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Lasers biomedical applications
Laser Systems & Applications
MSc in Photonics & Europhotonics
Cristina Masoller Research group on Dynamics, Nonlinear Optics and Lasers (DONLL)
Departament de Física i Enginyeria Nuclear Universitat Politècnica de Catalunya
[email protected] www.fisica.edu.uy/~cris
Goals
• To provide a broad overview of the applications of lasers
in the life sciences
• To describe a few examples
• To provide further reading
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Outline
• Introduction and overview
• Medical lasers and laser-tissue interactions
• A few examples
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Introduction
• In the early days of automotive mass production, Henry Ford
said: customers could buy a car in any color they wanted, as
long as it was black.
• In the first years of the laser age, users seeking visible lasers
could get any color they wanted, as long as it was red.
• We've come a long way since then.
Over the years the development of many different
lasers has spurred a wide-range of biomedical
applications.
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Firsts medical applications of lasers
Almost as soon as the laser was invented:
• Dermatology: in 1960 Leon Goldman tried to lighten
tattoos by aiming a ruby laser at the pigmented skin until
the pigment granules broke apart. He managed to
remove the marks with the laser (and also performed
pioneering research into the treatment of vascular
lesions with argon lasers).
• Ophthalmology: in 1963 Charles Campbell used a ruby
laser to treat a detached retina.
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Nowadays many laser applications: therapeutic & diagnostic
Examples
Ophthalmology
• Laser-based procedures to reshape the cornea, re-attach
retinas, etc.
• LASIK: laser-assisted in situ keratomileusis corrects several
vision problems. 23/01/2015 6
Dermatology
• Treatment of port wine stains
(skin birthmarks).
• Hair and tattoo removal.
• UV light to treat psoriasis,
eczema and other skin
diseases.
Laser use in dentistry
• High power infrared lasers are used to remove decay within a tooth
and prepare the surrounding enamel for receipt of the filling. Blue LEDs
“cure” and harden white composite fillings.
• Low power near-infrared diode lasers are used for gum surgery,
gently slicing soft tissue with less bleeding than would occur with a
scalpel (laser cauterization of tissue).
• Teeth whitening. A peroxide bleaching solution, applied to the tooth
surface, is ''activated" by laser energy, which speeds up of the whitening
process.
• Lasers are also used to remove bacteria during root canal procedures.
• Optical Coherent Tomography (OCT) uses near-infrared light to view
cracking and cavities in teeth more effectively than x-rays.
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Other laser biomedical applications
Laser-based 3D printers
• Allow doctors and dentists to
customize medical implants;
researchers are experimenting with
fabricating artificial organs.
• These printers can replicate 3D
forms because of laser-based
imaging techniques (laser
scanning).
• In several hospitals surgeons have
used 3D printed heart models to
prepare for major surgery.
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A 3D printer used by researchers at
Harvard University’s Wyss Institute
creates a model vascular network.
Lasers in cancer treatment
• Laser light can be used to remove cancer or precancerous growths or to
relieve symptoms of cancer.
• It is used to treat cancers on the surface of the body or the lining of internal
organs (in this case, laser light is delivered through an endoscope).
• Laser therapy causes less bleeding and damage to normal tissue than
standard surgical tools do, and there is a lower risk of infection.
• However, the effects of laser surgery may not be permanent, so the
surgery may have to be repeated.
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Cancer treatments
• Laser-induced interstitial thermotherapy (LITT, also referred as interstitial
laser photocoagulation): uses heat to shrink tumors by damaging or killing
cancer cells. Laser light at the tip of the fiber raises the temperature of the
tumor cells and damages or destroys them.
• Photodynamic therapy (PDT): a certain drug, called a photosensitizer or
photosensitizing agent, is injected into a patient and absorbed by cells all
over the patient’s body. After a couple of days, the agent is found mostly in
cancer cells. Laser light (of specific wavelength) is then used to activate
the agent and destroy cancer cells.
• Which lasers?
– CO2 and argon lasers can cut the skin’s surface without going into deeper
layers. Thus, they can be used to remove superficial cancers (skin cancer).
– The Nd:YAG laser is more commonly applied through an endoscope to treat
internal organs.
– Argon lasers are often used to activate the drugs used in PDT.
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Lasers in cancer diagnosis • Flow cytometry is a laser-based technology employed in cell counting,
cell sorting, biomarker detection and protein engineering.
• By suspending cells in a stream of fluid and passing them by an
detection apparatus, it allows simultaneous multi-parametric analysis of
the physical and chemical characteristics of up to thousands of particles
per second.
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• Flow cytometry is routinely
used in the diagnosis of health
disorders, especially blood
cancers.
• Instruments include several
lasers (for example, allowing to
see two UV, six violet, seven
blue and three red fluorescent
parameters). From Cancer Research UK Cambridge Institute
Laser-based optical imaging
• Two main operation principles:
– Light transport : absorption & scattering
– Bioluminiscence: fluorescent molecules (dyes, proteins)
• Raman spectroscopy used for cancer diagnosis (skin and internal
organs): is based on inelastic (Raman) scattering of laser light (visible,
near infrared, or near ultraviolet). The laser light interacts with
molecular vibrations, phonons or other excitations in the system,
resulting in a shift of the photons’ energy that gives information about
the molecular vibrational modes.
• Photo-acoustic: when laser light strikes the target tissue, some of the
energy is converted to heat by the cells, resulting in a mechanical
wave that can be processed into an image. Because different types of
tissue absorb laser energy at different rates, lasers can be tuned to
target specific tissues.
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Laser-based optical imaging
• Fluorescence microscopy: light-activated fluorescent molecules.
23/01/2015 13 BioOptics World june 2014
Other laser-based imaging techniques
• Spectral imaging: generates many images of the same object, each
measured at a different wavelength.
• Optical coherence tomography (OCT) is analogous to ultrasound,
measuring the intensity of back-reflected or back-scattered infrared light,
rather than acoustical wave. Light with short coherence length is used.
While ultrasound produces image resolution on the scale of a tenth of
a millimeter, OCT can achieve orders-of-magnitude finer resolution
(on the micrometer scale).
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• Confocal microscopy: a technique for increasing the contrast of
microscope images (better resolution that OCT but less tissue penetration).
• Plasmon imaging using nano-particles (gold): plasmons are collective
oscillation of electrons on a conductive surface that can be excited by light.
• Multimodal biomedical imaging systems: no single imaging modality can
provide all the information in one image.
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A laser scanning, or "confocal" microscope scans a sample point-by-point or
line-by-line at once, assembling the pixel information to generate one image.
This allows for a very high-resolution and high-contrast image in three
dimensions. The image is from a laser scanning microscope of a mouse
retina, where the cells have been stained with fluorescent dye.
http://lightexhibit.org
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Astrocytes are the star-shaped cells found in spinal cord and the brain. In
this image of astrocytes, the nucleus of each cell has been stained blue
while the cytoplasm (the fluid that fills the cell) has been colored green.
To achieve this, the process of immuno-fluorescence was used (antibodies
are used to attach fluorescent dyes to specific molecules in the cells).
http://lightexhibit.org
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Protozoa are single-celled animals found throughout the world in many
different habitats. They play a key role in maintaining and balance of bacteria,
algae, and other microbial life. This photograph illuminates one particular
type of protozoa called vorticella. In this image, a technique called "dark field
microscopy" was used. This technique blocks out the direct light from the
source, so that only light scattered by the specimen is observed, enabling
brilliant bright images to be seen again a dark background.
http://lightexhibit.org
Optical imaging
• Advantages:
– Relatively low cost.
– Wide range of spatial resolution: μm – mm
(molecular to physiological information).
– Time-resolved measurements.
• Main disadvantage:
– Limited depth penetration: about 10-fold loss in
photon intensity for every centimeter of tissue depth.
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Lasers and LEDs for sensors & safety
• Smart phones are integrated with optical sensors and apps for real-time
diagnosis and treatment in remote environments. These portable
devices can screen for diabetes, etc.
• Instruments employing lasers emitting a several frequencies can
identify different materials and verify the composition of different drugs.
• Optical sensors also used to ensure food quality and safety (to
measure the oxygen levels inside sealed packaged food to ensure that
the food inside the package will not spoil).
• UV light’s ability to alter virus DNA to halt its replication has made UV
germicidal irradiant (UVGI) devices possible for water purification and
medical equipment sterilization.
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Other applications
Dental DNA analysis
• Take sample by swiping teeth with a toothpick like piece
of paper.
• Placing the sample in a device that amplifies the DNA
with the polymerase chain reaction (PCR) fluorescent
labeling plus a laser diode and a photo-detector can
identify 11 types of bacteria.
• It can be used to select the best antibiotic treatment.
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Optical transfection • Is the process of introducing nucleic acids into cells using light.
• Lasers can be used to burn a tiny hole on the cell surface, allowing those
substances to enter.
• Typically, a laser is focused to a diffraction limited spot (~1 µm diameter)
using a high numerical aperture microscope objective. The membrane of a
cell is then exposed to this highly focused light for a small amount of time
(typically tens of milliseconds to seconds), generating a transient pore on
the cell membrane. The generation of a photopore allows exogenous
DNA, RNA, or larger objects (such as quantum dots) to enter the cell. In
this technique, one cell is treated at a time.
• This technique has been demonstrated using a variety of laser sources,
including 800 nm femtosecond pulsed Ti:Sapphire and 1064 nanosecod
pulsed Nd:YAG.
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Controlling neurons with light
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Optogenetics
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Six steps to optogenetics
Nature Vol. 465, 6 May 2010
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Optics and Photonics News, August 2011
With blue light: www.youtube.com/watch?v=88TVQZUfYGw
Which light source?
• Illuminating a small number of neurons in the brain
requires a low-noise laser.
• In coarser applications, power fluctuations of the laser
over micro- or milliseconds might not matter.
• Usually two laser wavelengths are required: one to
excite cells and one to inhibit them.
• LEDs can also be used in optogenetics:
– LEDs are more readily available in different colors
than laser diodes, and
– they are much cheaper.
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MEDICAL LASERS AND LASER-TISSUE INTERACTIONS
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Medical lasers • Ultraviolet: excimer lasers (no or limited fiber optic transmission)
– Xenon-Cloridel (XeCl)
– Argon-Fluoridel (ArFl)
• Visible and NIR (fiber optic transmission):
– Diode lasers
– Neodimium:YAG (Nd:YAG: frequency double, Q-switching, free-
running)
• Infrared :
– Holmium:YAG (Ho:YAG): fiber optic transmission
– Erbium:YAG (Er:YAG): special fibers
– CO2: no fiber optic transmission
• Femtosecond lasers (no fiber optic transmission)
– Neodimium:glass
– Titanium:Sapphire (Ti:Sa)
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23/01/2015 28 Source: R. Pini (Institute of Applied Physics-CNR, Florence)
Medical lasers
23/01/2015 29 Source: M. J. Leahy (University of Limerick)
Diode lasers for biomedical applications
• Gallium arsenide (GaAs) yield reds (above 630 nm)
• Indium phosphide (InP) yields blues (375-488 nm)
• Indium gallium nitride (InGaN) yields greens: (515- 536.6 nm)
• Power: up to 0.5 Watts
• Biomedical instruments often combine multiple lasers for
multiple wavelengths.
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Light delivery systems
23/01/2015 31 Source: R. Pini (Institute of Applied Physics-CNR, Florence)
Which light source?
Depends of:
- optical properties of cells/tissue and
- light – cells/tissue interactions.
• Chromophores are responsible for the color of a molecule,
absorb certain wavelengths and transmit or reflect the rest.
• The absorption properties of tissue are dominated by the
absorption of properties of the four principal chromophores of
tissue (proteins, DNA, melanin, hemoglobin), and water.
• Tissue optics: the most important factor for penetration depth
is wavelength.
Optical absorption properties of tissue
Chemical Reviews, 2003, Vol. 103, No. 2
Therapeutic window
Tissue
transparency is
maximum in the
near-infrared
(600–1000 nm):
light penetrates
several cms
because of low
absorption by
water.
Map of laser-tissue interactions
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The diagonals show
constant energy
fluences (J/cm2).
Source: R. Pini, Institute
of Applied Physics-CNR,
Florence
Examples
• Tumor ablation
– 590–1064 nm: maximum photo-thermal effect in human
tissue
– laser diodes emitting in the 800-980 nm range have been
used for kidney and brain tumors ablation
• Cardiac surgery
– for patients with blocked or narrowed coronary arteries,
photo-thermal treatments are commonly used in
angioplasty to remove blood-vessel plaque.
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Lasers -- applications
• Welding of tissue
– Lasers: CO2, Argon, Diode
– For welding of blood vessels, cornea, skin
• Photo-coagulation
– Lasers: Nd:YAG, Dye, Diode, Argon, frequency doubled Nd:YAG
– For photocoagulation of the retina; treatment of pigmented and
vascular lesions.
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23/01/2015 37 Source: R. Pini (Institute of Applied Physics-CNR, Florence)
A FEW EXAMPLES
-IMAGING (CELL,TISSUE)
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The Nobel Prize in Chemistry 2014
“for the development
of super-resolved
fluorescence
microscopy".
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Eric Betzig Stefan W. Hell William E. Moerner
The classical limit for visualizing the biological world
• About half the wavelength of light, i.e., about 0.2 m
(d=/2NA; =400 nm, NA = 1.4)
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Beating the diffraction limit via stimulated emission of fluorescent molecules
• Stimulate Emission Depletion (STED) microscopy
(developed by Stephan Hell):
molecules are first excited by a focused laser beam and
then de-excited by a second laser with a doughnut-
shaped focus, so that only few molecules in the center of
the doughnut remain in their excited state and their
fluorescence serves as measure signal.
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The path to single-molecule microscopy
• William E. Moerner discovered that it is possible to optically control
fluorescence of single molecules.
• The fluorescence of one variant of a green fluorescent protein (GFP)
could be turned on and off at will.
• The strong green color of a GFP protein appears under blue and
ultraviolet light.
• When Moerner excited the protein with light of wavelength 488
nanometres the protein began to fluoresce, but after a while it faded.
• Regardless of the amount of light he then directed at the protein, the
fluorescence was dead.
• But light of wavelength 405 nm could bring the protein back to life again.
• When the protein was reactivated, it once again fluoresced at 488 nm.
individual molecules act like tiny lamps with switches
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And by using light pulses
• Eric Betzig: a super-resolution image can be obtained by
using fluorescent molecules that fluoresce at different times &
superimposing the images.
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conventional microscopy
Science 313:1642–1645 (2006)
single-molecule microscopy
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Hell, Moerner & Betzig are still active mapping the secrets of the biological world
• Stefan Hell has studied the inside of living nerve cells in
order to better understand brain synapses.
• William E. Moerner has studied proteins in relation to
Huntington’s disease.
• Eric Betzig has tracked cell division inside embryos.
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Fluorescing live synapses shed light on learning, memory formation
By using a green fluorescent
protein (GFP), which glows
brightly when exposed to blue
light, researchers studied
structural changes in the brain
when we make a memory or
learn something (and found
that what gets changed is the
distribution of synaptic
connections).
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G.G. Gross et al., Neuron, 78, 6, 971–985 (2013).
Tissue-level imaging: tomography
• Slices of tissue can be imaged by focusing a source onto the
focal plane, while structures in other planes appear blurred.
• The source can be optical, x-ray, ultrasound, gamma rays,
electrons, or magnetic resonance, or a combination of them.
• A tomogram (2-D slice) is produced from image
reconstruction algorithms. 3-D reconstructions are made by
scanning a detector across the sample.
• Many types of tomography for many types of imaging.
• Type used is highly dependent on information desired.
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Based on optical scattering of tissue, typically uses infrared
light and offers m-resolution.
Optical coherence tomography (OCT)
Light sources in the 900 - 1500 nm can achieve sub-micron resolution.
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OCT fundus image
produced at a 1.2
MHz axial scan rate.
in vivo human retina
obtained with a 1050
nm VCSEL.
BioOptics World July 2013
400 kHz axial-scan-
rate 3D volume of in
vivo human retina
obtained with a 1050
nm VCSEL was
motion-corrected and
averaged from four
volumes.
1 MHz axial-scan-rate 3D
volume of in vivo rabbit
stomach was obtained with a
miniature endoscopic probe
with a 1310 nm VCSEL.
Photoacoustic tomography/microscopy • Based on the photo-acoustic (PA) effect.
• Radio-frequency or optical pulses are transmitted into the tissue. Upon
absorption of the pulse, a sound wave is produced due to energy deposition.
• The PA signal depends on optical properties, thermal diffusivity/expansion
and elastic properties.
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• Returning ultrasound is
detected at tissue surface by
a transducer (single
transducer, a line array, or a
circular array can be used for
detection).
• PA systems image cms into
tissue: can offer 100 μm
resolution at 4 cm.
Source: Wikipedia
23/01/2015 52
Researchers at Washington University (St. Louis, US) have
developed a hand-held photoacoustic microscopy device that
can be used directly on a patient and accurately measure
how deep a melanoma tumor extends into the skin.
BioOptics World 12/2014
EXAMPLES -LIGHT THERAPIES
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Light therapy
• Laser irradiation over chromophores (the part of a
molecule that is responsible for its color, that absorbs
certain wavelengths and transmits or reflects the rest).
• Results in: photon-induced chemical reactions and/or
photon-induced alterations (stimulating or inhibiting
cellular functions).
• Goal: to chose a wavelength to reach chromphores in
target cells without the photons being absorbed by other
substances.
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Photo-bio-modulation Therapy (also known as Low Level Laser Therapy, LLLT)
• Light therapy used on sports injuries, arthritic joints, back
and neck pain, nerve injuries, spinal cord injuries, etc.
• Uses lasers or LEDs to improve tissue repair, reduce
pain and inflammation wherever the beam is applied.
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Source: Lite Cure LLC Companion Laser Therapy
• Usually applied by a doctor,
therapist or technician,
treatments take about 10
minutes and should be applied
two or more times a week.
• videos at
http://www.thorlaser.com/video/
Light therapy (phototherapy)
• Therapeutic
radiation used to
treat skin conditions
(psoriasis)
• Also used to treat
circadian rhythm
(sleep) disorders,
depression.
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Treatment for neonatal jaundice (yellowish pigmentation of the skin):
photo-induced degradation of bilirubin molecules.
Source: H. Failache, UDELAR, Uruguay
Transcranial Laser (and LED) Therapy (TLT) boosts cognitive function following brain injury
• Two patients with chronic traumatic brain
injury (TBI) were treated with transcranial
LEDs.
• The patients showed significant
improvement in concentration and memory.
• Light source: a LED console device,
containing 52 near-infrared (870 nm) and
nine red (633 nm) diodes for a total output
power of 500 mW continuous wave.
• But the patients’ improvements vanished if
they stopped the treatment.
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Photomedicine & Laser Surgery (doi:10.1089/pho.2010.2814)
A transparent permanent window to the brain
• Yttria-stabilized-zirconia (YSZ)
is a ceramic material, which is
well tolerated and used in hip
implants and dental crowns.
• It was modified to make it
transparent.
• The modified YSZ prosthesis
provide a permanent window
through which doctors can aim
light-based treatments for the
brain without having to perform
repeated craniectomies.
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Y. Damestani et al, Nanomedicine: Nanotechnology, Biology and Medicine Volume 9, Issue 8 , Pages 1135-1138, November 2013
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Laser-control of single neurons
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• Fluorescence (YFP) image of a cultured
cortical neuron expressing YFP-tagged
CHR2(H134R) 48 hrs post-transfection.
Scale bar represents 30 mm.
• Representative trains of spikes evoked
by pulsed (T = 10 ms) blue light
excitation (=470 nm, P=0.45
mW/mm2).
• Spiking success rate at various
stimulation frequencies
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Infrared inhibition of electrically-evoked muscle contraction (Scientific Reports 2013)
Big money
Is being invested in the US and in the EU in brain research:
– in the US: 100 M proposed by Obama for the BRAIN Initiative (Brain Research through Advancing Innovative Neurotechnologies)
– In the EU: 10 year Human Brain Project (54 M€ for the rump up phase, 2013-2016)
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http://www.humanbrainproject.eu http://thebraininitiative.org
EXAMPLES -SENSORS
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Pulse Oximetry • Non-invasive method that gives information
of blood oxygenation.
• A clip on the fingertip (or earlobe) is used,
with a red (660nm) and IR (910 nm) LEDs
facing a photodetector.
• Can be operated in reflection or
transmission mode.
• Absorption at these wavelengths varies
significantly for oxy and de-oxy
haemoglobin.
• From the ratio of the absorption of the red
and infrared light the oxy/deoxyhaemoglobin
ratio can be calculated.
• Many conditions can be studied (depth of
anesthesia, blood loss).
23/01/2015 64 Source: M. Leahy (University of Limerick)
23/01/2015 65
Take home message
• Laser-based imaging methods provide early diagnoses of
diseases before the point when costly medical treatments are
necessary.
• The emerging fields of neurophotonics and optogenetics will
drastically advance our understanding of the brain.
• Nowadays drugs can be delivered and/or activated by light.
• Optical sensors are fast, reliable and sensitive for detection of
harmful substances, as well as valuable diagnostic tools: can
monitor through breath analysis; can test drinking water for
contamination, etc.
• Lab-on-a-chip devices and low-cost microscopes incorporated
to smart phones could revolutionize diagnosis and track
infectious diseases. 23/01/2015 66
THANK YOU FOR YOUR ATTENTION !
Universitat Politecnica de Catalunya
http://www.fisica.edu.uy/~cris/