1. Two-Photon fluorescence Light Microscopy
In The Name of GOD
An Introduction to:
Two-Photon fluorescence Light Microscopy
Nanophotonics Course Research
Dr S.M.Hamidi
Mohammad Reza Sharifimehr
Laser and Plasma Research Institute-SBU
April 23,2013.
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2. Two-Photon fluorescence Light Microscopy
Outline
An Introduction to Microscopy Classes
A brief history of TPA
Nature of Two-Photon Absorption (TPA)
Difference between TPA and SHG
Comparison of Two-Photon Absorption and One-Photon Absorption
The order of required intensity for Two-Photon Excitation (TPE)
Design of a Two-Photon Microscope
Conventional Confocal and Two-Photon light microscopy
Advantages and Disadvantages of TPA
Recent TPA applications
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3. An Example of Microscopy
Scanning electron microscope (SEM) image of pollen
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4. Microscopy classes Microscopy techniques has developed since 1590!
18th century microscopes
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5. Two-Photon fluorescence Light Microscopy
Microscopy classes
Microscopes can be separated into several different classes. One grouping is based on what interacts with the sample to generate the image
Microscopy
Optical (Photon) Microscopy
(EM wave interaction with sample)
Electron Microscopy
(Electron wave interaction with sample)
Scanning probe Microscopy
(force, voltage, current, … measurement, using a cantilever)
Other types
Common techniques
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6. Two-Photon fluorescence Light Microscopy
Microscopy classes
Optical (Photon) Microscopy
(EM wave interaction with sample)
White Light Microscopy
Fluorescence Microscopy
Dark field microscopy
Electron Microscopy
Bright field microscopy
(Electron wave interaction with sample)
Polarized light microscopy
Phase contrast microscopy
Dispersion staining microscopy
Microscopy
Scanning probe Microscopy
Oblique illumination microscopy
(force, voltage, current, … measurement, using a cantilever)
Other types
Confocal microscopy
Two-photon excitation microscopy
Light sheet fluorescence microscopy
Common techniques
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7. Two-Photon fluorescence Light Microscopy
Microscopy classes
Optical (Photon) Microscopy
(EM wave interaction with sample)
Electron Microscopy
Transmission Electron Microscope (TEM)
(Electron wave interaction with sample)
Scanning Electron Microscope (SEM)
Dark Field Microscopy
Microscopy
Scanning probe Microscopy
(force, voltage, current, … measurement, using a cantilever)
Other types
Common techniques
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8. Two-Photon fluorescence Light Microscopy
Microscopy classes
Optical (Photon) Microscopy
(EM wave interaction with sample)
Atomic Force Microscopy (AFM)
Electrostatic Force Microscopy (EFM)
Electron Microscopy
Scanning Tunneling Microscopy (STM)
(Electron wave interaction with sample)
Scanning Thermal Microscopy (SThM)
Scanning Capacitance Microscopy (SCM)
Microscopy
Scanning probe Microscopy
Scanning Near-Field Optical Microscopy
(SNOM)
Near-Field Scanning Optical Microscopy
(NSOM)
(force, voltage, current, … measurement, using a cantilever)
Other types
Common techniques
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9. Two-Photon fluorescence Light Microscopy
Microscopy classes
Optical (Photon) Microscopy
(EM wave interaction with sample)
Electron Microscopy
(Electron wave interaction with sample)
Microscopy
Scanning probe Microscopy
(force, voltage, current, … measurement, using a cantilever)
Other types
X-ray microscopy
Scanning acoustic microscopes
Ultrasonic Force Microscopy (UFM)
Common techniques
digital holographic microscopy (DHM)
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10. Two-Photon fluorescence Light Microscopy
Microscopy classes
Optical (Photon) Microscopy
(EM wave interaction with sample)
Electron Microscopy
(Electron wave interaction with sample)
Microscopy
Scanning probe Microscopy
(force, voltage, current, … measurement, using a cantilever)
Other types
Microscope Image Processing
Digital Microscopy (CCD+Microscope)
Multifocal Plane Microscopy
(Multiplane Microscopy)
Common techniques
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11. An Example of Fluorescence Microscopy
The microtubules are red
DNA is stained blue
A protein called INCENP is green
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12. An Example of Fluorescence Microscopy+
fluorescent imaging of the human cancer cell
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13. Fluorescence Microscopy
Schematic of a fluorescence microscope
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14. A brief history of Two-photon fluorescence light microscopy:
1929
Maria Goppert -Mayer- Theoretical basis of two-photon excitation was established
1963
Kaiser and Garret - two-photon excitation was verified experimentally
1990
Denk et al. - The invention of two-photon fluorescence light microscopy
Theory --> Experiment --> Application
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15. A brief history of Two-photon fluorescence light microscopy:
Milestones relevant to the development of two-photon microscopy
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16. Two-Photon Absorption and Fluorescence Definition
The familiar one-photon fluorescence process involves exciting a fluorophore from the electronic ground state to an excited state by a single photon. This process typically requires photons in the ultraviolet or blue/green spectral range. However, the same excitation process can be generated by the simultaneous absorption of two less energetic photons (typically in the infrared spectral range) under sufficiently intense laser illumination. This nonlinear process can occur if the sum of the energies of the two photons is greater than the energy gap between the molecule’s ground and excited states. Under sufficiently intense excitation, three-photon and higher-photon excitation is also possible and deep UV microscopy based on these processes has been developed.
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17. Two Photon Absorption (TPA) Process
Jablonski diagram of one-photon (a) and two-photon (b) excitation, which occurs as fluorophores are excited from the ground state to the first electronic states. One-photon excitation occurs through the absorption of a single photon. Two-photon excitation occurs through the absorption of two lower-energy photons via short-lived intermediate states. After either excitation process, the fluorophore relaxes to the lowest energy level of the first excited electronic states via vibrational processes. The subsequent fluorescence emission process for both relaxation modes is the same.
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18. Difference between TPA and SHG
Two-Photon Excitation (TPE) is different from SHG, because in SHG the optical output is fixed at 2ω and is a very sharp line at 2ω, related to the line width only of the fundamental at frequency ω. In the TPE, ω3 is a higher frequency compared to either ω1 or ω2, but it is not 2ω1, 2ω2, or ω1 + ω2. In most cases, ω3 < (ω1+ ω2).
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19. Two-Photon fluorescence Light Microscopy
TPA Equations
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20. Two (Multi)-Photon Absorption versus One-Photon Absorption
One-photon and two-photon excitation are fundamentally different quantum-mechanical processes and have very different selection rules.
A fluorophore that is one-photon active at wavelength λ can often be excited by two photons of twice the wavelength (2λ).
A fluorophore’s emission spectrum, is independent of the excitation mechanism, since the molecule relaxes to the same excited state through vibrational mechanisms before emission.
A fluorophore’s two-photon excitation spectrum scaled to half the wavelength is typically not equivalent to its one-photon excitation spectrum. Further, fluorophores designed for one-photon excitation are not necessarily optimized for good two-photon absorption characteristics (such as σ).
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21. Two (Multi)-Photon Absorption versus One-Photon Absorption
Comparison of one-photon (broken lines) and three-photon (solid lines) fluorescence excitation
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22. Other differences between one-photon and two-photon excitation
For a spatially uniform specimen, fluorescence signals are generated equally from each z-section above and below the focal plane for one-photon excitation. In contrast, over 80% of the total fluorescence signal can be confined to a region 1µm thick about the focal point using two-photon excitation. This property is a base for depth discrimination.
A demonstration of the localization of two-photon excitation volume
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23. Two Photon Absorption (TPA) Process
Question:
What’s the order of required intensity for two-photon excitation?
Answer:
A high-radiance light source on the order of is required for efficient two-photon excitation.
High repetition rate (100 MHz), ultrafast (femtosecond or picosecond pulse widths) lasers, such as titanium–sapphire and Nd:YLF lasers, are the most widely used light sources.
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24. Confocal versus Two-Photon Light Microscopy
Conventional Light microscope --> Confocal Microscopy (1960s) --> Two-Photon Microscopy (1990s) --> 3D Imaging
Confocal microscopy is a technique very similar to two-photon microscopy.
The resolution of a microscope system scales inversely with the wavelength of light used. For a given fluorophore, two-photon excitation requires the use of excitation at twice the one-photon wavelength, resulting in approximately half the resolution.
Two-photon excitation wavelengths are typically about twice the one-photon excitation wavelengths. This wide separation between excitation and emission spectrum ensures that the excitation light and the Raman scattering can be rejected while filtering out a minimum of fluorescence photons.
Near-infrared radiation used in two-photon excitation has orders of magnitude less absorption in biological specimens than UV or blue-green light. The attenuation of excitation light from scattering is also reduced, as the scattering cross-section decreases with increasing wavelength.
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25. Confocal versus Two-Photon Light Microscopy
Compared with confocal microscopy operating in the UV or blue-green excitation wavelengths, two-photon microscopy minimizes photobleaching and photodamage. The reduction in the photodamage volume results in a dramatic increase in viability of biological specimens.
Confocal microscopy obtains 3D resolution by limiting the observation volume, whereas two-photon microscopy limits the excitation volume.
The reduction in the photodamage volume results in a dramatic increase in viability of biological specimens.
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26. Typical two-photon microscopy design
Galvo Mirrors ?
Barrier filter
A schematic drawing of typical components in a two-photon microscope. This system typically consists of a high-peak-power pulsed laser, a high-throughput scanning microscope and high-sensitivity detection circuitry.
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27. TPA Imaging Disadvantages
A critical component in a two-photon microscope is its light source and very kind of light sources can’t be use in this method.
A High-sensitivity detection electronics, such as single-photon counting circuitry, is needed to ensure maximal detection efficiency.
Photodamage maybe caused by dielectric breakdown, resulting from the intense electromagnetic field of the femtosecond laser pulses.
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28. Concept --> Experiment --> Application
My Experiment
Concept --> Experiment --> Application
My Experiment
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29. Typical two-photon microscopy-Image Construction
3D Images are constructed by raster scanning the fluorescent volume in three dimensions using a galvanometer driven x–y scanner and a piezo-objective z-driver.
A two-photon laser-scanning fluorescence microscopy 3D image of sulphorhodamine-stained human epidermis in vitro.
A voxelgram generated from a two-photon image stack of a GFP-labeled cortical pyramidal neuron.
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30. Two-Photon fluorescence Light Microscopy
TPA microscopes
Carl Zeiss (over 160 years): Laser scanning microscope for multiphoton imaging
LSM 710 NLO & LSM 780 NLO
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31. TPA microscopes scanning microscope for multiphoton imaging Movie 1
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32. Recent TPA Application
- High-speed 3D printing
- Two-Photon Microscopy for 4D Imaging of Living Neurons
- 3D Photonic crystals Fabrication using Two-Photon lithography
- Single Molecule Detection
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33. Recent TPA Application
Two-Photon Microscopy for 4D Imaging of Living Neurons
Movies 2
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34. Recent TPA Application
3D Photonic crystals Fabrication using Two-Photon lithography
Tightly focused in a photosensitive material, femtosecond laser pulses initiate two-photon polymerization and produce structures with a resolution down to 200 nm.
SEM image of a photonic crystal fabricated by two-photon lithography. From Cumpston et al. (1999)
SEM image of structures produced by two-photon polymerization of Deso-Bond A resolution of ~200 nm was achieved.
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35. Recent TPA Application
High-speed 3D printing of tiny objects:
A race car model no larger than a grain of sand, created using the new high-speed two-photon lithography process
A model of St. Stephen's Cathedral, Vienna, created using the new high-speed two-photon lithography process
A model of London's Tower Bridge, created using the new high-speed two-photon lithography process
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36. Recent TPA Application
Single Molecule Detection
Single Molecules and Nanotechnology- R. Rigler-2008, Springer:
Single emitting molecules are currently providing a new window into nanoscale systems ranging from biology to materials science. The amount of information that can be extracted from each single molecule depends upon the specific photophysical properties of the fluorophore and how these properties are affected by the nearby environment.
Two-Photon Fluorescence scanning confocal image of single DCDHF-6 molecules in a PMMA film.
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37. Recent TPA Application
Single-molecule spectroscopy has two requirements:
1-There is only one molecule present in the volume probed by the light source. This condition is met by using appropriate dilution together with microscopic techniques to probe a small volume.
2-The signal-to-noise (SNR) ratio for the single-molecule signal is sufficiently greater than unity for a reasonable averaging time to provide adequate sensitivity. For this purpose, large absorption cross sections, high photostability, operation below saturation of absorption, and (in the case of fluorescence detection) a high fluorescence quantum yield are needed.
Fluorescence NSOM images of single molecules.
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38. We need a powerful software for Image processing
References
We need a powerful software for Image processing
Movies 3
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39. Two-Photon fluorescence Light Microscopy
References
[1] Topics in Fluorescence Spectroscopy- Volume 5- Nonlinear and Two-Photon-Induced Fluorescence-Joseph R. Lakowicz-kluwer academic publishers
[2] Two-Photon Fluorescence Light Microscopy-(2002),Peter TC So- ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group
[3] Single Molecules and Nanotechnology- R. Rigler-2008, Springer
[4] Multiphoton Fluorescence Light Microscopy -15th June 2012, Konstantinos Palikaras-John Wiley & Sons, Ltd
[5] Nanophotonics- Paras N. Prasad, 2004, John Wiley & Sons- Chapters 3, 9, 11
[6] Properties of single organic molecules on crystal surfaces, Peter Grütter, 2006-Imperial College Press
[7] Nonlinear and Two-Photon-Induced Fluorescence, JOSEPH R. LAKOWICZ, 2002 Kluwer Academic Publishers
[8] Photophysics of Molecular Materials, Guglielmo Lanzani, 2006 WILEY-VCH Verlag GmbH
[9] Photonics, Ralf Menzel, 2006 Springer
[10] Nonlinear Optics, second edition, Robert W..Boyd, 2003
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40. There is a big difference between knowing and understanding.
For example, you can “know” the sky is blue but “understanding” why it’s blue is so much more important.
Thanks
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