Light and tissue: 2. Two-photon, Optical Coherence Tomography, Photoacoustics

Transcription

1 Light and tissue: 2 Two-photon, Optical Coherence Tomography, Photoacoustics

2 Last lecture: Absorption and Scattering

3 Last lecture: Mean free path Mean free path Transport mean free path Ntziachristos (2010)

4 Outline Two-photon microscopy Optical coherence tomography Σ Photoacoustic tomography Summary

5 Two Photon Microscopy

6 Einstein and the Photoelectric Effect 1921

7 Fluorescence Excited state Ground state No Excita-on

8 Actually Excited state Virtual state Ground state Maria Göppert-Mayer Doctoral thesis: 1931 Nobel Prize: 1963 BUT this requires coincident absorption of two photons within a very short time: ~ 1 fs Probability I 2

9 Energy-time uncertainty principle ΔE Δt > h 4π Energy levels get uncertain ( blurred ) at short time intervals

10 How likely is that? Exposed to sunlight, a single molecule of Rhodamine absorbs about one green photon per second. Two photon absorption happens every 10 9 years

11 Pulses: concentrating photons in time Typical specs: 80MHz repe--on rate (12.5 ns interval), 100 fs pulse length, 2 W

12 Concentrating photons in space

13 I 2 dependence is very useful

14 No waste of emitted photons

15 Simple setup

16 Less out-of-focus excitation / bleaching

17 Greatly increased penetration depth surface 750 µm hgp://microscope.olympus- global.com/en/ga/product/fv1000mpe/

18 Structural imaging Trachtenberg et al 2002

19 Functional imaging 30% Dye injection Stosiek et al 2003 Relative fluorescence change 8x speed 0%

20 2P summary 2P Absorption is proportional to I 2 Requires specialized light sources (fs-pulsed Ti:sapphire laser) Much less out-of-focus excitation compared to confocal microscopy Longer excitation wavelengths penetrate deeper Less waste of emitted photons Imaging depth up to µm in brain tissue Can be used to study structures with sub-µm resolution in vivo Can be combined with functional dyes Limitations: Cost Penetration depth

21 Optical Coherence Tomography (OCT)

22 Interference For stable interference to occur, waves have to be coherent: Same wavelength, constant phase rela-onship

23 Coherence

24 Coherence length L λ 2 n Δλ Example: Bandwidth at 500 nm: L (in air): 1 nm 250µm nm 250mm

25 Temporal vs. spatial coherence 1 2 High coherence 3 4 Low spa-al and temporal coherence Spa-ally filtered by pinhole: High spa-al, low temporal coherence

26 Michelson interferometer λ/2 detector x s x r sample reference Detector Response x s - x r coherent source (narrow spectrum)

27 DEMO

28 Interferometry with low coherence low- coherence source (wide spectrum) reference Detector Response detector x r equals x s - x r x s sample Detector Response 0 x s - x r

29 Depth imaging Sample reflec-vity profile Detector response (ac part) Envelope of detector response posi-on in sample arm rela-ve posi-on of reference arm / -me rela-ve posi-on of reference arm / -me

30 Optical coherence tomography Usually IR Time domain OCT. Other variants of OCT (frequency domain, spectral domain) can be derived from this basic idea

31 OCT for in vivo eye imaging Cornea Sclera Aqueous Iris IzaG, et. al., Arch. Ophthalmol. 112: , Lens Log Reflection 4 mm Vitreous Fovea Choroid RNFL Optic Disk 250 µm Hee, et al., Arch. Ophthalmol. 113: , Sclera Log Reflection 250 µm

32 Human endoscopic OCT Esophagus Stomach Small Intes5ne Colon Rectum

33 OCT summary Interference Coherence length OCT: Depth sectioning based on short coherence length Contrast: Reflectance Interference make is highly selective for ballistic photons Resolution: 10 µm Penetration depth: 1-3 mm Limitations: Speckles Resolution No fluorescence

34 Photoacoustic imaging

35 The Photoacoustic Effect Bell, 1880

36 Photoacoustic tomography Ultrasound scagers much less in -ssues than light

37 Photoacoustic tomography (PAT) Op-cal Imaging Lab, WUSTL

38 Imaging oxygen saturation Op-cal Imaging Lab, WUSTL

39 Multispectral Optoacoustic Tomography (MSOT) Ntziachristos (2010)

40 Photoacoustic summary Photoacoustic effect Signal: Absorption-induced thermal expansion Contrast: Absorption Resolution: like ultrasound (usually µm) Penetration depth: like ultrasound (up to 10 cm and more) Limitations: Absorption contrast requires high concentrations No fluorescence Low Resolution

41 Comparison (fill in) Resolu-on 1 µm 10 µm 100 µm 100 µm (MFP) 1 mm (TMFP) 10 mm Max penetra-on depth 2P, OCT, Confocal, PAT