PH880 Topics in Physics

Transcription

1 PH880 Topics in Physics Modern Optical Imaging (Fall 2010)

2 Overview of week 8 Monday Nonlinear Microscopy Wednesday No class (Mid term week)

3 Quantum Optics Electro- magnetic Optics Wave Optics Ray Optics Wave (Fourier) Optics: effective to deal with imaging, interference, holography, and so on. Electromagnetic Optics: mostcomplete treatment of light within classicaloptics Quantum optics: interaction of photons with atoms

4 Nonlinear Microscopy Nonlinear methods approaches whereby output intensity is proportional to I n, where I is the input intensity and n is the number of photons involved in the interaction. e.g. two photon absorption Optical Sectioning i Deeper penetration depths (~600 μmcompared to 50 μm) Reduced photodamage Rd Reduced dphotobleaching hi

5 Nonlinear Microscopy Incoherent nonlinear microscopy techniques: 2, 3, ormulti photon microscopy Based on molecular absorption Emission is isotropic and unpolarized Phase information is lost, thus Power [radiating molecules]. Coherent nonlinear microscopy techniques: Second harmonic generation (SHG), third harmonic generation (THG), coherent anti Stokes Raman scattering (CARS) microscopy Based on photon scattering Emission is directional and polarized Phase information is maintained: rigorously prescribed by a variety of factors including the excitation light phase and the geometric distribution of the radiating molecules

6 1PE fluorescence microscopy Excitation wavelengths (<400 nm) result in photodamage of biological sample (DNA, protein) Risk of photobleaching in entire volume No optical (axial) sectioning w/o confocal pinhole Limited penetration depth (~50 μm) into tissues (absorption fromh2o H2O, melanin, Hb)

7 Two photon excitation (2PE) M. Oheim et al. / Advanced Drug Delivery Reviews 58 (2006)

8 Two photon excitation (2PE) : optical sectioning Brad Amos/Science Photo Library, London

9 Detector configurations for multiphoton microscopy

10 Probability for Multi photon absorption Probability that a fluorophore at the center of focus absorb 2 photons (paraxial approx) NA p a σ2pe P Fp τ p 2 cλ ex 2 photon X section Average Power Repetition rate Pulsewidth 2 For optimal fluorescence generation and avoiding saturation, p a <1, assuming F p = 100 MHz, τ p =100 fs, and NA =1.4, <P> = 1 10mW,

11 Probability for Multi photon absorption For tightly focused beam, p a outside focal region falls off with z^( 2n), where z is the distance from the focal plane Spatial confinement & optical sectioning htm

12 Absorption cross section Units of Goppert Mayer [1 GM=10 58 m 4 s/photon] Difficult to predict from 1PE cross section Measured experimentally (challenging) σ 2PE Rule of thumb is that 2PE cross section is peaked at 2x 1PE λ

13 Some common fluorophore for Two photon

14 Advantages of nonlinear fluorescence excitation over confocal detection Problems in Confocal microscopy 2PE microscopy multiple lasers/illumination sources for multi color imaging Loss of contrast by absorption & scattering of excitation photons Loss of resolution due to out of focus fluorescence excitation ti Photobleaching and photodamage Light collection efficiency Limited depth penetration 2PEF absorption spectra are much broader than 1PEF absorption spectra Infrared light used in 2PEF is subject to less absorption and scattering than UV or visible light so that excitation penetrates more deeply into a sample. Multi photon excitation confines fluorescence excitation to a small volume at the focus of the objective. Photobleaching and photodamage are limited to the zone of 2PEF (focused position) Since 2PEF is localized to a point, no confocal pinhole is needed. No requirement for de scanning, less alignment sensitive. IR photons travel deeper into tissue with less scattering and absorption.

15 Scattering in deep tissue imaging In most biological tissues absorption of light is negligible compared to scattering, particularly in the NIR range. Elastic scattering depends on RI inhomogeneities, which are strong in tissue (cells are a heterogeneous mixture of molecules and supramolecular structures with varying molecular polarizabilities.) The strength of scattering is described by the 'mean free path' (l s ), the average distance between scattering events. For objects comparable in size to the wavelength (as in cells) scattering is directed mostly in the forward ddirection. i This can be quantified by the anisotropy parameter (g) or by the 'transport mean free path', l t = l s / (1 g), which is the distance after which 'direction memory' is lost. Helmchen F & Denk W (2005) Deep tissue two photon microscopy. Nature methods 2(12):

16 Scattering in deep tissue imaging Measurements in brain gray matter yielded values for l s of um at 630 nm in extracted tissue and of about 200 um at 800 nm in vivo. In nonlinear optical microscopy only ballistic (non scattered) photons contribute to signal generation in the focal volume. The ballistic power follows a Lambert Beer like exponential ildecline with ihimaging i depth z with length constant l s and surfacepower P O. Because of the quadraticintensity intensitydependence in 2PLSM, the fluorescence signal declines as Helmchen F & Denk W (2005) Deep tissue two photon microscopy. Nature methods 2(12):

17 Scattering in deep tissue imaging Helmchen F & Denk W (2005) Deep tissue two photon microscopy. Nature methods 2(12):

18 Functions to describe resolution Spatial domain Spatial frequency domain E f field Point Spread Function (PSF) Amplitude Transfer Function (ATF) a.k.a. pupil function hxyz (,, ) Hk (, k, k ) x y z Incoherent Point Spread Function (ipsf) Optical Transfer function (OTF) ensity Int hxyz (,, ) 2 H Hk ( x, ky, kz) Modulation Transfer function (MTF) H Hk ( x, ky, kz)

19 Functions to describe resolution ipsf hxyz (,, ) 2 OTF H Hk ( x, ky, kz) Carl Zeiss webpage

20 Resolution in widefield illumination Back focal plane Image plane * In Kohler illumination, BFP is conjugate to filament plane

21 Köhler illumination * In Kohler illumination, BFP is conjugate to filament plane Before, Kohler illumination, the critical illumination had been used, which project filament image to the sample plane the filament could be visible in the sample plane

22 Resolution in widefield illumination Back focal plane Condenser Image plane For conventional fluorescence microscopy, sample is uniformly illuminated by light source and resolution is primarily determined by objective Image formation is primarily expressed by detection ipsf: h obj ( x, y, z) 2

23 Resolution in confocal microscopy Back focal plane Condenser Confocal fluorescence microscopy: point illumination and point detection Resolution is determined by both the condenser and objective lens confocal (,, ) = cond (,, ) obj (,, ) h xyz h xyz h xyz

24 Resolution in two photon microscopy (w/o pinhole) Back focal plane Condenser For two photon fluorescence microscopy there are 2 independent excitation events ; pa is proportional to square of excitation intensity Resolution is determined by excitation only (,, ), (,, ), (,, ) PE cond cond cond (,, ) =, λ λ = 1, 1 h xyz h xyz h xyz h xyz 2

25 Resolution in two photon confocal microscopy (w/ pinhole) Back focal plane Condenser For two photon fluorescence microscopy there are 2 independent excitation events ; pa is proportional to square of excitation intensity Resolution is determined by excitation only PE confocal = cond obj h2 ( xyz,, ) h ( xyz,, ) h ( xyz,, )

26 Summary Widefield fluorescence Confocal microscopy 2PE (no pinhole) Optical slice thickness not definable (optical sectioning is not possible) 1.4nλ NA 2 Axial resolution 2nλ 1.4n 14nλλ 2.3n 23nλλ (approximated) 2 NA 2 2 NA NA Lateral resolution 0.61λ 0.4λ NA NA 0.7λ NA * The addition of a pinhole can enhance resolution but at a cost of signal loss

27 Application: in vivo two photon imaging of neo cortex Helmchen F & Denk W (2005) Deep tissue two photon microscopy. Nature methods 2(12):

28 Application: two photon microscopy of in vivo brain function Kherlopian et al. BMC Systems Biology :74 doi: /

29 Application: Two photon imaging of capillary blood flow in olfactory bulb glomeruli PNAS October 28, 2003 vol. 100 no

30 Overview of week 8 Monday Nonlinear Microscopy Wednesday No class (Mid term week) Overview of week 9 Monday: mid term paper due No class (Mid term week) Wednesday Second Harmonic Generation

31 Reading List (wk 7 day 2) 1. Helmchen F & Denk W (2005) Deep tissue two photon microscopy. Nature methods 2(12): So P, Dong C, Masters B, & Berland K (2000) Two Photon Excitation Fluorescence Microscopy. Annual lreview of Biomedical Engineering i 2(1):