Two-Photon fluorescence Light Microscopy
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Two-Photon fluorescence Light Microscopy Mohammad Reza SharifimehrLaser and Plasma Research Institute-SBUApril 23,2013.Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.SharifimehrAn Introduction to:1/36In The Name of GOD
Nanophotonics Course ResearchDr S.M.Hamidi OutlineTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr2/36An Introduction to Microscopy ClassesNature of Two-Photon Absorption (TPA)Difference between TPA and SHGComparison of Two-Photon Absorption and One-Photon AbsorptionThe order of required intensity for Two-Photon Excitation (TPE)Design of a Two-Photon MicroscopeConventional Confocal and Two-Photon light microscopy Advantages and Disadvantages of TPARecent TPA applications A brief history of TPAAn Example of Microscopy
Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr3/36Scanning electron microscope (SEM) image of pollen
Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr4/36Microscopy techniques has developed since 1590!
18th century microscopesMicroscopy classesTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr5/36Microscopes can be separated into several different classes. One grouping is based on what interacts with the sample to generate the imageMicroscopyOptical (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 typesCommon techniques5Microscopy classesTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr6/36MicroscopyOptical (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 typesCommon techniquesDark field microscopyBright field microscopyPhase contrast microscopyDispersion staining microscopyOblique illumination microscopyPolarized light microscopyWhite Light MicroscopyFluorescence MicroscopyConfocal microscopyTwo-photon excitation microscopyLight sheet fluorescence microscopy6Microscopy classesTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr7/36MicroscopyOptical (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 typesCommon techniquesTransmission Electron Microscope (TEM)Scanning Electron Microscope (SEM)Dark Field Microscopy7Microscopy classesTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr8/36MicroscopyOptical (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 typesCommon techniquesAtomic Force Microscopy (AFM)Electrostatic Force Microscopy (EFM)Scanning Tunneling Microscopy (STM)Scanning Thermal Microscopy (SThM)Scanning Capacitance Microscopy (SCM)Scanning Near-Field Optical Microscopy(SNOM)Near-Field Scanning Optical Microscopy(NSOM)
8Microscopy classesTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr9/36MicroscopyOptical (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 typesCommon techniquesX-ray microscopyScanning acoustic microscopesUltrasonic Force Microscopy (UFM)digital holographic microscopy (DHM)9Microscopy classesTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr10/36MicroscopyOptical (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 typesCommon techniquesMicroscope Image ProcessingDigital Microscopy (CCD+Microscope)Multifocal Plane Microscopy (Multiplane Microscopy)10An Example of Fluorescence MicroscopyTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr11/36
The microtubules are red
DNA is stained blue
A protein called INCENP is greenAn Example of Fluorescence MicroscopyTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr12/36fluorescent imaging of the human cancer cell
++Fluorescence MicroscopyTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr13/36Schematic of a fluorescence microscope
A brief history of Two-photon fluorescence light microscopy:Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr14/361929Maria Goppert -Mayer- Theoretical basis of two-photon excitation was established 1963Kaiser and Garret - two-photon excitation was verified experimentally 1990Denk et al. - The invention of two-photon fluorescence light microscopyTheory --> Experiment --> ApplicationA brief history of Two-photon fluorescence light microscopy:Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr15/36
Milestones relevant to the development of two-photon microscopy Two-Photon Absorption and Fluorescence DefinitionTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr16/36The 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 molecules 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.Two Photon Absorption (TPA) ProcessTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr17/36
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.Difference between TPA and SHGTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr18/36Two-Photon Excitation (TPE) is different from SHG, because in SHG the optical output is fixed at 2 and is a very sharp line at 2, related to the line width only of the fundamental at frequency . In the TPE, 3 is a higher frequency compared to either 1 or 2, but it is not 21, 22, or 1 + 2. In most cases, 3 < (1+ 2).
TPA EquationsTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr19/36
Two (Multi)-Photon Absorption versus One-Photon Absorption Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr20/36A fluorophore that is one-photon active at wavelength can often be excited by two photons of twice the wavelength (2).One-photon and two-photon excitation are fundamentally different quantum-mechanical processes and have very different selection rules.A fluorophores 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 ).A fluorophores emission spectrum, is independent of the excitation mechanism, since the molecule relaxes to the same excited state through vibrational mechanisms before emission.Two (Multi)-Photon Absorption versus One-Photon Absorption Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr21/36Comparison of one-photon (broken lines) and three-photon (solid lines) fluorescence excitation
Other differences between one-photon and two-photon excitation
Two-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr22/36For 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 1m 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 volumeTwo Photon Absorption (TPA) ProcessTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr23/36Question:
Whats the order of required intensity for two-photon excitation?
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 titaniumsapphire and Nd:YLF lasers, are the most widely used light sources.Confocal versus Two-Photon Light MicroscopyTwo-Photon fluorescence Light Microscopy LAPRI-SBUM.R.Sharifimehr24/36Confocal microscopy is a technique very similar to two-photon microscopy.Conventional Light microscope --> Confocal Microscopy (1960s) --> Two-Photon Microscopy (1990s) --> 3D Imaging 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 an