In The Name of GOD :An Introduction to

In The Name of GOD :An Introduction to

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 LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 1/36 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 LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 2/36 An Example of Microscopy Scanning electron microscope (SEM) image of pollen

LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 3/36 Microscopy classes !Microscopy techniques has developed since 1590 18th century microscopes LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 4/36 Microscopy classes

Microscopes can be separated into several different classes. One grouping is based on what interacts with the sample to generate the image 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 Common techniques LAPRI-SBU Two-Photon fluorescence Light Microscopy

M.R.Sharifimehr 5/36 Microscopy classes Optical (Photon) Microscopy )EM wave interaction with sample( White Light Microscopy Dark field microscopy Electron Microscopy Bright field microscopy )Electron wave interaction with sample( Polarized light microscopy Phase contrast microscopy

Microscopy Scanning probe Microscopy )force, voltage, current, measurement, using a cantilever ( Dispersion staining microscopy Oblique illumination microscopy Fluorescence Microscopy Other types Confocal microscopy Two-photon excitation microscopy Light sheet fluorescence microscopy Common techniques LAPRI-SBU Two-Photon fluorescence Light Microscopy

M.R.Sharifimehr 6/36 Microscopy classes Optical (Photon) Microscopy )EM wave interaction with sample( Electron Microscopy )Electron wave interaction with sample( Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM) Microscopy Scanning probe Microscopy Dark Field Microscopy

)force, voltage, current, measurement, using a cantilever ( Other types Common techniques LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 7/36 Microscopy classes Optical (Photon) Microscopy )EM wave interaction with sample( Atomic Force Microscopy (AFM)

Electrostatic Force Microscopy (EFM) Electron Microscopy )Electron wave interaction with sample( Scanning Tunneling Microscopy (STM) Scanning Thermal Microscopy (SThM) Microscopy Scanning Capacitance Microscopy (SCM) Scanning probe Microscopy )force, voltage, current, measurement, using a cantilever ( Scanning Near-Field Optical Microscopy )SNOM( Near-Field Scanning Optical Microscopy ) NSOM( Other types

Common techniques LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 8/36 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 LAPRI-SBU digital holographic microscopy (DHM) Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 9/36 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

LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 10/36 An Example of Fluorescence Microscopy The microtubules are red LAPRI-SBU DNA is stained blue Two-Photon fluorescence Light Microscopy A protein called INCENP is green M.R.Sharifimehr

11/36 An Example of Fluorescence Microscopy + + fluorescent imaging of the human cancer cell LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 12/36 Fluorescence Microscopy Schematic of a fluorescence microscope LAPRI-SBU

Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 13/36 :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 LAPRI-SBU

Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 14/36 :A brief history of Two-photon fluorescence light microscopy Milestones relevant to the development of two-photon microscopy LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 15/36 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 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. LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 16/36 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. Twophoton 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. LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 17/36 Difference between TPA and SHG Two-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). LAPRI-SBU Two-Photon fluorescence Light Microscopy

M.R.Sharifimehr 18/36 TPA Equations ~ ~ ~ ~ P (t ) (1) E (t ) ( 2 ) E 2 (t ) (3) E 3 (t ) ... N l(,2m) I1 I 2 N l t ( 2) Rng

( 2) ( 2) Rng ng ( ) I 2 I 2 N total ( 2 ) I exc z ( 2) ng 3 8 ( ) 2 2

nc LAPRI-SBU 1 I ( z ) I 0 1 ( 2) N total I 0 z pn( 2 ) (t ) t dI I I 2 I 3 dz nc 2 I E 2

N m( 2,l) I1 I 2 N m t 2 nm mg m 2 ( ) f (ng 2 ) mg Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 19/36 Two (Multi)-Photon Absorption versus One-Photon Absorption One-photon and two-photon excitation are fundamentally different quantummechanical processes and have very different selection rules. A fluorophore that is one-photon active at wavelength can often be excited by two

photons of twice the wavelength (2). A fluorophores emission spectrum, is independent of the excitation mechanism, since the molecule relaxes to the same excited state through vibrational mechanisms before emission. 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 twophoton absorption characteristics (such as ).). LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 20/36 Two (Multi)-Photon Absorption versus One-Photon Absorption Comparison of one-photon (broken lines) and three-photon (solid lines) fluorescence excitation LAPRI-SBU

Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 21/36 Other differences between one-photon and two-photon excitation For a spatially uniform specimen, fluorescence signals are generated equally from each zsection 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 volume LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 22/36

Two Photon Absorption (TPA) Process Question: Whats the order of required intensity for two-photon excitation? Answer: A high-radiance light source on the order of 10 . photon excitation 10 1012 w / cm 2 is required for efficient two- 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 LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr

23/36 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. LAPRI-SBU

Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 24/36 Confocal versus Two-Photon Light Microscopy Compared with confocal microscopy operating in the UV or blue-green excitation wavelengths, twophoton 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 twophoton microscopy limits the excitation volume. The reduction in the photodamage volume results in a dramatic increase in viability of biological specimens. LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr

25/36 Typical two-photon microscopy design Barrier filter ? Galvo Mirrors A schematic drawing of typical components in a two-photon microscope. This system typically consists of a high-peakpower pulsed laser, a high-throughput scanning microscope and high-sensitivity detection circuitry. LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 26/36 TPA Imaging Disadvantages

A critical component in a two-photon microscope is its light source and very kind of light sources cant 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. LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 27/36 My Experiment Concept --> Experiment --> Application

My Experiment LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 35/36 Typical two-photon microscopy-Image Construction 3D Images are constructed by raster scanning the fluorescent volume in three dimensions using a galvanometer driven xy scanner and a piezo-objective z-driver. A two-photon laser-scanning fluorescence microscopy 3D image of sulphorhodaminestained human epidermis in vitro. LAPRI-SBU A voxelgram generated from a twophoton image stack of a GFP-labeled

cortical pyramidal neuron. Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 28/36 TPA microscopes Carl Zeiss (over 160 years): Laser scanning microscope for multiphoton imaging LSM 710 NLO & LSM 780 NLO http://microscopy.zeiss.com LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 29/36

TPA microscopes scanning microscope for multiphoton imaging Movie 1 LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 29/36 Recent TPA Application - Two-Photon Microscopy for 4D Imaging of Living Neurons - 3D Photonic crystals Fabrication using Two-Photon lithography - High-speed 3D printing - Single Molecule Detection

LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 30/36 Recent TPA Application Two-Photon Microscopy for 4D Imaging of Living Neurons Movies 2 LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr

31/36 Recent TPA Application 3D Photonic crystals Fabrication using Two-Photon lithography Tightly focused in a photosensitive material, femtosecond laser pulses initiate twophoton polymerization and produce structures with a resolution down to 200 nm. SEM image of structures produced by two-photon polymerization of Deso-Bond 956-105. A resolution of ~200 nm was achieved. LAPRI-SBU SEM image of a photonic crystal fabricated by twophoton lithography. From Cumpston et al. (1999) Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 31/36 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 LAPRI-SBU A model of London's Tower Bridge, created using the new high-speed two-photon lithography process Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 32/36 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. LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 33/36 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 LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 33/36 References We need a powerful software for Image processing Movies 3 LAPRI-SBU Two-Photon fluorescence Light Microscopy

M.R.Sharifimehr 34/36 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 Grtter, 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 LAPRI-SBU Two-Photon fluorescence Light Microscopy

M.R.Sharifimehr 34/36 There is a big difference between knowing and understanding. For example, you can know the sky is blue but understanding why its blue is so much more important. Thanks LAPRI-SBU Two-Photon fluorescence Light Microscopy M.R.Sharifimehr 36/36

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