History Santiago Ramn y Cajal Staining method Golgi

History Santiago Ramón y Cajal • • 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 k. Hz 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 • • • 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) 1 P in IR 2 P 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 m. W, focused on a diffraction-limited spot • Area of the spot = 2 × 10− 9 cm 2 • Average laser power in the spot = 0. 1 W /(2 × 10− 9 cm 2) = 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 modulator

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

Beam expander Reversed telescope The radius of the spot at the focus (aberration-free microscope objective, at distance z): a(z) = lf/pa 0 where f - focal length of the lens l- the wavelength emitted by the laser a 0 - the beam waist radius at the laser exit aperture Beam expander increases a 0 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 - % 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