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Single particle tracking: principles and applications

Valeria LeviLaboratorio de Dinámica IntracelularFacultad de Ciencias Exactas y NaturalesUniversidad de Buenos Airesvlevi12@gmail.com

1) Main 3D-conformation(s) of the molecule in different conditions2 ) Quantification of the docking/undocking dynamics

Why single molecule experiments?: An example

Hodak J. et al. PNAS (2005)

Studying folding of individual structural elements of RNA

FRET donor

FRET acceptor

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Why single molecule experiments?

What information are we missing in "cuvette" assays?

output

solution 1 M 1015 molecules

Hodak et al. PNAS (2005)

Mg2+

FRET trajectorieslow Mg2+ high Mg2+

donoracceptor

Studying RNA structure and dynamics

FRET donor

FRET acceptor

FRET donor

FRET acceptorno Mg2+

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molecules are not synchronizedaverage properties

Bulk vs. single molecule measurements

Limitations of bulk measurements

Temporal evolution of each molecule/particleNo need to synchronize processes Detection of different populations

DisadvantagesMore expensive equipmentsStatistics!

Single molecule/particle assays

Dynamics of intracellular processes: Tracking

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Protein function in the cellular context

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1. Measuring motion in cells: FRAP

FRAP (fluorescence recovery after photobleaching) (Axelrod, 1976) and related techniques (FLIP, etc)

• Photodamage

• Photobleaching is not always irreversible

timein

tens

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IoIf

2. Measuring motion in cells: photoactivation

Patterson and Lippincott-Schwartz, Science 2002

PA-GFP (photoactivatable GFP) photoconverts to a fluorescent specie after irradiation at = 400 nm

before photoconversion after photoconversion

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FCS (fluorescence correlation spectroscopy)

3. Measuring motion in cells: FCS

• Low laser power

• Low concentration of fluorescent molecules

• Equilibrium

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It is hard to study complex processes by “averaging”de behavior of multiple molecules

Why single particle tracking experiments?

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Single particle tracking (SPT)

Position of the particle as a function of time

mechanism of motion

• interactions

• populations

• switches (no synchronization) z

y

x

Single particle tracking

1. Acquisition of single particle trajectories

2 .Quantitative analysis of single particle trajectories

3. Applications to intracellular transport

4. 3D particle tracking

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Image-based tracking techniques

•How can we obtain the trajectory of the particle from the movie? •What is the accuracy on the position determination?

Locating a particle with nanometer precision

point-like object

microscope

diffraction-limited spot

deconvolution

r = resolution = wavelengthNA = numerical aperture of the objective

r~250 nm, visible light

r λ/2NA

x or y position

Inte

nsity

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An example of a simple SPT method: FIONA (fluorescence imaging with nanometer accuracy)

Imaging single fluorophore

from http://physics.berkeley.edu/research/yildiz/technology.html

the center of this distribution can be found with 1.5 nm

precision!

We have to be sure that there is a single molecule/particle !!

The trajectory of the particle is then recovered after locating the particle in every frame of the movie

Pattern-recognition tracking method

j)w(i,B)j)(i,Ij)(i,(Iδji,

2patternimage

B = background differencew(i,j) = weight factor= 2)),(( borderpattern IjiI

Levi et al. Biophys J (2006)

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Tracking performance

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piezo step (nm)Levi et al. Biophys J (2006)

500 nm fluorescent bead

piezo stageair

nm steps

Improving SPT: Error in the particle position

Thompson et al. Biophys J (2002)

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24222 812

Na

BsN/a

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x = error in the particle positions = standard deviation of the PSFN= number of photons detecteda = pixel sizeB = background noise

photon noise background noise

B increasespixelization noise

a increases

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Application: molecular motors

microtubules

actin filaments

How the motor moves along the filament?

A simple answer from SPT experiments

Adapted from Rief et al., PNAS (2000)

36 nm steps !!

explicar con flechitas

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We can observe steps of motors in living cells!

Levi et al, Biophys J (2006b)Bruno et al, submitted

1 m

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Transport of melanosomes by myosin V

Myosin-V in living cells

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200 ± 4070dwell time (ms)

in cellsin vitro

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Levi et al, Biophys J (2006b)Bruno et al, submitted

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Understanding the stepping mechanism

Yildiz et al. Science, 2003www.studiodaily.com

low signal/noiselast forever20-50nanoparticles

low brightness,bleaching (<10s)

size0.5-10single dye molecules

low brightness,high concentration, bleaching

endogenously expressed

10-20fluorescent proteins

blinkingbrigthness, spectra

10-20quantum dots

sizebrigthness5-1000fluorescent beads

DisadvantageAdvantage Size (nm)Probe

Probes for SPT experiments

In SPT experiments, probes may:1) delay the motion of the particle2) change the properties of the tracked molecule

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Single particle tracking

Motor stepping dynamics is just a very specific case..

How can we obtain information of complex intracellular processes?

Obtaining quantitative information from trajectories

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the distance traveled by the particle after a given time period is different for the different motion regimes !

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Calculation of the mean square displacement (MSD)

22 )t(y-)t(y)t(x-)t(x)(MSD

What information can we obtain from MSD analysis?

lag time lag time

MSD

MSD

D6

random diffusion

2)v( 1D transport

D6anomalous subdiffusion

( < 1)constrained diffusion

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“Single things” analysis means that we have to be extremely careful with the statistics

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transport ??

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Hirokawa, Science 1998

in cellsin vitro

Applications: MSD analysis helps us to understand the mechanisms involved in organelle transport

Electron micrograph of mouse axon

The conditions in which transport develops in cells are completely different from those of in vitro assays

vs.

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MSHdispersion

melatonin aggregation

Gelfand´s lab - Wallin, bioscience-explained.org

Melanophores: a beautiful system to study transport

kinesindynein

myosin

microtubulesactin filaments

How do these transport systems work together to target organelles to their final destination?

3 families of motors

2 different tracks

Organelle transport in living cells

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Properties of myosin-V dependent transport

microtubule-dependenttransport

actin-dependenttransport

Why trajectories along actin filaments are not curvilinear?

microtubules actin filaments

Properties of transport driven by myosin-V

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α1o τAMSDMSD

1.33 ± 0.024100 ± 1000600 ± 30dispersion1.37 ± 0.031500 ± 50560 ± 30aggregation

A1 [nm2]MSD0 [nm2]Condition

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Numerical simulations

MSD

(m

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fitting:A1 and

Model

How are these parameters related to the transport mechanism?

The transport-diffusion model

4100 ± 1000dispersion1500 ± 50aggregationA1 [nm2]Condition

4000 ± 600301700 ± 30090

A1 [nm2]diffusion time (s)

Experimental Model

Brunstein et al. Biophys J 2009; Bruno et al. Phys Rev E 2009

Intersection: switching and detaching probabilities (Ali et al, PNAS 2007)Lower number myosin motor/melanosome in aggregation (Gross et al. JCB, 2004)

transport intersection? diffusion transport

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Diffusion increases the probability of switching to the microtubule network during aggregation

aggregation

actin

microtubule

actin

microtubule

longer diffusion period in aggregation

During aggregation, organelles need to be transferred from the actin to the microtubule networks

3D Particle tracking

1 z-plane ~ 0.1-1 s

Problems: low temporal resolution photobleaching

Making use of the z-sectioning capability of confocal and two-photon microscopes

Alternative: spinning disk confocal microscopy

Lange et al, Traffic, 2008

Eg: tracking ribonucleoprotein(RNP)time resolution: 0.3 s

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Image-based 3D methods

Z = 0m Z = 1m Z = 2m Z = 3mZ = 0m Z = 1m Z = 2m Z = 3m

Problems: limited z rangeerror in (x,y) increases when the particle moves far from the focal plane

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the radius of the diffraction ring is related

to the z-position

(Tilli and Massaccesi)

Image-based 3D methods: Bifocal imaging

Toprak et al, Nano lett 2007in-focus

(x,y position)out-of-focus(z-position)

2-5 nm accuracy

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Particle tracking in a 2-photon microscope

Motivations: particles could move far from the field of viewwidefield microscopy involves photobleachingRaster imaging is slow

2-photon

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Particle tracking in a 2-photon microscope

the laser follows the particle in real time

Levi et al Biophys J 2005a, 2005b

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step size (nm)

data from Diana Wetzler

50 nm

8 nm steps !

time resolution = 4.1 msaccuracy = 2 nm

Mitochondria transport in NIH3T3 cells

3D-Particle tracking in a 2-photon microscope

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Levi et al Biophys J 2005a

more on 3D tracking at the Weber Conference

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More SPT talks in the Gregorio Weber Conference

Thursday, December 15

S2-3) ANDRE GOMES (Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil)

12:40-13:00

S2-2) ENRICO GRATTON (LFD, University of California Irvine, USA)

12:20-12:40

S2-1) LUCIANA BRUNO (Universidad de Buenos Aires, Argentina)

12:00-12:20