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

cristina.masoller@upc.edu 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

23/01/2015 2

Outline

• Introduction and overview

• Medical lasers and laser-tissue interactions

• A few examples

23/01/2015 3

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.

23/01/2015 7

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.

23/01/2015 8

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.

10

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).

14

• 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

23/01/2015 17

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.

23/01/2015 19

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

23/01/2015 23

Six steps to optogenetics

Nature Vol. 465, 6 May 2010

23/01/2015 24

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

23/01/2015 26

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)

23/01/2015 27

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.

23/01/2015 30

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

23/01/2015 34

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.

23/01/2015 35

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.

23/01/2015 36

23/01/2015 37 Source: R. Pini (Institute of Applied Physics-CNR, Florence)

A FEW EXAMPLES

-IMAGING (CELL,TISSUE)

23/01/2015 38

The Nobel Prize in Chemistry 2014

“for the development

of super-resolved

fluorescence

microscopy".

23/01/2015 39

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)

23/01/2015 40

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.

23/01/2015 41

23/01/2015 42

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

23/01/2015 43

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.

23/01/2015 44

conventional microscopy

Science 313:1642–1645 (2006)

single-molecule microscopy

23/01/2015 45

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.

23/01/2015 46

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).

23/01/2015 47

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.

23/01/2015 48

23/01/2015 49

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.

23/01/2015 50

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.

23/01/2015 51

• 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

23/01/2015 53

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.

23/01/2015 54

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.

23/01/2015 55

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.

23/01/2015 56

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.

23/01/2015 57

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.

23/01/2015 58

Y. Damestani et al, Nanomedicine: Nanotechnology, Biology and Medicine Volume 9, Issue 8 , Pages 1135-1138, November 2013

23/01/2015 59

Laser-control of single neurons

23/01/2015 60

• 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

23/01/2015 61

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)

23/01/2015 62

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 !

<cristina.masoller@upc.edu>

Universitat Politecnica de Catalunya

http://www.fisica.edu.uy/~cris/