Two-Photon Microscopy for Nanoparticle Imaging in Porous Material

Christian Honsaker Dr. Chunqiang Li 2012-2013

Overview

● Nanoparticles

● Microscopes

● 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 risksImportant to understand how nanoparticles interact with the environment.

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

Nanoparticles

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

● Not limited by opticaldiffraction barrier

● Wavelength 1000xshorter than visible light

● Magnification 1000xlarger than optical microscope

Types

● Transmission Electron Microscope

● Scanning Electron 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 twomore 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

● Precise three-dimensional imaging by scanning multiple thin sections of a sample

● Reduces out-of-focus light

● Better resolution

Disadvantages

● Limited depth due to scattering of light

● Photobleaching and Phototoxic effects even when not being visualized

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

Two-Photon Microscopy

● Optical cross sectioning ability (i.e. 3D imaging)

● Reduced photobleaching and phototoxicity

● Increased penetration depth of samples from the excitation beam

● No out-of-focus fluorescence generated

○ Increased efficiency of fluorescence collection

● Cellular/Subcellular resolution

● In Vivo imaging

Reasons why we chose two-photon microscopy

Confocal Two-Photon

What are some research applications with a two-photon microscope?

Research Applications

Biological

● Sub-cellular/organelles

● Dynamic cellular imaging

● Imaging within intact organs

● Imaging whole organisms

Medical

● Alzheimer's Disease

● Detection of skin cancers

● Metabolic Disorders

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 objectiveTo study the uptake, transport, and accumulation of zinc oxide nanoparticles in porous material.

ZnO Nanoparticle Aggregate

SummaryUnderstand the optical principles behind the two-photon microscope, it's applications in research, and the role nanoparticles play in our everyday lives.

Thank You

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 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 materialare those of the author(s) and do not necessarily reflect the views of the National Science Foundation.