Nanophotonics Course Research
Dr S.M.Hamidi
Mohammad Reza Sharifimehr
Laser and Plasma Research Institute-SBU
April 23,2013.
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Two-Photon fluorescence Light Microscopy
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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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Two-Photon fluorescence Light Microscopy
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Scanning electron microscope (SEM) image of pollen
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Two-Photon fluorescence Light Microscopy
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Microscopy techniques has developed since 1590!
18th century microscopes
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Two-Photon fluorescence Light Microscopy
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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
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Two-Photon fluorescence Light Microscopy
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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
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Two-Photon fluorescence Light Microscopy
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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
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Two-Photon fluorescence Light Microscopy
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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
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Two-Photon fluorescence Light Microscopy
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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
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digital holographic microscopy (DHM)
Two-Photon fluorescence Light Microscopy
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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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Two-Photon fluorescence Light Microscopy
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The microtubules are red
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DNA is stained blue
Two-Photon fluorescence Light Microscopy
A protein called INCENP is green
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+
+
fluorescent imaging of the human cancer cell
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Two-Photon fluorescence Light Microscopy
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Schematic of a fluorescence microscope
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Two-Photon fluorescence Light Microscopy
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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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Two-Photon fluorescence Light Microscopy
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Milestones relevant to the development of two-photon microscopy
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Two-Photon fluorescence Light Microscopy
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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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Two-Photon fluorescence Light Microscopy
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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.
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Two-Photon fluorescence Light Microscopy
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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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Two-Photon fluorescence Light Microscopy
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~
~
~
~
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

8
( )  2 2
nc
3
( 2)
ng
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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
 f (ng  2 )

2
m  ( mg   )
Two-Photon fluorescence Light Microscopy
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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 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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Two-Photon fluorescence Light Microscopy
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Comparison of one-photon (broken lines) and three-photon (solid lines) fluorescence excitation
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Two-Photon fluorescence Light Microscopy
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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 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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Two-Photon fluorescence Light Microscopy
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Question:
What’s the order of required intensity for two-photon excitation?
Answer:
10
A high-radiance light source on the order of 10
photon excitation.
 1012 w / cm 2 is required for efficient two-
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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Two-Photon fluorescence Light Microscopy
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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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Two-Photon fluorescence Light Microscopy
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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.
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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.
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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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Two-Photon fluorescence Light Microscopy
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Concept --> Experiment --> Application
My Experiment
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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 sulphorhodaminestained human epidermis in vitro.
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A voxelgram generated from a twophoton image stack of a GFP-labeled
cortical pyramidal neuron.
Two-Photon fluorescence Light Microscopy
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Carl Zeiss (over 160 years): Laser scanning microscope for multiphoton imaging
LSM 710 NLO & LSM 780 NLO
http://microscopy.zeiss.com
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scanning microscope for multiphoton imaging
Movie 1
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- Two-Photon Microscopy for 4D Imaging of Living Neurons
- 3D Photonic crystals Fabrication using Two-Photon lithography
- High-speed 3D printing
- Single Molecule Detection
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Two-Photon Microscopy for 4D Imaging of Living Neurons
Movies 2
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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.
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SEM image of a photonic crystal fabricated by twophoton lithography. From Cumpston et al. (1999)
Two-Photon fluorescence Light Microscopy
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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
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A model of London's Tower Bridge, created using the
new high-speed two-photon lithography process
Two-Photon fluorescence Light Microscopy
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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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Two-Photon fluorescence Light Microscopy
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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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We need a powerful software for Image processing
Movies 3
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[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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