Visible light Infrared light Two-photon excitation requires IR laser IR penetrates tissue much deeper

16. 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

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

18. Imaging laboratory

19. 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

20. 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

21. 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

22. 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 femtosecondsevery 10 nanoseconds; therefore, the pulse duration to gap duration ratio = 10−5 • Instantaneous power when laser is on = 5 × 1012 W cm−2

23. Acusto-optic modulator

24. Acusto-optic modulator No RF signal 0-order beam RF signal diffraction

25. 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

26. Scanner Focal plane Line scan

27. 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

28. 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

29. Epi and trans-fluorescence

30. Second harmonic generation and transmitted fluorescence 810 nm 810 nm 405 nm 500 nm SHG Transmitted fluorescence

31. Second harmonic generation

32. Second harmonic generation and fluorescence imaging

33. Second harmonic generation and fluorescence image of C.elegance SHG and fluorescence images of C.elegance

34. Computers Specialized computer Computer with user interface Scanner PMTs Scanning control Image reconstruction

35. Computer software

36. Imaging laboratory CCD Scanners Ext. PMTs Microscope Electrophysiologymonitors Imagingmonitors Manipulators Remote controls, keyboards Antivibrationtable