1931-6

Preparatory School to the Winter College on Micro and Nano

Photonics for Life Sciences

4 - 8 February 2008

Introduction to biophotonics

Dan COJOC
Laboratorio TASC
INFM, Italy
 Introduction to Biophotonics

Dan Cojoc

CNR-Instituto Nazionale per la Fisica della Materia

Laboratorio Nazionale TASC Trieste, Italy

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Preparing this course I considered the following:
The audience is non-homogeneous (from physics and/or biology)
You have basic understanding of optics and biology (Preparatory lectures
on cell biology, diffraction theory, optical microscopy were provided)
Advanced lectures on Biophotonics topics will be presented at Winter
College next week
Lot of information can be found in internet
Not all the information can be included in a 3 h lecture

The goal of the course is to provide you a basic introduction to Biophotonics

with some history and examples and to open your appetite for this
fascinating field of science.
Motto:
“We should know and we will know”, David Hilbert, 1930
(expressing his disagreement with the
“ignoramus et ignorabimus / we do not know and will not know”
conclusion of Emil du Bois-Reymond, in his
“On the limits of our understanding of nature” of 1872
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 OUTLINE

What is Biophotonics ?

A bit of modern science history (with “Biophotonics” in mind)

Interaction of light with biomaterials

Imaging and spectroscopy of biomaterials

Various types of optical microscopy and contrast mechanisms

Laser micro and nano surgery of biomaterials

Optical tweezers, basics

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 What is Biophotonics?
Three definitions of Biophotonics among of many other:

Biophotonics is the science of generating and harnessing light (photons) to

image, detect and manipulate biological materials.

1 Biophotonics is used in BIOLOGY to probe for molecular mechanisms, function

and structure. It is used in MEDICINE to study tissue and blood at the macro
(large-scale) and micro (very small scale) organism level to detect, diagnose and
treat diseases in a way that are non-invasive to the body.
T. Husser IEEE-LEOS 2004

Interdisciplinary science studying the interaction of light with biological

2 material – where “light” includes all forms of radiant energy whose quantum
unit is the photon.
D. Matthews, Optik & Photonik June 2007 No. 2

3 The application of light and other forms of radiant energy to the life sciences.

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 Life Sciences

BioPhotonics
Subject areas:
Biochemistry, Cell Biology,
Developmental Biology, Ecology,
Photonics Evolution and Diversity of Life,
Functional and Comparative,
Morphology, Genetics and Disease,
Genetics and Molecular Biology
Immunology, Microbiology, Neuroscience,
Plant Science, Science and Society,
Structural Biology, Virology

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 The E-M Spectrum

http://enews.lbl.gov/MicroWorlds/teachers/alsposter.pdf

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 “Light” Spectrum

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 A bit of modern science history
(with “Biophotonics” in mind)

Some important discoveries in Microscopy

• 1590 – Hans and Zacharias Janssen make the first microscope.
• 1675 – Anton van Leeuwenhoek uses a simple microscope with only one lens to look at blood,
insects and many other objects. He was first to describe cells and bacteria through
microscope.
• 18th century – Several technical innovations make microscopes better and easier to handle,
which leads to microscopy becoming more and more popular among scientists (e.g. Lens
doublet reduces the chromatic abberations).
• 1878 – Ernst Abbe formulates a mathematical theory correlating resolution to the wavelength
of light. Abbes formula make calculations of maximum resolution in microscopes possible.
• 1903 – Richard Zsigmondy develops the ultramicroscope and is able to study objects below
the wavelength of light.
• 1932 – Frits Zernike invents the phase-contrast microscope that allows the study of colorless
and transparent biological materials.
• 1938 – Ernst Ruska develops the electron microscope. The ability to use electrons in
microscopy greatly improves the resolution and greatly expands the borders of exploration.
• 1981 – Gerd Binnig and Heinrich Rohrer invent the scanning tunneling microscope that gives
three-dimensional images of objects down to the atomic level.
http://nobelprize.org/
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 Ultramicroscopy
an example of instrumentation development, starting in 1900 …

Sample:
colloidal gold solution

NO

The sun beams fell upon a mirror S, were reflected from this
into the lens L, which brought them to the focus at 6, over
which was arranged a low power microscope.
(total magnification about 100 diameters)

N.B.: With ordinary illumination, even with the best

objectives, they were not perceptible.

1903 – Zsigmondy and Siedentopf develop the first ultramicroscope,

being able to study objects werll below the wavelength of light.

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 Ultramicroscopy: three-dimensional visualization of
… 2007
neuronal networks in the whole mouse brain

Working principle:
The sample is illuminated from two sides by a blue laser forming a thin sheet
of light. Fluorescent light is thus emitted only from a thin optical section and
collected by the objective lens. Stray light is blocked by a GFP filter and the
image is projected through the tube lens onto the camera target.

H.U. Dodt et al, Nature Methods 4 331 (2007)

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 Ultramicroscopy 2007 Results:
Brain imaging

Scale bar 1 mm Scale bar 500 um Scale bar 200 um Scale bar 5 um

(a) Surface of a whole mouse brain reconstructed from 550 optical sections. Both the GFP and
autofluorescence signal are imaged.
(b) Excised whole hippocampus reconstructed from 410 optical sections. Single cell bodies are
visible.
(c) 3D reconstruction of part of a whole hippocampus using 132 optical sections.
(d) 3D reconstruction of dendritic spines of CA1 pyramidal neurons obtained with a higher
resolution objective (20; NA, 0.4) in a whole hippocampus (430 optical sections, deconvolved).

H.U. Dodt et al, Nature Methods 4 331 (2007)

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 Major events in Cell Biology & Imaging

http://www.biology.arizona.edu/
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 Cell Theory

Old (1838) Scleiden & Schwann

1. The cell is the unit of structure, physiology, and organization in living things. OK
2. The cell retains a dual existence as a distinct entity and a building block in the
construction of organisms. OK
3. Cells form by free-cell formation, similar to the formation of crystals
(spontaneous generation). WRONG

Modern Cell Theory

1. All known living things are made up of cells.
2. The cell is structural & functional unit of all living things.
3. All cells come from pre-existing cells by division.
4. (Spontaneous Generation does not occur).
5. Cells contains hereditary information which is passed from cell to cell during cell
division.
6. All cells are basically the same in chemical composition.
7. All energy flow (metabolism & biochemistry) of life occurs within cells.

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 Discoveries in the field of X rays
(Nobel Prizes – incomplete list)

1901 - W. C. Röntgen: the discovery of X-rays.

1914 - M. von Laue: the discovery of X-rays by crystals.
1915 - W. H. Bragg and W. L. Bragg: the determination of crystal structures using X-rays.
1927 - A. H. Compton: revealing the particle nature of X-rays in scattering experiments on
electrons.
1936 - P. Debye: for determining molecular structures by X-ray diffraction in gases.
1962 - M. F. Perutz and J.C. Kendrew: determining the structure of hemoglobin and
myoglobin.
1962 - F. Crick, J. Watson and M. Wilkins: their discoveries concerning the molecular
structure of nucleic acids and its significance for information transfer in living material.
1964 - D. Crowfoot Hodgkin: the determination of the structure of penicillin and other
important biochemical substances.
1979 - A. M. Cormack and G. N. Hounsfield: the development of computerized
tomography.
1985 - H. A. Hauptman and J. Karle: for the development of direct methods for X-ray
crystallographic structure determination.
1988 - J. Deisenhofer, R. Huber and H. Michel: the determination of protein structures
crucial to photosynthesis.
http://nobelprize.org/
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 Milestones in DNA History

1869 Johann Friedrich Miescher identifies a weakly acidic substance of unknown function in the
nuclei of human white blood cells. This substance will later be called deoxyribonucleic acid, or
DNA.
1912 Physicist Sir William Henry Bragg, and his son, Sir William Lawrence Bragg, discover that
they can deduce the atomic structure of crystals from their X-ray diffraction patterns. This
scientiFic tool will be key in helping Watson and Crick determine DNA's structure.
1924 Microscope studies using stains for DNA and protein show that both substances are
present in chromosomes.
1928 Franklin Griffith, a British medical officer, discovers that genetic information can be
transferred from heat-killed bacteria cells to live ones. This phenomenon, called transformation,
provides the first evidence that the genetic material is a heat-stable chemical.
1944 Oswald Avery, and his colleagues Maclyn McCarty and Colin MacLeod, identify Griffith's
transforming agent as DNA. However, their discovery is greeted with skepticism, in part because
many scientists still believe that DNA is too simple a molecule to be the genetic material.
1949 Erwin Chargaff, a biochemist, reports that DNA composition is speciesspecific; that is, that
the amount of DNA and its nitrogenous bases varies from one species to another. In addition,
Chargaff finds that the amount of adenine equals the amount of thymine, and the amount of
guanine equals the amount of cytosine in DNA from every species.
1953 James Watson and Francis Crick discover the molecular structure of DNA.

http://nobelprize.org/
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Biophotonics has already played a central role in the most famous paper
in modern biology: J.D. Watson and F.H.C. Crick, Nature, 25 April 1953

1953 James Watson and Francis Crick

discover the molecular structure of DNA

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 See/Read/Think-on their Nobel Lectures:
http://nobelprize.org/nobel_prizes/medicine/laureates/1962/

The molecular configuration

On the Genetic Code
of nucleic acids
The involvement of RNA in
the synthesis of proteins

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“We wish to suggest a structure for the salt
of deoxyribose nucleic acid (DNA). This
structure has novel features which are of
considerable biological interest …
The two ribbons symbolize the two
We wish to put forward a radically different
phosphate - sugar chains, and the
structure for the salt of DNA. horizonal rods the pairs of bases
This strucure has two helical chains holding the chains together.

each coiled round the same axis.” The vertical line marks the fibre axis.

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 Fibre diagram of DNA
from B. coli.
Fibre axis vertical.

“The purpose of this communication

is to describe, in a preliminary way, Diffraction pattern of system of helices
some of the experimental evidence corresponding to structure of DNA.
for the ploy-nucleotide chain The squares of Bessel functions are plotted about 0 on
the equator and on the first, second third and fifth
configuration being helical, and layer lines for half of the nucleotide mass at 20 A.
existing in this form when in the A diameter and remainder distributed along a radius,
natural state.” the mass at a given radius being proportional to the
radius. About C on the tenth layer the similar
functions are plotted for an outer diameter of 12 A.
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 Life Sciences

BioPhotonics
Subject areas:
Biochemistry, Cell Biology,
Developmental Biology, Ecology,
Photonics Evolution and Diversity of Life,
Functional and Comparative,
Morphology, Genetics and Disease,
Genetics and Molecular Biology
Immunology, Microbiology, Neuroscience,
Plant Science, Science and Society,
Structural Biology, Virology

Investigated Analysis
Light source Detector
Subject Instrumentation

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 Interaction of light with biomaterials

Investigated
Light Biomaterial - Subject Detector
source

• Reflection • Transmission
• Absorption • Refraction
• Scattering

In complex materials, any combination of interactions are possible.

The exact nature of each process depends on the
physical and chemical structure of the biomaterial

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 Light-Induced Processes

Tissue light interaction

Radiative Nonradiative
Tissue autofluorescence
Fluorescence from various
constituents of the tissue

Photochemical Photoablation Photodisruption

•Excited state reaction •Direct breaking of cellular structure •Shockwave generation .at high
•Occurs even at low optical •Performed by high energy UV radiation pulse intensity
.
power density •Fragmentation and cutting of the
tissue by mechanical force of
shockwave

Thermal Plasma – induced ablation

•Induced by high intensity short pulse
•Light absorption converted to heat
•Dielectric breakdown creats ionized
•Can produce coagulation,
plasma that interacts with light yo
vaporization,carbonization and melting
produce ablation

Prasad, Introduction to Biophotonics, John Wiley & Sons © 2003

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 Light Scattering Processes

Light Scattering

Elastic Scattering Inelastic Scattering

Incident and scattered photons Incident and scattered photons
are of the same frequency are of different frequencies

Rayleigh scattering Mie scattering Brillouin Scattering Raman Scattering

•Scattering by particles of size •Scattering by particles of The difference in energy The difference in energy
smaller than the wavelength size comparable with . generates acoustic generates a vibrational
of light. •Weaker wavelength phonons excitation in the molecule
•Scattering depens on the -4 dependence: -X where
hence significantly more for 0.4 < X < 0.5.
blue than for red light. •Forward scattering
•Forward and backward preffered.
scattering are the same

Introduction to Biophotonics – Prasad - John Wiley & Sons © 2003

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 Imaging and spectroscopy of bio-materials

Scattering and absorption

Fluorescence
Optical Microscopy
Transmission and Absorption
Phase contrast
Confocal
Fluorescence Correlation Spectroscopy
Fluorescence Lifetime Imaging
Multiphoton Excitation
Spontaneous Raman Spectroscopy
Coherent Anti-Stokes Raman Scattering Mcroscopy (CARS)
Second Harmonic Generation Microscopy
Stimulated Emission Depletion, Image Deconvolution Microscopy
Nearfiled Optical Microscopy
Single Molecule Detection
Courtesy Prof. T. Huser (slides: 25-30, 32-35,4, 5152,55, 63,64,69, 85-88)
NSF Center for Biophotonics Science and Technology, University of California, Davis, USA

http://cbst.ucdavis.edu/education/short-courses/osabiophotonicscourse.pdf/download
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 Main sources for light loss in biological materials:
absorption and light scattering

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 Scattering in tissue

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 Absorption spectroscopy

routinely used technique in the biosciences

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 Application for absorption spectroscopy

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 Light microscopy enables live imaging at the cellular level

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 Some relevant length scales

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Various types of optical microscopy and contrast mechanisms

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 Transmission/absorption microscopy of biological samples
requires preparation and staining

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 The most important light microscopy imaging technique:
Phase contrast microscopy avoids staining

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 Phase contrast microscopy

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 Differential Interference Contrast (DIC) Microscopy

Nomarski DIC Sénarmont

In DIC microscopy, the spatial relationship and phase difference between ordinary
and extraordinary wavefronts is governed either by the position of the objective
prism (Nomarski DIC) or the relationship between the polarizer and a thin quartz
retardation plate in a de Sénarmont design.
http://www.microscopyu.com/tutorials/java/dic/wavefrontcomparison/index.html
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 Phase contrast - DIC microscopy

Phase Contrast Image HeLa Cell Culture DIC Image

HeLa cells have been cultured continuously for scientific use since they were first taken from
the tumor of a woman suffering from cervical cancer in the 1950s. They have been utilized for
many purposes, including the development of a polio vaccine, the pursuit of a cure for diseases
such as leukemia and cancer, and the study of the cellular effects of drugs and radiation.

http://www.microscopyu.com/galleries/dicphasecontrast/index.html
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 Polarization microscopy

http://www.microscopyu.com/articles/polarized/polarizedintro.html

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 Example: Identification of Asbestos Fibers

Asbestos is a generic name for a group of naturally occurring mineral fibers, which have been
widely used, for example, in insulating materials, brake pads and to reinforce concrete.
They can be harmful to health when inhaled and it is important that their presence in the
environment be easily identified. Samples are commonly screened using scanning electron
microscopy and x-ray microanalysis, but polarizing microscopy provides a quicker and easier
alternative that can be utilized to distinguish between asbestos and other fibers and between
the major types asbestos – chrysotile, crocidolite and amosite. With the use of crossed polars
it is possible to deduce the permitted vibration direction of the light as it passes through the
specimen, and with the whole wave plate, a determination of the slow and fast vibration
directions. Under crossed polars, chrysotile shows pale interference colors - low order whites
(a). When a full wave plate is added (530-560 nanometers), the colors are transformed. Aligned
Northeast-Southwest, the wave plate is additive and gives blue and yellow in the fiber (b).
When aligned Northwest-Southeast (c) the plate is subtracting to give a paler yellow fiber with
no blue. From this it is possible to deduce that the slow vibration direction is parallel with the
long axis of the fiber.
http://www.microscopyu.com/articles/polarized/polarizedintro.html
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 Fluorescence
is an electronic process

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Excitation / Emission spectroscopy of native fluorophores
in tissue (autofluorescence)

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 Fluorescence microscopy

More specific information requires Fluorescent probes, fluorochromes:

optical labeling: Molecules capable of undergoing electronic
Fluorescent dyes spread transitions that result in fluorescence.
throughout the entire optical spectrum Fluorophore:
The structural domain or specific region
of a molecule that is capable of
exhibiting fluorescence.

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 Fluorescence microscopy

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 DIC and fluorescence X-ray microscopy

Topography Chlorine Potassium Phosphorus

Scanning x-ray microscope picture of human liver cell. (a) topographic image. By
varying the incoming photon energy according to the absorption edge of selected
elements, and by inserting a fluorescence detector, we were able to map the
distribution of some elements inside the cell.

See also the lecture on X-ray diffractive elements by D. Cojoc next week

E. Di Fabrizio et al J. Electron Spectrosc. Relat. Phenom., 144-147, 957, 2005.

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Fluorescence resonance energy transfer (FRET) enables
measurements of molecular association/dissociation and lengths

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 Fluorescent dyes can be linked to biomolecules

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 Fluorescent proteins can be “fused” to other proteins
and co-expressed to intrinsically label proteins in cells

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 Fluorescence microscopy

Some drawbacks:

See the lectures next week for pros and cons

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Total intern reflection fluorescence (TIRF) microscopy excites
only a very narrow region right above the glass interface

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Confocal fluorescence microscopy efficiently suppresses
background signals and enables three-dimensional sectioning

Pinhole High axial resolution

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 Examples of the value of confocal fluorescence microscopy

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 Scanning confocal fluorescence microscopy

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Fluorescence correlation spectroscopy (FCS) measures
molecular diffusion times and association/dissociation rate

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Time-correlated single photon counting (TCSPC) enables
fast fluorescence lifetime measurements

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Pulsed laser sources combined with TCSPC enable lifetime analysis
per pixel in confocal images: fluorescence lifetime imaging

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 2-photon fluorescence microscopy

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 2-photon absorption

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 Multi-photon fluorescence microscopy

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 Multi-photon fluorescence microscopy

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 Multi-photon fluorescence microscopy

Living neurons

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 Raman spectroscopy

Drawback of Fluorescence: is limited by the need to label and photobleaching

-> Raman provides molecular information

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 Raman scattering is the interaction of photons and
intrinsic molecular bonds

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 Confocal Raman microscopy

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 Example: Raman spectroscopy can distinguidh
between ds DNA and protein-DNA compexes

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 An example for micro-Raman analysis of single cells
optically trapped cells

Real-time detection of hyperosmotic stress response in a

single Saccharomyces yeast cell

• the cell stress response is the reaction of a living cell to ambient

changes which are potentially harmful: for example, an increase in
temperature, pH, saline concentration, the presence of toxins

• the basic reaction at the fermentation process is the response of the

Saccharomyces yeast cells on the hyperosmotic stress

ethanol
C2H5OH
+ glucose =
glycerol
C3H8O3

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 Real-time detection of hyperosmotic stress response in a
single Saccharomyces yeast cell

10% pure ethanol

and glycerol in
water

difference spectrum of a
single yeast cell under
the hyperosmotic
pressure

30 min
after the experiment starts

Detection area 1 femtoL

Detectable concentration 100 atto_mol
Detectable number of molecules 108

G. P. Singh et al. Analyt. Chemistry, 2005, 77, 2564

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 An example for micro-Raman analysis of single cells:

The biochemical changes taking place during

the lag phase and G1 phase of a single
S. cerevisiae yeast cell

(A) The growth curve shows the phases of yeast cell

population.
(B) The yeast cell cycle diagram shows the phases of the
cell cycle. The abbreviations used for yeast cell cycle
stand for the standard terms: 1 M, mitosis; S, synthesis;
G1 and G2, gap phases. For our experiments cells that
did not have a bud were chosen.
(C) Upper row shows the budding of a yeast cell and its
subsequent growth while in the optical trap. Lower row
shows the same cell at a moment when it is released from
the optical trap

Singh G P, Creely C M, Volpe G, Grotsch H and Petrov D,

Anal. Chem. 77 2564–8, (2005)

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An example of combined Raman and fluorescence microscopy

Huang et. al., Biochemistry, V44, 10009-10019 (2005)

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 Advantages and limitations of spontaneous Raman imaging

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 Coherent Anti-Stokes Raman Scattering (CARS) microscopy
The newest member to the optical microscopy family

Two laser frequencies interact resonantly CARS signals are generated at wavelengths
with a specific molecular vibration: shorter than the excitation wavelengths
(anti-Stokes)

Why develop CARS ?

• Contrast signal based on vibrational characteristics, no need for fluorescent tagging
• CARS signal is at high frequency (lower wavelength) – no fluorescence interference
• Higher resolution
• More sensitive (stronger signals) than spontaneous Raman
• Microscopy – faster, more efficient imaging for real-time analysis
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 CARS microscopy setup
requires two synchronized ultrashort pulsed laser sources

Key components in a CARS setup

with two tunable synchronized
Lasers (5 psec, 80 MHz rep rate)

X.S. Xie et al., LNNL and Harvard

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 CARS microscopy: application to live cell imaging
Example 1

Lasers @ 853 nm (100 W) and 1135 nm (100 W)

tuned to Raman shift of 2913 cm-1 C-H vibration

Unstained live bacterial cells. Unstained live HeLa cells.

Signal due to cell membranes. Bright spots due to mitochondria.

Zumbusch et. al., Phys. Rev. Lett. V82, 4142 (1999)

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 Tracking trajectories of organelles inside single living cells
Example 2

X. L. Nan, E. O. Potma, and X. S. Xie, "Nonperturbative chemical imaging of organelle

transport in living cells with coherent anti-stokes Raman scattering microscopy," Biophys.
J. 91, 728-735 (2006).

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 Raman spectroscopy for single cell cancer detection

Spontaneous Raman spectra takes

2 minutes per cell !!!

cbst.ucdavis.edu/education/courses/winter-2007-ead-bim-289/chan_cars-lecture.pdf
Chan et. al., Biophys. Journal. V90, 648 (2006)
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 Future applications : CARS cytometry for rapid, labelless
cancer cell detection and sorting

Optical trapping combined with

CARS for faster spectral analysis

Trapped polystyrene bead

using two CARS beams

Potential solution for faster

chemical analysis of cells

Chan et. al., IEEE J. Sel. Topics. Quant. Elec. V11 858 (2005)
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 Other future directions for CARS
– Fiber based CARS for endoscopy
– CARS optical coherence tomography

See the CARS lectures by H. Rigneault next week

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 Second Harmonic Generation (SHG)
Another nonlinear optical imaging technique

SHG: Nonlinear process in which incident light at a

given frequency ( 1) is converted into light at twice
the frequency ( SHG = 2 1)
It occurs:
• only at beam focus (intense field), eliminating out-of-
plane signal and enabling sub-micron spatial resolution
• when intense light interacts with matter organized on
the scale of the wavelength with no inversion symmetry

To break centrosymmetry, SHG is allowed in:

• interfaces or surfaces (weak)[1]
• membranes with potentials (due to directional electrical field) [2,3]
• biophotonic crystal structures [4]
1. Y. R. Shen, Nature 337, 519 (1989)
2. L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, Opt. Lett. 25, 320 (2000).
3. G. Peleg, A. Lewis, M. Linial, and L. M. Loew, Proc. Natl. Acad. Sci . 96, 6700 (1999).
4. S. W. Chu, C. K. Sun, and B. L. Lin et. al., to be published in J. Microscopy (2002)

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 Example: SHG microscopy in collagen

The triple helical structure of collagen, the most abundant structural

protein in the cornea and the entire body, satisfies this requirement
SHG signal is uniquely sensitive to collagen structure

Optical Setup

P. Stoller et all Appl. Opt. 42 5209 (2003)

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 Example: SHG microscopy in collagen

Applications
Many pathological conditions are
Polarization microscope image characterized by abnormal collagen
of a section of rat tail tendon. structure
• Cornea: keratoconus, bullous
keratopathy, trauma
• Skin: melanoma, Ehlers-Danlos, scarring,
burns
• Cartilage: osteoarthritis, post-traumatic
degeneration
• Spinal column: intervertebral disk disease
Second-harmonic signal as a function of • Liver: fibrosis/cirrhosis
transverse position in a section of rat tail • Bone: osteogenesis imperfecta
tendon obtained with a 1um scan
resolution and the 40 objective with the
100-mm collimating lens
P. Stoller et all Appl. Opt. 42 5209 (2003)
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 Stimulated Emission Depletion (STED) Fluorescence Microscopy
Optical super-resolution 20 nanometers

The million dollar

microscope
http://www.nanowerk.com/news/newsid=1568.php
Working principle: Excite with a short pulse (10^-9s) Quickly follow with a second
synchronised pulse (shaped so that it illuminates a ring around the sample rather
than a spot) that stimulates emission.
Those molecules hit by the body of the ring are forced to dump their energy, while
those that sit in the hole in the middle of the ring are allowed to fluoresce as normal.
Careful tuning of the second pulse's intensity can narrow the size of the central hole
to (in theory) the size of a molecule. This ring-shaped pulse effectively provides a tiny
aperture for studying samples.
Observe the residual spontaneous emission by the detector.
S.W. Hell, and J. Wichmann
"Breaking the diffraction resolution limit by stimulated emission.“
Opt. Lett. 19 780 (1994).
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 STED

Neurofilaments in human neuroblastoma recorded in the confocal mode and with

STED after nonlinear deconvolution displaying a focal plane resolution of 20 to 30 nm
G. Donnert et al., Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
Stefan W. Hell, Far-Field Optical Nanoscopy, Review Science, 316 1153 (2007)

See the “Super-resolution” lectures by Volker Westphal

next week at the Winter College

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 Nonlinear structured-illumination microscopy
Wide-field fluorescence imaging with theoretically unlimited resolution

Principle: Resolution extension through the moire´ effect. If an unknown

sample structure (a) is multiplied by a known regular illumination pattern
(b), a beat pattern (moire´ fringes) will appear (c). The moire´ fringes occur at
the spatial difference frequencies between the pattern frequency and each
spatial frequency component of the sample structure and can be coarse
enough to observe through the microscope even if the original unknown
pattern is unresolvable. Otherwise-unobservable sample information can be
deduced from the fringes and computationally restored.
Mats G. L. Gustafsson, PNAS 102 13081 2005

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 Resolution extension by nonlinear structured illumination

(a)The region of frequency space that is observable by conventional microscopy

(b)An example of a sinusoidal illumination pattern.
(c)The illumination pattern has three frequency components: one at the origin (black), representing
the average intensity, and two at k1, representing the modulation (dark gray). These are also the
frequency components of the effective excitation under linear (i.e., nonsaturating) structured
illumination. Under conditions of saturation, or other nonlinear effects, a theoretically infinite
number of additional components appear in the effective excitation; the three lowest harmonics
are shown here (light gray).
(d)Observable regions for conventional microscopy (black), linear structured illumination (dark
gray), and nonlinear structured-illumination microscopy (light gray) based on those three lowest
harmonics.
(e)Corresponding observable regions if the procedure is repeated with other pattern orientations.
M.G.L. Gustafsson, PNAS 102 13081 2005
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 Near-field scanning optical microscopy

Near-field scanning optical microscopy….

… conceived in 1928 - realized in 1984

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 Near-field optics relies on the production of
sub-wavelength lightsources: fiber tips

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 The strong confinement of light by near-field optics has
enabled single molecule fluorescence excitation

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 Logical consequence: Ultrasensitive confocal fluorescence
micro-spectroscopy enables single molecule detection

Single Molecule Detection provides

• Fluorescence lifetime
• Triplet state lifetime
• Spectral shifts (jumps)
• Intensity fluctuations
• Orientation
• Diffusion
• Molecular distances (FRET)
• Conformational changes
• Reaction kinetics

Single Molecule Detection is limited by:

• no information on molecular identity
• photobleaching
• long-lived excited states limit # photons
• specific labeling necessary

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 Laser micro and nano surgery of biomaterials

Tissue light interaction

Radiative Nonradiative
Tissue autofluorescence
Fluorescence from various
constituents of the tissue

Photochemical Photoablation Photodisruption

•Excited state reaction •Direct breaking of cellular structure •Shockwave generation at high
•Occurs even at low optical •Performed by high energy UV radiation pulse intensity .
power density •Fragmentation and cutting of the
. 10 - 100 ns pulses
tissue by mechanical force of
sec-min exp
shockwave
Low intensity

Thermal Plasma – induced ablation

•Light absorption converted to heat •Induced by high intensity short pulse
•Can produce coagulation, •Dielectric breakdown creats ionized
vaporization,carbonization and melting plasma that interacts with light yo
produce ablation
1 ms pulses 100 fs - 10 ns pulses
10-106 Watt/cm2 107-106 Watt/cm2

Prasad, Introduction to Biophotonics, John Wiley & Sons © 2003

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 Plasma formation: laser induced ionization

La réaction induisant l’ionisation d’un milieu exposé à une impulsion laser se divise en
deux phases:
- l’absorption simultanée de plusieurs photons incidents provoque la libération d’un
électron, ou électron quasi libre.
- l’ionisation en cascade. L’ionization d’impact produit deux electrons libres qui
pourront être de nouveau accélérés par absorption de nouveaux photons. Le
phénomène d’impact peut ainsi se reproduire en cascade pendant toute la durée de
l’impulsion laser, provoquant une avalanche d’électrons libres qui forment le plasma.

J. Colombelli et al, Rev Sci Instr 75 472 (2004)

A. Vogel et al. Appl Phys B 81 1015 (2005) J. Colombelli et al, Medicine/Scineces 22 651 (2006)
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 Plasma formation: laser induced ionization

La formation d’un plasma survient l’évolution spatiale d’un plasma

au delà d’une énergie de seuil. en trois temps

t1 : Ionisation du milieu,
formation du plasma
t2 : Expansion spatiale du plasma
t3 : Expansion maximale

Dans un milieu aqueux, l’intensité de seuil augmente

de 3 ordres de grandeur lorsque la durée d’impulsion
diminue de 10 ns à 100 fs. Cependant, l’énergie
déposée n’est autre que la puissance multipliée par la
durée d’impulsion et, par conséquent, il faut 100 fois
moins d’énergie pour produire un plasma en utilisant
des impulsions ultra-courtes. C’est donc autant
d’énergie qui ne sera pas simultanément absorbée par
le plasma.

A.Vogel et al, Chem Rev 103 577 (2003) J. Colombelli et al, Medicine/Scineces 22 651 (2006)

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 Laser micro and nano surgery: cell biology applications

En biologie cellulaire et du développement, il y a deux manières

d’appliquer la chirurgie au laser:
1. tire avantage des effets secondaires induits par l’expansion d’un
plasma sur une échelle de quelques micromètres afin de détruire une
cellule ou un groupe de cellules dans un organisme vivant en
développement. Cette ablation est déjà possible en utilisant des
impulsions longues de l’ordre de quelques nanosecondes et on parle
alors encore de microchirurgie.
2. nanochirurgie s’adresse plus particulièrement à l’étude d’éléments
subcellulaires. La destruction ciblée de structures subcellulaires ou
d’organelles est ainsi possible sans affecter les parties superficielles
(membranes) seulement si le procédé ablatif est parfaitement
contrôlé, c’est-à-dire sans perturbation mécanique ou thermale.

J. Colombelli et al, Medicine/Scineces 22 651 (2006)

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 Laser nano surgery: cell biology applications

Nous considérons trois modes d’utilisation de la nanochirurgie en biologie

cellulaire, en fonction de la cible à étudier :
L’ ablation a première est la simple miniaturisation de la chirurgie classique qui
consiste à éliminer une partie de la cellule tout en préservant l’imperméabilité de sa
membrane et d’observer, comme en biologie du développement, les mécanismes par
lesquels la cellule réagit localement ou glo-balement. Ainsi, il est possible de détruire
spécifiquement des mitochondries, un centriole ou une neurite.
La dissection permet d’avoir accès à des structures cellulaires normalement
masquées. On peut ainsi accéder à la membrane plasmique des cellules de plantes
pour effectuer des mesures par patch-clamp] ou y insérer des bactéries]. La dissection
à l’échelle intracellulaire permet également de disséquer des sous régions
chromosomiques.
L’induction concerne plus spécifiquement l’étude de phénomènes dynamiques.
L’utilisation du laser permet d’induire des modifications structurales et de changer
l’équilibre dynamique de façon ciblée et non invasive, permettant l’observation du
comportement hors équilibre ou du retour vers l’équilibre. Cela concerne les différents
éléments du cytosquelette, les flux cytoplasmiques, la motilité cellulaire et la
morphogenèse.
J. Colombelli et al, Medicine/Scineces 22 651 (2006)
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 Example: Nanosurgery Cytoskeleton in Interphase Cells

5 m

A. cellule Ptk-2 transfectée de façon stable avec une construction -tubuline-YFP. Les
microtubules peuvent êtres sectionnés par un laser UV à impulsions (470 ps). L’irradiation à
basse énergie (minimum 50 nJ) le long d’une ligne (pointillés bleus) forme un front de
catastrophes artificielles, créant ainsi de nouvelles pointes plus (+) et moins (-) des
microtubules présents dans le volume de dissection. Chaque microtubule réagit de manière
différente.
La dépolymérisation (flèches rouges en B, C, D) de la pointe (+) est le phénomène le plus
fréquent après la catastrophe et le sauvetage alors que la pointe (-) reste relativement stable.

J. Colombelli et al, Traffic 6 1093 (2005) J. Colombelli et al, Medicine/Scineces 22 651 (2006)
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 What is an optical tweezers ?

A single-beam gradient force trap obtained by tightly focusing a

cw laser beam through a high NA objective - 3D trap

Founder:
Arthur Ashkin,
Bell Labs, USA

nmW
F=Q
c
F – trapping force
Q – dimensionless efficiency coefficient
W – power of the laser beam
nm – refractive index of the medium
c – light speed www.bell-labs.com/user/feature/archives/ashkin/
A. Ashkin, et al Optics Letters 11 288 1986
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 Ray optics explanation of optical trapping

Sir Isac Newton:

“For every action force, there is a
corresponding reaction force which is equal input ray
in magnitude and opposite in direction”.

output ray

input ray

force exerted
output ray on the sphere
change of
optical momentum
bead pushes light light pushes bead
to left to right

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 Trapping with Gaussian beams

2D trapping with single

3D trapping with single
Gaussian beam
Gaussian beam

3D trapping with counter

propagating beams

Particle size:
High NA (NA > 1)
Low Numerical aperture (NA < 0.8) d>
Long Working Distance (WD > 1 mm) Short WD (WD < 1 mm)
High index: High resolution: r <
Limited resolution: r=1.2 /NA> 1.5 n=np/nm>1

A. Ashkin et al, PRL 24 156 1970 A. Ashkin et al, Opt. Lett. 11 288 1986
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 Characteristics of optical traps

• Material:
Dielectric (polystyrene, silica); Metallic (gold, silver, copper),
Biological (cells, macro-molecules, intracellular structures,
Types of particles: DNA filaments), Low index (ultrasound agent contrast)
• Size: 5 nm – 20 µm
• Shape: spherical, cylindrical, arbitrary

z axis propagation
Types of laser beams: x-y intensity profile
non diffracted beam
• Gaussian • Bessel
x-y intensity profile
• Laguerre-Gaussian
LG carries also orbital angular momentum that can
be transferred to the trapped particles

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 Characteristics of optical traps

Micrometer sized glass or polystyrene beads are commonly

used as attachment handles of the materials under
investigation.
The advantage of this approach is the clear and uniform
interaction between the beads and the laser beam.
Typical stiffness: 100 pN/micrometer

Typical displacements: 1-500 nm

Typical forces: 0.1-100 pN

Measurable speeds: ~1 kHz

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 Characteristics of optical traps

Comparison of forces with other techniques and biological processes:

Optical traps 0.1 - 100 pN

Electric fields (electrophoresis) 0-1 pN

AFM 10 - 10000 pN

Kinesin step 3-5 pN

RNA polymerase stalling 15-30 pN

Virus motor stalling ~50 pN

DNA conformational change ~65 pN

Biotin-streptavidin binding 300-400 pN

Courtesy Prof. D. Petrov, ICFO, Barcelona, Spain

http://users.icfo.es/Dmitri.Petrov/Teaching/lectures.htm
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 The physics behind optical tweezers

Radiation pressure is the force per unit area on a object due to

change in light momentum

The light momentum of a single photon is:

The change is momentum can be calculated by the difference is

momentum flux between entering and leaving a object

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 Some physics behind optical tweezers

A 1 mW laser beam reflecting from a mirror gives a pressure of:

= 10 −11 Ν
The weight of a 10 um water drop is 5x10-12 kG.
Gravity pulls the drop with 5x10 -11 N.
Hence, the gravity force and the light force are comparable.

light pressure
gravity

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 10 µm

The weight of a 10 µm water drop is 5x10-13 kG.

Imagine being able to pick up and move a single cell without

physically touching it. Moreover to measure mechanical properties
of single cells, or making its spectroscopical studies.
Such a technique could give enormous advantage for better
understanding of behavior of living cells, micromachines,
microfluides, colloid physics.

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 SELECT AND MOVE A GIVEN CELL

REMOTELY APPLY CONTROLLED FORCES

ON LIVING CELLS, INTERNAL PARTS OF CELLS,
AND LARGE BIOLOGICAL MOLECULES WITHOUT
OPTICAL DAMAGE

OPTICAL TRAP MEASURE MECHANICAL PROPERTIES

(FOR INSTANCE, ELASTICITY) OF DIFFERENT
PARTS OF CELLS

MEASURE THE FORCES GENERATED BY SINGLE

MOTOR MOLECULES IN THE PICONEWTON RANGE

USING OPTICAL SPECTROSCOPY TO MEASURE

TEMPERATURE, DNA STRUCTURE
CELL VIABILITY, INTRACELLULAR pH
OF A GIVEN SINGLE CELL
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 Pro’s and Con’s optical traps for Biophysics

Pro’s:
•Measurable forces and distances are well suited for enzyme dynamics
and molecular motors
•They work in normal buffer conditions

Con’s:
Radiation damages of samples

See the lectures (5) on Optical Tweezers given by K. Dholakia,

E. Di Fabrizio and D. Cojoc next week at Winter college

On line (free lectures)

http://users.icfo.es/Dmitri.Petrov/Teaching/lectures.htm

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 Further reading

Web sites (examples)

http://www.cbst.ucdavis.edu/
http://vlib.org/Science/Cell_Biology/index.shtml
http://omlc.bme.ogi.edu/classroom/ece532/
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookTOC.html
Books
• Alberts, Johnson, Lewis, Raff, Roberts, Walter, “Molecular Biology of the Cell”,
4th Edition, Garland Science, 2002
• Prasad, P.N., “Introduction to Biophotonics”, John Wiley & Sons, Inc., 2003
• Lakowicz, J.R., “Principles of Fluorescence Spectroscopy”, 2nd Edition, Kluwer
Academic, 1999
• Pawley, J.B., “Handbook of Biological Confocal Microscopy”, 2nd Edition,
Plenum Press, 1995
• Marriott, G., Parker, I., Methods in Enzymology, Vol. 360 (Part A) and Vol. 361
(Part B), “Biophotonics”, Academic Press, 2003
Journals (examples)
Biophotonics International (free)
Journal of Biomedical Optics
Single molecules
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 Acknowledgements

• Prof. J. Niemjela from ICTP for inviting me to give this lecture

• Prof. T. Huser from NSF CBST, University of California, Davis, and
Prof. D. Petrov from ICFO Barcelona, for permission to use their
lectures/slides
• To all the people not mentioned here, whose material about
Biophotonics I could find and use
• Special thanks to Prof. G. Scoles from University of Princeton and
Elettra/SISSA – Trieste, who ‘pushed’ and encouraged me
constantly toward this marvelous field of science

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 I hope that you agree with David Hilbert:
“We should know and we will know”
(with Biophotonics in mind )

Thank You for Your Patience !

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