5/3/2012

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Supplement (videos)

Ruben’s tube (sound):Ruben s tube (sound):http://www.youtube.com/watch?v=gpCquUWqaYw

Doppler US (diagnostic use):http://www.youtube.com/watch?v=FGXZG-j_Hfwhttp://www.youtube.com/watch?v=UpsmEnYOju8

High Intensity, focused US (therapy):http://wn.com/High-intensity_focused_ultrasoundhttp://www.youtube.com/watch?v=f6vqqHD8Vh0http://www.youtube.com/watch?v=unDJVQI2cuM&

UltrasoundBiophysics 2nd semester

József Orbán

Dep. of Biophysics

Biophysics 2nd semester

April 2012.

Source of images: www.robaid.com/bionics/bat-biosonar-biomimicry-for-improved-sonar-technology.htm

5/3/2012

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? ! ?

How can we determine the distance of the thunderbolt?

What defines the brightness of any pointon the US image/screen?

distance ~ time: d=vtvair= 320 m/s

Ultrasound instrument

transducers

Source of images: http://e-discountmedical.com/wordpress/?page_id=129https://www.eemedicals.com/ultrasound-c-50.html?page=3&alpha_filter_id=71&sort=3a

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Color doppler:Toward to Transducer: warm colour (red)Away from Transducer: cold colour (blue)

Slower: darkerSlower: darkerFaster: brighter

Umbilical cortVideos:http://www.youtube.com/watch?v=FGXZG-j_Hfwhttp://www.youtube.com/watch?v=UpsmEnYOju8

Doppler

http://ircamera.as.arizona.edu/NatSci102/NatSci102/lectures/spectroscopy.htm

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Vascular Doppler Ultrasound:

Doppler shift (f-f0) is in the audible range

0 0

'2 cos

US

vf f f f

v

Ultrasound

f0 = 8 MHz

f = 7.994 MHz

∆f = 5.4 kHz

Loudspeaker

0

' 751540

458

US

v cm sv m s

f MHz

v’vUSf0

Skin

Vessel

ΘTransducer

f

∆f

'

Ultrasound - Doppler

Doppler ultrasound: Frequency of US reflected from a moving surface is changed relative to the original frequency:

f=frequency of reflected US

1842: Christian Doppler

0

'1

US

vf f

v

0' USv f fv

q y

f0=original frequency

vUS=propagation velocity of US in the medium

v'=velocity of the reflecting surface

v'=velocity of blood flow

vUS=propagation velocity of US in the medium

f f D l hift

Continous ultrasound should be used

2 cosf Applications:1. Doppler echocardiography2. Vascular Doppler, blood flow 3. Fetal ultrasound

f-f0=Doppler shift

=angle between US beam and the blood flow axis

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Colour Doppler echocardiography

Ultrasound – Doppler echocardiography

LV

LA

RV

RA

Ao

Aorta insuffitienty

Sound

20 Hz 20 kHz

infra audible u l t r a

Sound wave: longitudinalmechanical wave! (vibration)

20 MHz2 MHz US diagnosticrange

0 Hz f (Hz)

infra audible u l t r a

lunglung bile bonebone

1000 2000 700030000 4000 5000 6000

v (m/s)glass/metal

1400 1500 1600 1700

• propagation of sound requires elastic medium (particles): gas, liquid, solidBUT! vacuum

fatfat w bloodblood

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2D B-scan

Sequence of one‐dimensional B(rightness)‐images.

Multiple reflection →echo shadow artefact

Stretching/conractionof area(s) on screenof area(s) on screen

3D reconstruction

With a sequence of 2D B‐images, one can collect information from all the volume elements of a selected volume within the body.

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Transducer

polarity cabel

other polarity cabel

Image source : http://www.genesis.net.au/~ajs/projects/medical_physics/ultrasound/index.html

Generation of US: Inverse piezoelectroic effect

Piezoelectric crystals: • Natural crystal: quartz

――

• Artificial crystal: ceramic wafer, PZT (lead zirconate titanate)

Direct piezoelectric effect(mechanic deformation chargeseparationelectric potential)

+

――――+

+

Inverse piezoelectric effect(alternating voltagemovement of ions/chargesmechanic deformation US)

―+

――――+

+――++

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Ultrasound pulse

Skin Pulse separation timei dPulse separation:

transducer

lifetime of a pulse

pPropagation speedof US in soft tissues 

is 1450 m/s

Pulse separation:1 ms «» 1 kHz repetition rate

p

μs

Propagation of (ultra) sound in medium

v= 1500 m/s, in water

Pulse-echo principle

Reflection at thefirst interphase.

Propagation time: t= 2d/v

US source

What can we see on screen?

US machine measures thetime of pulse-echo (back and

Propagation time: t= 2d/v forth)! Then calculates with thepropagation speed in water.

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Propagation of (ultra) sound in medium

Water

Vair = 331 m/sVwater = 1550 m/sViron = 5100 m/s

US attenuates in air (absorption)

UH forrás

Water

Iron

Air

f = 2000 Hzλair = 16.55 cmλwater = 77.5 cmλiron = 2.55 m

(absorption).

Reflection on interphase.Attenuation in 2nd medium.

I0 = Ireflected+Irefracted

v1< v2, ρ ~ v??? What happens at the 2nd interphase ???

Ultrasound tomography - basics

Why don’t we use US for total body section?

falciform ligament FL lienorenal ligament LR gastrolienal ligament GL lesser omentum LO

The answer is given by the properties of US:absorbance, resolution, detection depth

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1. Sound pressure:The ultrasound wave exerts pressure on an objects in its direction of propagation. The pressure is directly proportional to ultrasound intensity.

2 Absorption

Ultrasound

2. Absorption:Energy absorption by the medium that leads to an increase in its temperature. Absorption increases with frequency and distance travelled:

A= amplitude= absorption coefficient

A(x) A0ex Typical frequency of devices:

8 MHz: superficial vasculature

4 MHz: deep vasculature absorption coefficientx= distance (layer thickness) 2 MHz: fetal ultrasound

T = 1-RR (reflected)

2

1 2z zR

3. Reflection

Ultrasound

4. Axial resolution

z1=v1*ρ1

R (reflected)

1 2

Rz z

z2=v2*ρ2

z: acoustic impedance (resistance)

tw d

vtw 2dIn order to resolvethe d axial distance:

For a given frequency, the axial resolution improves with decreasing Q.For a given Q, the axial resolution improves with increasing frequency.

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Resolution of ultrasound images

Resolution threshold: the distance between two points that may still be distinguished by their detected ultrasound image.

Axial resolution threshold: the half of the pulse length.

The higher the f, the shorter the pulse.

The higher the f, the higher the absorption of the tissue. 

Pulse frequency

high

FrequencyHigh Low

low

monitor

The choice of the appropriate frequencyis always a compromise betweenresolution power and detection depth.

Resolution of ultrasound images

Lateral resolution threshold: ≈ essentially the same as the beam width

US beam

Always greather than the axial threshold, so the lateral resolutionis worst than the axial one.

Best near the focus region

US beam

Best near the focus region.

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Focusing 

1. Fixed focus solution: e.g.  acoustic lenses

Focusing 

2. Electronic focusing

i i i /d i

Electric signals

During emission/detection.

The focus region can be set to any desired depth.

Multi‐element transducer, all of them contain an electronic delay unit to form the shape of US beam.

Transducers

unit to form the shape of US beam.

Interference of the waves, maximal sound pressure at given distance.

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Scanning(always with focused beam)

1. Mechanic: • Single piezo crystalSingle piezo crystal• Sector scan

out of date!

2. Electronic: Transducer arraylinear arraycurved array

• Several ceramic wafer side by side (pl. 512)

• 1D lines• Shift to the next transducers

Ultrasound – operation modesA-mode (Amplitude modulated): Single transducer. US beam propagates in a straight line.

The echo is displayed in the form of

transducer US pulse

a voltage peak on an oscilloscope.

B-mode (Brightness): Voltage

pulse is displayed as a grayscale

spot.

AmplitudeA mode

BrightnessB mode

2d B-mode: scanningReflection:tissue/bone 35%air/skin 100%gel/skin 0,1%

Important to use gel!

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Ultrasound modesM-mode (time Motion)

Temporal display of periodically moving objects in 1 dimensional (line) section(e.g., echocardiography).

Based on B mode linear scans in time.

X-axis: time.Y-axis: 1D B-mode image (line).

LA

Mitralis stenosis

LA

LV

septum

time

sys dias sys dias

Other areas of application

• As an effect of US, dust can aggregate, so in factories can be applied to get rid of dust.applied to get rid of dust.

• Similarly, at airports it can be used for dismiss the fog

• Structural test of metals (holes, cracks)

• Sonar: determination of sea/river depth, surface scanning

• It can kill microorganisms, it is appropriate for sterilization

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Other areas of application

http://spinoff.nasa.gov/Spinoff2008/hm_8.htmlhttp://sonohouse.co.kr/products.htmhttp://www.diytrade.com/china/pd/9262342/Dental_Handpiece.htmlhttp://www.omni-inc.com/omni-sonic-ruptor-400-ultrasonic-homogenizer-p-45.html

Therapic application US

The attenuation of US is due to the absorption mainly.The absorbed vibrational energy can have- Heat effect (increased particle motion)- Non thermal effect (cavitation, cell membrane permeability change)

CavitationFormation of short-lived cavities (small bubbles) upon the breakage of intermolecular cohesion forces.

MicromassageTissue vibration with different frequences – friction force – heat production

Physics‐Biophysics 2 2012.04.26.

1

CALORIMETRIC METHODS

DSC - Differential Scanning CalorimetryITC - Isothermal Titration Calorimetry

Dr. Beáta Bugyi – University of Pécs, Medical School, Department of Biophysics2012. 04. 26.

OVERVIEW  – THERMODYNAMICS ‐ HEAT

TCQ Δ=Δ

[ ]K

JC

T

QC =→

Δ=

LmQ=Δ

kg

JL

m

QL =→= ][

INCREASE IN TEMPERATURE NO INCREASE IN TEMPERATURE

HEAT CAPACITY LATENT HEAT

OVERVIEW – THERMODYNAMIC POTENTIAL FUNCTIONS

ENTHALPY

HELMHOLTZ FREE ENERGY

GIBBS FREE ENERGYFREE ENTHALPY

INTERNAL ENERGY

U pVUH +=

TSUF −= pVTSUG +−=

pV+

TS−

ENERGY BALANCE AND EQUILIBRIUM OF THERMODYNAMIC PROCESSESspontaneous direction equilibrium

U = constant V = constant ΔS > = 0 S maximumT = constant V = constant ΔF < = 0 F minimumT = constant p = constant ΔG < = 0 G minimum

OVERVIEW – THERMODYNAMICS OF CHEMICAL REACTIONS

THERMODYNAMIC NATURE OF CHEMICAL REACTIONSwhy potential functions are useful!!!

TYPEΔH < 0 EXOTHERM energy release, heat producedΔH > 0 ENDOTHERM energy absorption, heat absorbedDIRECTION: A + B ↔ AB

spontaneous directionΔG < 0 →ΔG > 0 ←ΔS > 0 ΔH < 0 →ΔS > 0 ΔH > 0 T ↑ →ΔS < 0 ΔH < 0 T ↓ →ΔS < 0 ΔH > 0 ←

HOW CAN WE „MEASURE” POTENTIAL FUNCTIONS?

THERMOANALYTICS

CALORIMETRY

allows measuring the absorbed / released heat of chemical reactions or physical changes using a

CALORIMETER

CALORIMETRY : calor (Latin) = heat

Physics‐Biophysics 2 2012.04.26.

2

ISOTHERM: T = constant (temperature)ISOBAR: p = constant (pressure)ISOCHOR: V = constant (volume)ADIABATIC: ΔQ=0

DIFFERENTIALtwo calometers built togethertwo cells: reference / sample

SCANNINGthe temperature of the system is regulated by a temperature program

TITRATIONthe sample is injected into the cell

CALORIMETER ‐ TYPES

isochor: CONSTANT VOLUME„bomb”

isobar: CONSTANT PRESSURE”coffe-cup”

ΔQ = ΔUchange in the internal energy

ΔQ = ΔHchange in the enthalpy

REACTIONexotherm/endotherm

↓heat is generated/consumed

↓REFERENCE

(water, known heat capacity: CR)T increses/decreases

↓Measure: T before and after the

reaction: ΔT↓

calculate: ΔQ = CRΔT

CALORIMETER ‐ PRINCIPLES

DIFFERENTIAL SCANNING CALORIMETRY ‐ DSC

DSC – APPLICATIONS ‐ ADVANTAGES

APPLICATIONScharacterization of molecules

(small molecules, proteins, antibodies, nucleic acids, membranes, lipids, …)the effects of structural change on a molecule’s stabilitymeasurement of molecular interactionsliquid biopharmaceutical formulations.process developmentprotein engineeringrank order bindingantibody domain studiesassessment of biocomparability during manufacturing

ADVANTAGESsensitivedoes not disturb the moleculesstudy molecules in their native state in solution, without labeling or immobilizationcan be use with solutions that interfere with optical methods including turbid or colored solutions or particulate suspensions

DSC – PRINCIPLES

referencesample

the sample and the reference cells are heated according to a temperature program:SCANNING

HEATING RATE: ΔT / tdue to the reaction in the sample cell heat is absorbed or released

endotherm: T decreases in the sample cell heating of the sample cell (ΔQ)exotherm: T increases in the sample cell heating of the reference cell (ΔQ)

HEAT FLOW: ΔQ / t THERMOGRAM

ΔT = constantbetween the sample and the reference cells

two calorimeters built together: DIFFERENTIAL

DSC ‐ THERMOGRAM

TEMPERATURE: T

HEAT FLOWΔQ / t

Physics‐Biophysics 2 2012.04.26.

3

TEMPERATURE: T

DSC ‐ PARAMETERS DERIVED FROM THE THERMOGRAM

HEAT FLOWΔQ / t

ENDOTHERM

EXOTHERM

+heat to the sample

-heat to the reference

ENTHALPY CHANGE: ΔHarea under the curve

PHASE TRANSITION TEMPERATURE: Tpeak

ENTROPY CHANGE: ΔS = ΔH / TFREE ENTHALPY: ΔG = ΔH - TΔS Parameters

we can directly calculate from the datawe can further calculate

CHANGE IN HEAT CAPACITYshift of the baseline

DSC – CHARACTERISTIC TRANSITIONS ON THE THERMOGRAM

TEMPERATURE: T

HEAT FLOWΔQ / t

TcCRYSTALLISATION

exotherm peak

Tg GLASS TRANSITION

amorph polymersrigid glass like soft rubber like

step

TmMELTING

endotherm peak

barnasebacterial protein (Bacillus amyloliquefaciens)ribonuclease activity

DSC – AN EXPERIMENT: thermal stability of proteins

http://www.microcal.com/technology/dsc‐animation.asp

PHASE TRANSITION TEMPERATURE: Tmpeakthermodynamic stability50% native 50% denatured

ENTHALPY CHANGE: ΔHarea under the curve

further calculate:ENTROPY CHANGE: ΔS = ΔH / Tm

FREE ENTHALPY: ΔG = ΔH - TmΔS

DSC – AN EXPERIMENT: thermal stability of proteins

ISOTHERMAL TITRATION CALORIMETRY ‐ ITC

ITC – APPLICATIONS ‐ ADVANTAGES

APPLICATIONScharacterization of molecules(small molecules, proteins, antibodies, nucleic acids, membranes, lipids, …)measurement of molecular interactionsenzyme kineticsassessment of biological activity

ADVANTAGESsensitivedoes not disturb the moleculesstudy molecules in their native state in solution, without labeling or immobilizationcan be use with solutions that interfere with optical methods including turbid or colored solutions or particulate suspensions

Physics‐Biophysics 2 2012.04.26.

4

ITC – PRINCIPLES

molecule 2 reference

syringemolecule 2

injection

ITC = DCS + INJECTION

ITC – AN EXPERIMENT: ligand ‐ protein interaction

http://www.microcal.com/technology/itc‐animation.asp

TIME (minute)

ABSO

RBED

/ REL

EASE

D HE

AT IN

THE

REA

CTIO

N (μ

cal/s

)

„spikes”: injection

saturation

ITC – AN EXPERIMENT: ligand ‐ protein interactionTIME (min)

HEAT

FLO

W / I

NJEC

TION

(μca

l/s)

MOLAR RATIO

TOTA

L AMO

UNT

OF H

EAT

/ INJ

ECTI

ON(k

cal/m

ol)

RAW DATA

ANALYSIS

area under the curve

integrated heat

ITC – AN EXPERIMENT: ligand ‐ protein interaction

R: universal gas constant

FREE ENTHALPY: ΔGENTROPY CHANGE: ΔSΔG = - RTlnKD = ΔH -TΔS

FREE ENTHALPY: ΔGENTROPY CHANGE: ΔSΔG = - RTlnKD = ΔH -TΔS

ITC – AN EXPERIMENT: ligand ‐ protein interaction

AFFINITY:1/KD

BINDING CONSTANT:KD

STOCHIOMETRY:N

ENTHALPY CHANGE:

ΔH

MOLAR RATIO

TOTA

L AMO

UNT

OF H

EAT

(kca

l/mol

)