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
5/3/2012
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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
)
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