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Two-Photon Microscopy for Nanoparticle Imaging in Porous Material

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Two-Photon Microscopy for Nanoparticle Imaging in Porous Material Two-Photon Microscopy for Nanoparticle Imaging in Porous Material Christian Honsaker Dr. Chunqiang Li 2012-2013 ● Nanoparticles ● Microscopes Overview ● Fluorescence ● Confocal vs Two-Photon ● Applications Nanoparticles What’s a nanoparticle? A particle with a typical size between 1 and 100 nanometers (nm). Nanoparticles Examples ● Fullerenes ● Nanotubes ● Silica Powder ● Quantum Dots ● Noble Metals Standard Gold Nanoparticles How are nanoparticles utilized? Nanoparticles Applications ● Agricultural Products ● Automotive Industry ● Ceramics ● Fuel Cell Materials ● Paints Why study nanoparticles? Nanoparticles Concerns ● Environmental ● Health ● Safety https://www.youtube.com/watch?v=Ggy9FuNHGvs Assess potential risks Important to understand how nanoparticles interact with the environment. Nanoparticles Example 1. End up in soil by various means 2. Absorbed by plant roots or reach ground water 3. Enter the food chain and impact the ecosystem How do we observe the transport of nanoparticles? Microscopes Are there different types of microscopes? Scanning Probe Microscope ● Uses fine probe that is scanned over a surface; rather than a beam of light or electrons ● Not restrained by the wavelength of light or electrons ● True 3D maps Electron Microscope ● Powerful magnification ● Applications in various scientific fields ● Destroys sample ○ Ex Vivo Electron Microscope Advantages Types ● Not limited by optical ● Transmission Electron diffraction barrier Microscope ● Scanning Electron ● Wavelength 1000x Microscope shorter than visible light ● Magnification 1000x larger than optical microscope Transmission Electron Microscope ● Electrons are emitted from hot cathode and accelerated by a potential difference ● Electrons are focused into a parallel beam using a condensing "lens" that uses magnetic fields ● Beam passes through two more magnetic lenses ● Final image projected onto fluorescent screen Optical Microscope ● Objective lens - forms real and enlarged image ● Eyepiece - acts as a simple magnifier and forms a final virtual image ● Fluorescence Microscopes ○ Confocal ○ Two-Photon What’s fluorescence? Fluorescence ● Molecule absorbs a photon to reach an excited level and drops back down to ground level in steps ● Emits a photon with lower energy and longer wavelength ● Fluorescence is the emission of light when a sample absorbs external electromagnetic radiation How do fluorescence microscopes work? Fluorescence Microscopes ● Light source tries to excite fluorophores to emit photons ● Fluorophores are the chemical compound responsible for fluorescence Confocal Two-Photon Confocal vs Two-Photon Confocal Microscope ● Single photons are excited and energy is absorbed ● An electron in the fluorophore jumps from the ground state to the excited state ● Fluorescence emission occurs at a longer wavelength than the excitation light Confocal Microscope Advantages from Pinhole Disadvantages ● Precise three-dimensional ● Limited depth due to imaging by scanning multiple scattering of light thin sections of a sample ● Photobleaching and Phototoxic effects even when ● Reduces out-of-focus light not being visualized ● Better resolution Two-Photon Microscope ● Two photons are excited and energy absorbed by a molecule simultaneously ● Each photon contributes one half of the total energy required to induce fluorescence ● Fluorescence emission occurs at a shorter wavelength than the excitation light ● Optical cross sectioning ability (i.e. 3D imaging) ● Reduced photobleaching and phototoxicity Two-Photon ● Increased penetration depth of samples from the excitation beam Microscopy ● No out-of-focus fluorescence generated Reasons why we chose two-photon microscopy ○ Increased efficiency of fluorescence collection ● Cellular/Subcellular resolution ● In Vivo imaging Confocal Two-Photon What are some research applications with a two- photon microscope? Research Applications Biological Medical ● Sub-cellular/organelles ● Alzheimer's Disease ● Dynamic cellular imaging ● Detection of skin cancers ● Imaging within intact organs ● Metabolic Disorders ● Imaging whole organisms Living and highly motile lymphoid cells deep within an intact isolated lymph node. Reticular fibers (red) and T cells (green) In vivo multiphoton laser scanning imaging of senile plaques Plaques (red) & blood vessels (green) Research objective To study the uptake, transport, and accumulation of zinc oxide nanoparticles in porous material. ZnO Nanoparticle Aggregate Summary Understand the optical principles behind the two-photon microscope, it's applications in research, and the role nanoparticles play in our everyday lives. Bibliography 1. Li, Chunqiang, et al. "Multiphoton Microscopy of Live Tissues with Ultraviolet Autofluorescence." IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS 16 (2010): 516-21. Print 2. Mertz, Jerome "Nonlinear Microscopy: New Techniques and Applications." ELSEVIER (2004): 610-4. Print. 3. Zhao, Lijuan, et al. "Effect of Surface Coating and Organic Matter on the Uptake of Ce02 NPs by Corn Plants Grown in Soil: Insight into Thank You the Uptake Mechanism." ELSEVIER (2012): 131-7. Print. 4. Beaurepaire, E., et al. "Ultra-deep two-photon fluorescence excitation in turbid media." ELSEVIER (2001): 25-29. Print. 5. Zipfel, Warren, et al. "Nonlinear magic: multiphoton microscopy in the biosciences." Nature (2003): 1369-1375. Print. 6. Li, Chunqiang, et al. "Imaging immune response of skin mast cells in vivo with two-photon microscopy." Proc. of SPIE (2012): Vol. 8207 1- 5. Print. This material is based upon work supported by the National Science Foundation under Grant Number DUE-1140469. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation..
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