Lecture 12 - Optoacoustic and Brillouin Microscopy PDF

BIOPHOTONICS LECTURE 12 – OPTOACOUSTIC AND BRILLOUIN MICROSCOPY Seite 1 Resolution und sample size of imaging technologies 10 cm 100000 MRT...

BIOPHOTONICS LECTURE 12 – OPTOACOUSTIC AND BRILLOUIN MICROSCOPY Seite 1 Resolution und sample size of imaging technologies 10 cm 100000 MRT CT ultrasound PET 110000 cm FMT 1 mm sample size 1000 OPT M T/PA 100 µm 100 PA multiphoton OCT 10 µm 10 confocal Super- resolution 1 µm1 1 2 3 1 µm 10 µm 100 4µm 5 1 mm resolution Seite 2 Optoacoustics is often used as tomography What is “Tomography”? greek τομή, tome, „cut“ and γράφειν, grafëin, „drawing/writing“ } sketch of tomography (wikipedia): upper left: volume below: selected sections at S1 and S2 upper right: projection P the imaging procedure provides sections (“cuts”) through the sample and thereby allows 3D imaging. Seite 3 Example of tomography: Computed Tomography Principle Reconstruct the interior of a specimen from parallel ray projections. Setup Seite 4 Example of tomography: Computed Tomography Principle Reconstruct the interior of a specimen from parallel ray projections. d ф 0 360 Setup ° Projection Data ° Seite 5 Example of tomography: Computed Tomography Principle Reconstruct the interior of a specimen from parallel ray projections. d ф 0 360 Setup ° Projection Data ° Digital Reconstruction Seite 6 Building a CT with visible light: SLOT: Scanning Laser Optical tomograph: A laser is scanned through the specimen and „projects“ the sample properties (absorption&scattering) allong one line (just like the X-ray do in CT) Laser D xy- scanner Photodetector Filter 1 R.-A.Lorbeer et al.: “Highly efficient 3D fluorescence microscopy with a scanning Seite 7 laser optical tomograph.”, Optics Express, Vol. 19, Issue 6, pp. 5419-5430 (2011) Animated setup showing fluorescence detection Laser D xy- scanner Photodetector Fluorescence Filter Seite 8 Lab setup and example of a large specimen Seite 9 Animated 3D Reconstruction of Cochlea Seite 10 Conversion of sound into light... First sketch of the photophone by Bell (inventor of the telephone) www.wikipedia.org Seite 11...and vice versa! www.wikipedia.org Seite 12 Pioneer of optoacoustics: Alexander Graham Bell - 1880 „...probably one of my greatest inventions, the photophone!!!“ from today’s point of view we’d says it’s the telephone!!! www.wikipedia.org Seite 13  known from Lasers in Medicine-Lecture: Photoablation is often accompanied by acoustiv phenomena! acoustic phenomena ‘Optoacoustics’ cavitation Seite 14 Pioneers of optoacoustics today Alexander Oraevsky Lihon Wang Vasilis Ntziachristos Tomowave Washington University Helmholtz München iThera Medical Seite 15  Photoacousting imaging/tomography – PAT How it works combination of light & sound The image on the right shows laser ablation of a model substance, the ejection of ablated material leads to formation of a shockwave/pressure wave (light spherical shape in the lower part) H. Lubatschowski, Dissertation Seite 16 Underlying mechanisms: Thermoelastic effects s = 3ÀDT × K A temperature rise in tissue leads to a thermoelastic expansion, which is proportional to the temperature rise and compression module (K: compression module) with: À = 6 K-1 K = 2,2*109 Nm-2 DT » 80°C è s » 300 bar (! ) in water in biological tissue s is 3 times lower (compared to pure water) due to the compression module but: detectable sound waves can be achieved already at ONLY: DT » 10mK Seite 17  Optoacoustic signal generation: absorbing absorbierende Gewebe- light distribution Lichtausbreitung Schicht layer sample probe pressure Druck- sensor Laser- absorption Absorption sensor laser puls pulse thermoelastic Absorption thermoelastische expansion optoacoustic optoakustische Expansion transient Transiente pressure pressure wave propagation Absorption Druckausbreitung detection Detektion Zeit time Seite 18  Excitation Photoacoustic and detection of thetomography optoacoustic effect Ultrasound$scaGers$much$less$in$-ssues$than$light$ Lecture Judkewitz, Caltech Seite 19 ging process and stores the data. Typical setup A laser is absorbed in the specimen, leading to thermoelastic expansion and consequently to a sound wave traveling out of the specimen, which is collected by a detector and used for image reconstruction Seite 20  Photoacoustic tomography (PAT) Example of a first demonstration in living animals: A laser is illuminating the head of a living mouse, embedded in a water tank, the transducer (acoustic wave detector) is rotated around the head Op-cal$Imaging$Lab,$WUSTL$ Seite 21  Stimulation of whiskers: blood dynamic/saturation in the brain measured by optoacoustics in a living animal! stimulation left stimulation right 2.0 2.0 (cm) (cm) 1.5 1.5 1.0 1.0 0.5 0.5 0 0 0.5 1.0 1.5 2.0 0 0 0.5 1.0 1.5 2.0 (cm) (cm) Min Differential absorption Max Min Differential absorption Max Nature Biotech. 21, 803 (2003). Seite 22  Photoacoustic angiography through the skull in vivo (rat) (A) Without (B) With ICG-PEG ICG-PEG (cm) (cm) Speckle free 0 1 2 3 0 1 2 3 Min Optical absorption Max Min Optical absorption Max High resolution: 60 mm High sensitivity: ~fmol (C) B – A (D) Open- skull photo (cm) 0 1 2 3 Min Differential absorption Max Optics Letters 8, 608 (2004). Seite 23 Reflection-mode Dark-field Confocal Photoacoustic Microscopy x Tunable laser Nd:YAG pump laser z y Photodiode Motor driver Translation Optical illumination Ultrasonic stages Amplifier transducer Conical lens Sample holder AD Mirror Base Computer Heater & Dual foci Annular illumination temperature with a dark center controller Sample Optics Letters 30, 625 (2005). Nature Biotech. 24, 848 (2006). Seite 24  Volumetric Imaging of Rat Microvasculature In Vivo Maximum amplitude projection onto the skin 1 mm Volume: 10 mm x 8 mm x 3 mm Optics Express 14, 9317 (2006). Seite 25  Molecular imaging Spectral imaging (multispectral optoacoustic tomography) Hb The spectral dependence of the absortion HbO2 is different for different substances GoldNR The distribution of a given substance AF750 (component) is estimated by unmixing images at several wavelengths Razansky D. et al. Multispectral photoacoustic imaging of fluorochromes in small animals. Optics Letters 32:2891-3 (2007) Seite 26 Multispectral detection at four wavelengths (578, 584, 590, and 596 nm) Total hemoglobin Oxygen saturation Arteries and veins 1 mm Change in oxygenation 1 Artery Imaged SO2 0.8 Vein 0.6 Appl. Phys. Lett. Hypoxia Normoxia Hyperoxia Physiological states 90, 053901 (2007). Seite 27  Imaging of Melanoma In Vivo Composite photoacoustic image B-scan image at 764 nm acquired at 584 and 764 nm Melanoma Photograph 1 mm Melanoma Melanoma Histology Melanoma Movie 1 mm Surface rendering Contrasts: x Vessel: 13 z y Skin surface Melanoma: 69 3 mm 8 mm Nature Biotech. 8 mm 24, 848 (2006). Seite 28 Since then lots of research work on Optoacoustic tomography Example of oxygen saturation measurements around a tumor (blood vessels can primarily be seen) Seite 29  Imaging of Human Palm In Vivo Photo Maximum amplitude projection onto the skin Skin surface 3 5 0.3 mm 1 2 4 6 7 0.13 mm B-scan image Skin surface Stratum corneum 2 3 4 6 7 1 5 1 mm Optical absorption Nature Biotech. 24, 848 (2006). Seite 30 Application of optoacoustics in humans? Mammography for breast tumor detection Alexander Oraevsky, Tomowave Seite 31 Good detection success in first trials!!! (Lasermedicine/Fairway Medical) X-ray Mammogram Tumors detected: 29/30 Seite 32 Ntziachristos group (TUM/Helmholtz) transgenic zebrafish, mCherry-labeled hindbrain region, 6 mm diameter Seite 33 Use of fluorescent proteins like GFP or YFP for contrast in optoacoustics!!! Usually we use the emitted light of a fluorophore for fluorescence microscopy!!! Here, we use the energy loss (leading to the Stokes shift) for contrast! (the emitted fluorescence light is not used) Razansky et al. 2009, Nature Photonics Seite 34 Imaging with 30µm resolution in a cm-sized sample!!! Razansky et al. 2009, Nature Photonics Seite 35 Imaging with 30µm resolution in a cm-sized sample!!! Razansky et al. 2009, Nature Photonics Seite 36 Now commercialized by this group: Optoacoustic tomography in the animal model MSOT Imaging – reconstruction of light distribution in tissue improves the resolution of the device Seite 37 asio #, A.A. Oraevsky* Competing company: TomoWave (by Oraevsky) at St. Louis, Missouri, USA 3D OA Imaging of Tissue Morphology s 2D Projections of 3D Images Showing Mouse Anatomy LOIS performance was validated in live mice showing physiologically relevant tissue structures through the entire mouse body. The system demonstrated its capability to display quantitatively Seite 38 Brillouin Microscopy Seite 39  Brillouin Microscopy Relies on scattering -> inelastic scattering (like CARS) -> kind of Doppler shift new contrast modality -> insight into mechanical/elastic properties of tissue Seite 40 Last lecture: Scattering & Raman (inelastic: CARS) What kind of scattering can occur? Elastic scattering Inelastic scattering } energy is not changing à no change in } energy is changing à change in wavelength } wavelength } Rayleigh scattering } Brillouin scattering } Mie scattering } Raman scattering E E E Stokes scattering Anti-Stokes scattering Seite 41 What is Brillouin Scattering? Brillouin scattering, named after Léon Brillouin, occurs when light, transmitted by a transparent carrier interacts with that carrier's time-&-space- periodic variations in refractive index. Seite 42  Order of magnitude of scattering processes Bergmann Schaefer „Optik“ (1993) Seite 43 The effect was demonstrated quite early in the 60ties!!! Seite 44 The order of magnitude of the effect is quite small (see X(3)) Seite 45 46 For the detection, high resolution spectrometers have to be used : Fabry-Perot Interferometers!!! Seite 46 Hard materials have a large Brillouin shift, however biological substances are hard to detect!!! 20 Seite 47 Pioneers of Brillouin Microscopy in Tissue (in soft biological substances): Andy Yun and Giuliano Scarcelli https://loop.frontiersin.org/people/275457/overview https://bioe.umd.edu/clark/faculty/199/Giuliano-Scarcelli 18.02.2019 Seite 48  How can the density and E-module be deduced? longitudinal acoustic wave The scattered signals interfere similar like in the Bragg-setup (X-ray diffraction in crystals) 18.02.2019 Seite 49 How signals in tissue are generated Prevedel, Nat. Meth. 2019 Seite 50 Vectorial view of Brillouin signal (energy) The Rayleigh peak (middle peak, elastic scattering) is of very high intensity, whereas the Brillouin peaks are much smaller. Seite 51  Central element of the setup from Scarcelli et al was the VIPA (virtually imaged phased array), a highly dispersive element, acting like a tilted etalon/ Fabry Perot Scarcelli 2007, Nature Photonics Seite 52  Resolving spectral signals in Brillouin Microscopy Prevedel et al., Nature Methods 2019 18.02.2019 Seite 53 First experimental results from Scarcelli 2007, Nature Photonics Scarcelli 2007, Nature Photonics Seite 54 Brillouin shift in an eye/lens tissue Central part of the lens at approx. 1mm depth is harder (larger Brillouin-shift) than the rest. Scarcelli 2007, Nature Photonics Seite 55  Results of Brillouin in living human eyes (lenses) The overall stiffness in human lenses seems not to change, however, a broader part is harder in older lenses. 18.02.2019 Seite 56  Resolving spectral signals in Brillouin Microscopy Prevedel et al., Nature Methods 2019 18.02.2019 Seite 57 Results of Brillouin microscopy § The results of Brillouin measurements are still up to discussion, as the values are acquired at one frequency and deducing the mechanic properties for a different regime. § Some research groups argue, the shift mostly reflects water content and refractive index § This field stays a very active field with probably several innovations to be made in the near future 18.02.2019 Seite 58  bulk modulu s c Δν B νL K=( )2 ρ θ 2 n cos( ) 2 ρ = (1+0.35C)10³ density Brillouin- refractive shift index protein concen- tration Seite 59