Biophotonics

TEAM Pranav Negi Sarthak Kala Tanish Garg Tushar Swami Vikas Prasad

ACKNOWLEDGEMENT We would like to express our special thanks of gratitude and respect to our teachers Dr. Chhavi Bhatnagar and Ms. Sangeeta Yadav

who gave us the golden opportunity to present this wonderful presentation on the topic (Biophotonics), which also helped us in doing a lot of Research and we came to know about new things. We

are really thankful to them.

WHAT IS

BIOPHOTONICS ?

Biophotonics - Biology + Photonics development & application of optical techniques to study biological molecules, cells and tissue. Physists, Biologists, Mathematicians and Chemists – working together. allows researchers to see, measure, and analyse living tissues that wasn’t possible before. years of research lead to the invention of several new medical procedures - X-Rays - Laser Eye Surgery - Endoscopy

Sources: http://www.photonics.com/Splash.aspx?PID=1

AREAS OF APPLICATION

LIFE SCIENCESMEDICINE

AGRICULTURE

ENVIRONMENTAL SCIENCE

MICROSCOPY

WHAT IS MICROSCOPY Microscopy- microscopes to view objects and

areas of objects that cannot be seen with the naked eyes.

Three branches of microscopy: 1. Optical 2. Electron, and 3. Scanning Probe Optical microscopy involves passing visible

light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample.

Sources: http://microscopy.duke.edu/learn/introtomicroscopy/confocalpinhole.jpg

CONFOCAL MICROSCOPY• A laser is used to provide the

excitation light• The laser light (blue) reflects off a

dichroic mirror• mirrors scan the laser across the

sample• The emitted light passes through the

dichroic and is focused onto the pinhole

• the microscope is really efficient at rejecting out of focus fluorescent lightSources: http://microscopy.duke.edu/learn/introtomicroscopy/confocalpinhole.jpg

Total Internal ReflectionFluroscence Microscope

• A TIRFM uses an evanescent wave to selectively illuminate and excite fluorophores

• Incident light is totally internally reflected at the glass interface

• The evanescent electromagnetic field decays exponentially from the interface.

TIRFM diagram

1. Objective2. Emission beam

(signal)3. Immersion oil4. Cover slip5. Specimen6. Evanescent

wave range7. Excitation beam8. Quartz prism

Sources: https://upload.wikimedia.org/wikipedia/commons/thumb/5/54/Tirfm.svg/350px-Tirfm.svg.png

OPTICAL TWEEZERS

OPTICAL TWEEZERS invented by Arthur Ashkin in 1970. used for trapping and manipulating micro particles.Idea to trap or manipulate single cells like bacteria and manipulating organelles inside cells.Principles Light exerts force on matter. Dielectric particles with high refractive indexes such as glass, plastic beads and oil droplets are attracted to intense regions of laser beam.

Sources: An Introductory Information about Optical Tweezers by Mustafa Yorulmaz

SİNGLE BEAM OPTİCAL TWEEZERS

DUAL BEAM OPTİCAL TWEEZERS


The Mechanism of trapping can be described in two different regimes 1) When wavelength of light is greater than size of

particle.2) When wavelength of light is smaller than size of

particle.

Forces to hold the particle in trap

GRADIENT FORCEdue to refraction of light

(to pull object).

SCATTERING FORCEdue to reflection of light

(to push object)


CASE 1 When λ >> d (λ = wavelength of light , d= size of particle)

Due to the Electric Field of the light there is a induced electric dipole moment in the molecule. Electric Dipole moment is given by :- μ=αE

Sources: https://www.youtube.com/watch?v=MxmDzUSpXsY

Stable Trapping requires that the Gradient force must be greater than the Scattering force.

Increase in the N.A (Numerical Aperture) of the lens reduces the dimensions of the focal spot and increasing the gradient strength.

For a trapped particle the effect of scattering force is to move the equilibrium.

Balancing the two Forces:-

Sources: https://upload.wikimedia.org/wikipedia/commons/thumb/5/54/Tirfm.svg/350px-Tirfm.svg.png

CASE 2 When λ << d

In this case it is possible to describe the interaction using Ray optics.

Conservation of linear momentum.

Sources: https://www.youtube.com/watch?v=MxmDzUSpXsY

Sources: https://en.wikipedia.org/wiki/Optical_tweezers

OPTICAL TOMOGRAPHY

OPTICAL TOMOGRAPHY Optical tomography - a form of CT (computed tomography). creates a digital volumetric model of an object by reconstructing images made from light transmitted and scattered through an object. uses near infrared (NIR) light as the probing radiation.

Optical tomography is used mostly in medical imaging research.

Sources: https://upload.wikimedia.org/wikipedia/commons/3/3e/FEL_principle.png

Optical tomography relies on the object under study being at least partially light-transmitting or translucent. hence, works best on soft tissue, such as breast and brain tissue. soft tissues are highly scattering but weakly absorbing in the near-infrared and red parts of the spectrum, so that this is the wavelength range usually used.

PRINCIPLE

Sources: https://upload.wikimedia.org/wikipedia/commons/3/3e/FEL_principle.png

OPTICAL COHERENCE TOMOGRAPHY one of a class of optical tomographic techniques. OCT helps in a non contact, non invasive, micron resolution cross-sectional study of retina which correlates very well with the retinal histology

Sources: https://www.researchgate.net/profile/Alexei_Gorbatov/publication/

based on the principle of Michelson Interferometry.

Low Coherence infra red light coupled to a fiber optic travels through a beam splitter and is directed through a ocular media to the retina and a reference mirror. The distance between the beam splitter and the reference mirror is continuously varied.

When the distance between the light source and the retina tissue = distance between the light source and the reference mirror and interacts to produce an interference pattern. The Interference is measured by a photodetector and processed into a signal. A 2D image is built as the light source moves along the retina .


Highly Reflective structures are shown in bright colours (white & red) and those with low reflectivity are represented by dark colours (black & blue).

Intermediate reflectivity is shown in green.


DOI & DOT Diffuse optical imaging (DOI) is a method of imaging using near-infrared spectroscopy (NIRS) or fluorescence-based methods. When used to create 3D volumetric models of the imaged material DOI is referred to as diffuse optical tomography, whereas 2D imaging methods are classified as diffuse optical topography.

DOI techniques monitor changes in concentrations of oxygenated and deoxygenated hemoglobin and may additionally measure redox states of cytochromes.

Sources: http://www.ee.iitkgp.ac.in/ispschool/ispws2015/

LASER The acronym laser stands for "light amplification by stimulated emission of radiation." A laser is a coherent and focused beam of photons, means that it is all one wavelength, unlike ordinary light which showers on us in many wavelengths. Lasers work as a result of resonant effects.

Sources: http://hyperphysics.phy-astr.gsu.edu/hbase/optmod/qualig.html

TYPES OF LASERS1. Gas Laser : A gas laser is a laser in which an electric current is

discharged through a gas to produce coherent light. Like helium–neon laser (HeNe), carbon dioxide (CO2) lasers , Argon-ion lasers.

Red HeNe lasers have many industrial and scientific uses. Widely used in laboratory demonstrations in the field of optics because of their relatively low cost and ease of operation.


2. Excimer lasers : sometimes more correctly called an exciplex laser, is a form of ultraviolet laser which is commonly used in the production of microelectronic devices, semiconductor based integrated circuits or “chips”, eye surgery, and micromachining.

3. Chemical lasers : Chemical lasers are powered by a chemical reaction permitting a large amount of energy to be released quickly. Such very high power lasers are especially of interest to the military

4. Free-electron lasers : It consists of an electron beam propagating through a periodic magnetic field. Today such lasers are used for research in materials science, chemical technology, biophysical science, medical applications, surface studies, and solid-state physics.


Foster Resonance Energy Transfer (FRET)

What is FRET ?FRET DEFINITION FRET is a mechanism describing energy transfer between two light sensitive molecules (chromophores).

An energy transfer through non-radiative dipole–dipole coupling.

The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.

CHROMOPHOREA chromophore is the part of a molecule responsible for its color.The color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others.

Sources: http://micro.magnet.fsu.edu/primer/java/fluorescence/

THEORETICAL INTRODUCTION The FRET efficiency (E) is the quantum yield of the energy transfer transition:

E = Fret efficiency, r = donor to acceptor separation and

R = Foster distance of the pair (at which efficiency is 50%)

The FRET efficiency depends on :

•The distance between the donor and the acceptor (typically in the range of 1–10 nm).

•The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum.

•The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.Sources: http://micro.magnet.fsu.edu/primer/java/fluorescence/

MEASURING EFFICIENCYSENSITIZED EMISSION

To measure the variation in acceptor emission intensity, when the donor and acceptor are in proximity (1–10 nm) Due to the interaction of the two molecules, the acceptor emission will increase because of the intermolecular FRET from the donor to the acceptor. When a twist or bend of the protein brings the change in the distance or relative orientation of the donor and acceptor, FRET change is observed.

Sources: https://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer

PHOTOBLEACHING EFFECT Measuring photobleaching rates of the donor in the presence and absence of an acceptor. This method can be performed on most fluorescence microscopes: Shine the excitation light (of a frequency that will excite the donor but not the acceptor significantly) on specimens with and without the acceptor fluorophore and monitors the donor fluorescence over time.

Sources: https://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer

APPLICATIONS OF FRET FRET has countless applications in biology and chemistry. FRET can be used to measure distances and detect interactions between proteins. FRET has been used to detect of genes and cellular structures. Based on the mechanism of FRET a variety of novel chemical sensors and biosensors have been developed e.g., PHOGEMON.

Sources: http://www.fret.lif.kyoto-u.ac.jp/e-phogemon/phomane.htm

BIOPHOTONICS – MOVING AHEAD Microscopy equipment can not only useful when dealing with tissues or performing tests on free cells. used extensively in microelectronics, nanophysics, and mineralogy.

MICROELECTRONICS PCB designing and Semiconductor fabrication require high precision and accuracy. and optical microscopes are capable of that. Therefore, preferred in industries because of true depth perception, increasing the production efficiency, and also causes less eyes straining after a long use.


OPTICAL TWEEZERS confinement and organization (e.g. for cell sorting), tracking of movement (e.g. bacteria), application and measurement of small forces, and altering of larger structures (such as cell membranes).

NEW TECHNIQUES LIKE OCT ARE TAKING IT EVEN FURTHER AHEAD because of its resolution power being 10 times higher than other existing modalities such as ultrasounds and x-ray. Fiber-based OCT systems are particularly adaptable to industrial environments.

as they can access and scan interiors of hard-to-reach environments like radioactive and hot rooms.


Appendix Works cited Additional supporting data

Engineering

Biophotonics