Difference between revisions of "Optical Microscopy Part 4: Particle Tracking"

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{{Template:20.309}}
 
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==Introduction and Background==
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In this part of the lab, you will follow microscopic objects throughout a series of movie frames: small, fluorescent microspheres first diffusing in purely viscous solutions of glycerol-water, and next moving in fibroblast cells after endocytosis.
 +
Calculating the mean squared displacement of their motion as a function of time interval will allow you to characterize their physical environment and behavior, first in terms of diffusivity and viscosity coefficients of the glycerol-water mixtures, next recognizing other material or transport properties in fibroblast cells.
  
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===Microrheology===
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==Contextual background==
 +
===Background references===
 +
** R. Newburgh, [http://scitation.aip.org/content/aapt/journal/ajp/74/6/10.1119/1.2188962 Einstein, Perrin, and the reality of atoms: 1905 revisited], Am. J. Phys. (2006). A modern replication of Perrin's experiment. Has a good, concise appendix with both the Einstein and Langevin derivations.
 +
** A. Einstein, On the Motion of Small Particles Suspended in Liquids at Rest Required by the Molecular-Kinetic Theory of Heat, Annalen der Physik (1905).
 +
** M. Haw, [http://stacks.iop.org/JPhysCM/14/7769 Colloidal suspensions, Brownian motion, molecular reality: a short history], J. Phys. Condens. Matter (2002).
 +
** E. Frey and K. Kroy, [http://arxiv.org/PS_cache/cond-mat/pdf/0502/0502602v1.pdf Brownian motion: a paradigm of soft matter and biological physics], Ann. Phys. (2005).
 +
** [http://www.youtube.com/watch?v=FAdxd2Iv-UA Random Force & Brownian Motion — 60 Symbols]
  
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Many cellular functions such as migration, differentiation, and proliferation are regulated by the
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===Brownian motion===
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mechanical properties of cells, specifically, their elasticity and viscosity. Rheology is the science of
+
This section was adapted from http://labs.physics.berkeley.edu/mediawiki/index.php/Brownian_Motion_in_Cells.
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measuring materials' mechanical properties. Microrheology is a subgroup of techniques that are
+
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capable of measuring mechanical properties from microscopic material volumes. Clearly, given the
+
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typical size of biological cells, microrheology is the technique needed to measure their elasticity and
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viscosity.
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The elastic and viscous properties of cells can be characterized by a complex-valued shear
+
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modulus (with units of Pa) <i>G*(ω) = G'(ω) + iG"(ω)</i>. The real part <i>G'(ω)</i>, referred to as the
+
−
storage modulus, is a measure of cell elasticity, while the imaginary part <i>G"(ω)</i>, the loss modulus,
+
−
is a measure of their viscosity. A generalized Hookean relationship can be written as:
+
  
−
[[File: Eq1.PNG]]
+
If you have ever looked at an aqueous sample through a microscope, you have probably noticed that every small particle you see wiggles about continuously. Robert Brown, a British botanist, was not the first person to observe these motions, but perhaps the first person to recognize the significance of this observation. Experiments quickly established the basic features of these movements. Among other things, the magnitude of the fluctuations depended on the size of the particle, and there was no difference between "live" objects, such as plant pollen, and things such as rock dust. Apparently, finely crushed pieces of an Egyptian mummy also displayed these fluctuations.  
  
−
where <i> Δr</i> is a generalized displacement, and <i>F(ω)</i> is a force linearly proportional to it via the
+
Brown noted: ''[The movements] arose neither from currents in the fluid, nor from its gradual evaporation, but belonged to the particle itself''.  
−
shear modulus. Therefore, we can measure the shear modulus if we can measure the deformation
+
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of the cell under a known force. (Note that all these quantities are frequency-dependent).
+
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Particle-tracking microrheometry is based on measuring the displacement of a particle with
+
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radius <math>a</math> embedded in a cell driven by thermal forces. One complication is that this relationship is frequency dependent - this is because in complex fluids, such as the cellular cytoskeleton, there are different energy dissipation mechanisms over different time scales.
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To approach the derivation of the relevant formulas, it is more convenient to think in terms of
+
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energy, rather than force. The relationship between stored energy and displacement has a familiar
+
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form, similar to a spring-mass system (recall <math> KE \ \alpha \ k(\omega)^2</math> ):
+
  
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[[File: Eq2.PNG]]
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This effect may have remained a curiosity had it not been for A. Einstein and M. Smoluchowski. They realized that these particle movements made perfect sense in the context of the then developing kinetic theory of fluids. If matter is composed of atoms that collide frequently with other atoms, they reasoned, then even relatively large objects such as pollen grains would exhibit random movements. This last sentence contains the ingredients for several Nobel prizes!
  
−
What is the driving thermal energy <i>U(ω)</i>? Recall also that thermal energy is "white,"
+
Indeed, Einstein's interpretation of Brownian motion as the outcome of continuous bombardment by atoms immediately suggested a direct test of the atomic theory of matter. Perrin received the 1926 Nobel Prize for validating Einstein's predictions, thus confirming the atomic theory of matter.
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''i.e.'' it contains equal power at all frequencies and is equal to 0.5*<i>k<math>_B</math>T</i> for each degree of freedom in a
+
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second-order system, where <i>k<math>_B</math></i> is Boltzmann's constant and <i>T</i> is the absolute temperature. From
+
−
this relationship (since we're observing motion in two dimensions), we have
+
  
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[[File: Eq3.PNG]]
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Since then, the field has exploded, and a thorough understanding of Brownian motion is essential for everything from polymer physics to biophysics, aerodynamics, and statistical mechanics. One of the aims of this lab is to directly reproduce the experiments of J. Perrin that lead to his Nobel Prize. A translation of the key work is included in the reprints folder. Have a look – he used latex spheres, and we will use polystyrene spheres, but otherwise the experiments will be identical. In addition to reproducing Perrin's results, you will probe further by looking at the effect of varying solvent molecule size.
  
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Our argument is obviously very approximate.  A complete (and much more difficult) derivation results
+
===Diffusion coefficient of microspheres in suspension===
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in the following equation (see Mason <ref>T. G. Mason, "Estimating the viscoelastic moduli of complex fluids using the generalized Stokes-Einstein equation" Rheol. Acta, 39, pp. 371-378 (2000). </ref> for details):
+
According to theory,<ref>A. Einstein, [http://www.math.princeton.edu/~mcmillen/molbio/papers/Einstein_diffusion1905.pdf On the Motion of Small Particles Suspended in Liquids at Rest Required by the Molecular-Kinetic Theory of Heat], Annalen der Physik (1905).</ref><ref>E. Frey and K. Kroy, [http://www3.interscience.wiley.com/cgi-bin/abstract/109884431/ Brownian motion: a paradigm of soft matter and biological physics], Ann. Phys. (2005). Published on the 100th anniversary of Einstein’s paper, this reference chronicles the history of Brownian motion from 1905 to the present.</ref><ref>R. Newburgh, [http://scitation.aip.org/journals/doc/AJPIAS-ft/vol_74/iss_6/478_1.html Einstein, Perrin, and the reality of atoms: 1905 revisited], Am. J. Phys. (2006). A modern replication of Perrin's experiment. Has a good, concise appendix with both the Einstein and Langevin derivations.</ref><ref>M. Haw, [http://stacks.iop.org/JPhysCM/14/7769 Colloidal suspensions, Brownian motion, molecular reality: a short history], J. Phys. Condens. Matter (2002).</ref> the mean squared displacement of a suspended particle is proportional to the time interval as: <math>\left \langle {\left | \vec r(t+\tau)-\vec r(t) \right \vert}^2 \right \rangle=2Dd\tau</math>, where <i>r</i>(<i>t</i>) = position, <i>d</i> = number of dimensions, <i>D</i> = diffusion coefficient, and <math>\tau</math>= time interval.
  
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<math>\left\vert G^*(\omega) \right\vert = {k_B T \over \pi a \left \langle \Delta r^2 (\omega) \right \rangle \Gamma[1 + \alpha(\omega)]} </math>
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==Instructions==
  
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Some key additional details to help you make sense of this equation:
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===Estimating the diffusion coefficient by tracking suspended microspheres===
  
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<b>1.</b> As you can see, the dependence on displacement is more accurately expressed as the mean squared displacement (MSD) <i> <Δr<math>^2</math>(ω)></i>:
+
[[Image: 20.309_130924_GlycerolChamber.png|right|thumb|200px|Imaging chamber for fluorescent microspheres diffusing in water:glycerol mixtures]]
 +
1. Track some 0.84&mu;m Nile Red Spherotech polystyrene beads in water-glycerin mixtures (Samples A, B and C contain 0%, 30% and 50% glycerin, respectively).
  
−
[[File: Eq5.PNG]]
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:''Notes'': Fluorescent microspheres have been mixed for you by the instructors into water-glycerin solutions A, B, C, and D. (a) Vortex the stock Falcon tube, and then (b) transfer the bead suspension into its imaging chamber (consisting of a microscope slide, double-sided tape delimiting a 2-mm channel, and a 22x40mm No. 1.5 coverslip, and sealed at both ends nail polish).
  
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where < > denotes a time-average of the particle's displacement trajectory <i>r(t)</i>, at discrete
+
:''Tip'': Do not choose to monitor particles that remain stably in focus: these are likely to be 'sitting on the coverslip' and their motion will not be representative of diffusion in the viscous water-glycerol fluid.
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times <i>t = t<math>_1</math>,.... t<math>_n</math></i> (as sampled by a digital system like the PC and camera). Additionally, &tau;
+
−
is a characteristic lag/delay time for the measurement. corresponding to the frequency ω.
+
  
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<b>2.</b>  <math>\alpha(\omega)=\frac{\partial \ln \left \langle \Delta r^2(\tau) \right \rangle }{\partial \ln \tau}</math>
+
2. Estimate the diffusion coefficient of these samples: MSD = <math>\left \langle {\left | \vec r(t+\tau)-\vec r(t) \right \vert}^2 \right \rangle=2Dd\tau</math>, where <i>r</i>(<i>t</i>) = position, <i>d</i> = number of dimensions, <i>D</i> = diffusion coefficient, and <math>\tau</math>= time interval. Use Sample A to verify that your algorithm correctly calculates the viscosity of water at the lab temperature (check the temperature on the clock on the wall or by other means).
 +
:* '''Consider how many particles you should track and for how long. What is the uncertainty in your estimate?'''
 +
:* '''From the viscosity calculation, estimate the glycerin/water weight ratio.''' (This [https://dl.dropboxusercontent.com/u/12957607/Viscosity%20of%20Aqueous%20Glycerine%20Solutions.pdf chart] is a useful reference.)
 +
:* See: [http://labs.physics.berkeley.edu/mediawiki/index.php/Simulating_Brownian_Motion this page] for more discussion of Brownian motion and a Matlab simulation.
  
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<b>3.</b> The radius of the particle <math>a</math> plays a role in the formula.
 
  
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<b>4.</b> Γ(.) is the Gamma function (the generalized form of the factorial function, which can be
+
===Live cell particle tracking of endocytosed beads===
−
looked up in a mathematical table). Mason suggests that for our range of &alpha;,
+
  
−
<math>\Gamma[1 + \alpha] \approx
+
We can also use particle tracking to probe cell samples. 0.84 μm diameter red fluorescent microspheres were mixed with the growth medium and added to the plated cells for a period of 12 to 24 hours for bead endocytosis.  
−
= 0.457(1 + \alpha)^2 - 1.36(1 + \alpha) + 1.90.</math>
+
  
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This equation may look complicated but there is a simple approximation to calculate the elastic
+
You will be given two plates of cells for these experiments:
−
and viscous moduli:
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* <u>Dish 1</u> will be used to monitor particles in untreated cells, while
 +
* <u>Dish 2</u> will be reserved to track microspheres after adding CytoD.
  
−
[[File: Eq7.PNG]]
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# Pre-warm your DMEM++ and CytoD to 37&deg;C
 +
# Carefully pipet out the medium from Dish 1. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed. Then, place 2 mL of fresh medium in dish.
 +
# Choose cells in Dish 1 with 3 or 4 particles embedded in them and capture movies of the samples. Take as many movies as you can with about 3-5 cells in the field of view in each movie. Make sure to do this quickly, as the cells become unhealthy without the temperature and carbon dioxide regulation.
 +
# Next, carefully pipet out the medium in Dish 2. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed.
 +
# Treat the cells in Dish 2 with the cytoskeleton-modifying CytoD: Pipet out remaining medium, add 1 mL pre-warmed CytoD solution at 10 μM (pre-mixed for you) to the dish, and incubate for 20 minutes at 37&deg;C. It's a good idea to check on your cells after 15 minutes: sometimes they are in bad shape at that point but sometimes they still look very healthy. Wash 2X with 2 mL of pre-warmed DMEM++, leaving 2 mL in the dish when imaging.
 +
# Perform and repeat the particle tracking measurements again in Dish 2 as quickly as you are able. It would be good to image the beads in only one cell at a time, since different cells may have different degrees of cytoskeletal disruption. Take as many videos as you can before the cells become sad. The cells' physiology has now been significantly disrupted by the toxin CytoD, and they will die within a couple of hours.
  
−
A detailed discussion of particle tracking microrheology can be found in the papers by Mason
+
==Report==
−
and Lau<ref>A. W. C. Lau et al., "Microrheology, Stress Fluctuations, and Active Behavior of Living Cells," Phys. Rev. Lett.,
+
−
91(19), p. 198101 (2003)</ref> An application of the work of Mason et al to 3T3 cells in particular can be found in the work of Tseng et al. <ref> Tseng et al. :[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1302394/  Yiider Tseng, Thomas P Kole, and Denis Wirtz, "Micromechanical mapping of live cells by multiple-particle-tracking microrheology." Biophys J. 2002 December; 83(6): 3162–3176.]</ref>
+
  
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===Cell culture and labeling protocols===
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Find and follow all guidelines on the [[Microscopy report outline]] wiki page. Remember that all numerical quantities must be reported with an associated level of uncertainty or significance.
 +
{{:Optical Microscopy: Part 4 Report Outline}}
  
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Read [[Optical Microscopy Part 3c: Protocols for cell culture, cell fixing and cell imaging]].
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{{:Optical microscopy lab wiki pages}}
  
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===Effects of cytochalasin D on actin cytoskeleton===
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{{Template:20.309 bottom}}
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This last part of the lab module seeks to illustrate the effect of cytochalasin D (CytoD) on cells. Since there are significant rheological changes in 3T3 fibroblasts treated with CytoD, it is reasonable to assume that this chemical may modify the cells' cytoskeleton.  We will image actin (one of the most important component of the mammalian cell cytoskeleton) structures in these cells, with and without CytoD treatment.
+
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+
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The actin cytoskeleton is visualized using the toxin phalloidin labeled with Alexa Fluor 568 (excitation maximum: 578nm, and emission maximum: 600nm). Phalloidin is a fungal toxin (small organic molecule that hinders actin assembly) that binds only to polymerized filamentous actin (F-actin), but not to actin monomers, G-actin.
+
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+
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+
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==Instructions==
+
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+
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===Actin imaging===
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{{Template:Safety Warning|message=Wear gloves when you are handling biological samples.}}
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Since actin filaments and stress fibers are nanometer-scale objects, they are much dimmer than fluorescent
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beads or the dye solution - care must be taken to get good images of the cytoskeleton. You may
+
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need to cover the microscope to reduce room light contamination.
+
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+
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# Adjust the gain and exposure of the camera to get the best picture. Be sure to keep the same exposure conditions, however, for both untreated and CytoD-treated cells.
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# Using the 40x objective, take some good fluorescence images of Alexa Fluor phalloidin-labeled cells, both untreated and CytoD-treated.
+
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+
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===Live cell microrheology by particle traking===
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+
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Now that you have verified microscope stability and performance through the [[Optical Microscopy Part 3a: Particle Tracking|particle tracking lab]], you can apply its methods on cell samples. A key technique to keep in mind when working with live cells - to avoid shocking them with "cold" at 20°C, be sure that any solutions you add are pre-warmed to 37°C. We will keep a warm-water bath running for this purpose, in which we will keep the various media.
+
−
 
+
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[[File: 3T3.PNG|thumb|200px|3T3 Swiss Albino]]
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You are provided with NIH 3T3 fibroblasts, which were prepared as follows:
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Cells were cultured at 37°C in 5% CO<math>_2</math> in standard T75 flasks in a medium referred to as DMEM++. This consists of Dulbecco's Modified Eagle Medium (DMEM - Invitrogen) supplemented with 10% fetal bovine serum (FBS - Invitrogen) and 1% of the antibiotic penicillin-streptomycin (Invitrogen). The day prior to the microrheology experiments, fibroblasts were plated on 35 mm glass-bottom
+
−
cell culture dishes (MatTek). On the day of the experiments, the cell confluency should reach about 60%. 1 μm diameter orange fluorescent microspheres (Molecular Probes) were mixed with the growth medium (at a concentration of 5 x 10<math>^5</math> beads/mL) and added to the plated cells for a period of 12 to 24 hours for bead endocytosis.
+
−
 
+
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# Carefully pipet out the medium from both dishes. Replace with 2 mL of fresh, pre-warmed DMEM++ in each dish.
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# Choose cells with 3 or 4 particles embedded in them and capture movies of the samples. Take multiple movies with about 3-5 cells in the field of view in each movie.
+
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# In one of the dishes, treat the cell with the cytoskeleton-modifying chemical cytochalasin D (CytoD): Pipet out the medium, add 1 mL CytoD solution at 10 μM (pre-mixed for you) to the dish, and wait for 20-30 minutes. It's a good idea to check on your cells after 20 minutes: sometimes they are in bad shape at that point but sometimes they still look very healthy. Wash 2X with 2 mL of pre-warmed DMEM++, leaving 2 mL in the dish when imaging.
+
−
# Perform and repeat the particle tracking measurements again as quickly as you are able. The cells' physiology has now been significantly disrupted by the toxin CytoD, and they will die within a couple of hours. It's very unlikely that you'll be able to find the exact same cells you've already tracked; however it's very much advisable to use the same dish for the "before" and "after" so you're aren't also comparing between different cell populations.
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+
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[[Image:20.309_130813_ActinExampleImage.png|center|thumb|400px|Example image included by past students in their Part 3b report: Alexa Fluor phalloidin-stained F-actin and fluorescent beads in fibroblast cells.]]
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==Lab manual homepage==
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[[Lab Manual: Optical Microscopy]]
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==References==
 
==References==
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<references/>
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<References/>
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{{Template:20.309 bottom}}
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Latest revision as of 01:01, 10 March 2016

20.309: Biological Instrumentation and Measurement

ImageBar 774.jpg


In this part of the lab, you will follow microscopic objects throughout a series of movie frames: small, fluorescent microspheres first diffusing in purely viscous solutions of glycerol-water, and next moving in fibroblast cells after endocytosis. Calculating the mean squared displacement of their motion as a function of time interval will allow you to characterize their physical environment and behavior, first in terms of diffusivity and viscosity coefficients of the glycerol-water mixtures, next recognizing other material or transport properties in fibroblast cells.

Contextual background

Background references

Brownian motion

This section was adapted from http://labs.physics.berkeley.edu/mediawiki/index.php/Brownian_Motion_in_Cells.

If you have ever looked at an aqueous sample through a microscope, you have probably noticed that every small particle you see wiggles about continuously. Robert Brown, a British botanist, was not the first person to observe these motions, but perhaps the first person to recognize the significance of this observation. Experiments quickly established the basic features of these movements. Among other things, the magnitude of the fluctuations depended on the size of the particle, and there was no difference between "live" objects, such as plant pollen, and things such as rock dust. Apparently, finely crushed pieces of an Egyptian mummy also displayed these fluctuations.

Brown noted: [The movements] arose neither from currents in the fluid, nor from its gradual evaporation, but belonged to the particle itself.

This effect may have remained a curiosity had it not been for A. Einstein and M. Smoluchowski. They realized that these particle movements made perfect sense in the context of the then developing kinetic theory of fluids. If matter is composed of atoms that collide frequently with other atoms, they reasoned, then even relatively large objects such as pollen grains would exhibit random movements. This last sentence contains the ingredients for several Nobel prizes!

Indeed, Einstein's interpretation of Brownian motion as the outcome of continuous bombardment by atoms immediately suggested a direct test of the atomic theory of matter. Perrin received the 1926 Nobel Prize for validating Einstein's predictions, thus confirming the atomic theory of matter.

Since then, the field has exploded, and a thorough understanding of Brownian motion is essential for everything from polymer physics to biophysics, aerodynamics, and statistical mechanics. One of the aims of this lab is to directly reproduce the experiments of J. Perrin that lead to his Nobel Prize. A translation of the key work is included in the reprints folder. Have a look – he used latex spheres, and we will use polystyrene spheres, but otherwise the experiments will be identical. In addition to reproducing Perrin's results, you will probe further by looking at the effect of varying solvent molecule size.

Diffusion coefficient of microspheres in suspension

According to theory,[1][2][3][4] the mean squared displacement of a suspended particle is proportional to the time interval as: $ \left \langle {\left | \vec r(t+\tau)-\vec r(t) \right \vert}^2 \right \rangle=2Dd\tau $, where r(t) = position, d = number of dimensions, D = diffusion coefficient, and $ \tau $= time interval.

Instructions

Estimating the diffusion coefficient by tracking suspended microspheres

Imaging chamber for fluorescent microspheres diffusing in water:glycerol mixtures

1. Track some 0.84μm Nile Red Spherotech polystyrene beads in water-glycerin mixtures (Samples A, B and C contain 0%, 30% and 50% glycerin, respectively).

Notes: Fluorescent microspheres have been mixed for you by the instructors into water-glycerin solutions A, B, C, and D. (a) Vortex the stock Falcon tube, and then (b) transfer the bead suspension into its imaging chamber (consisting of a microscope slide, double-sided tape delimiting a 2-mm channel, and a 22x40mm No. 1.5 coverslip, and sealed at both ends nail polish).
Tip: Do not choose to monitor particles that remain stably in focus: these are likely to be 'sitting on the coverslip' and their motion will not be representative of diffusion in the viscous water-glycerol fluid.

2. Estimate the diffusion coefficient of these samples: MSD = $ \left \langle {\left | \vec r(t+\tau)-\vec r(t) \right \vert}^2 \right \rangle=2Dd\tau $, where r(t) = position, d = number of dimensions, D = diffusion coefficient, and $ \tau $= time interval. Use Sample A to verify that your algorithm correctly calculates the viscosity of water at the lab temperature (check the temperature on the clock on the wall or by other means).

  • Consider how many particles you should track and for how long. What is the uncertainty in your estimate?
  • From the viscosity calculation, estimate the glycerin/water weight ratio. (This chart is a useful reference.)
  • See: this page for more discussion of Brownian motion and a Matlab simulation.


Live cell particle tracking of endocytosed beads

We can also use particle tracking to probe cell samples. 0.84 μm diameter red fluorescent microspheres were mixed with the growth medium and added to the plated cells for a period of 12 to 24 hours for bead endocytosis.

You will be given two plates of cells for these experiments:

  • Dish 1 will be used to monitor particles in untreated cells, while
  • Dish 2 will be reserved to track microspheres after adding CytoD.
  1. Pre-warm your DMEM++ and CytoD to 37°C
  2. Carefully pipet out the medium from Dish 1. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed. Then, place 2 mL of fresh medium in dish.
  3. Choose cells in Dish 1 with 3 or 4 particles embedded in them and capture movies of the samples. Take as many movies as you can with about 3-5 cells in the field of view in each movie. Make sure to do this quickly, as the cells become unhealthy without the temperature and carbon dioxide regulation.
  4. Next, carefully pipet out the medium in Dish 2. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed.
  5. Treat the cells in Dish 2 with the cytoskeleton-modifying CytoD: Pipet out remaining medium, add 1 mL pre-warmed CytoD solution at 10 μM (pre-mixed for you) to the dish, and incubate for 20 minutes at 37°C. It's a good idea to check on your cells after 15 minutes: sometimes they are in bad shape at that point but sometimes they still look very healthy. Wash 2X with 2 mL of pre-warmed DMEM++, leaving 2 mL in the dish when imaging.
  6. Perform and repeat the particle tracking measurements again in Dish 2 as quickly as you are able. It would be good to image the beads in only one cell at a time, since different cells may have different degrees of cytoskeletal disruption. Take as many videos as you can before the cells become sad. The cells' physiology has now been significantly disrupted by the toxin CytoD, and they will die within a couple of hours.

Report

Find and follow all guidelines on the Microscopy report outline wiki page. Remember that all numerical quantities must be reported with an associated level of uncertainty or significance.

  1. Viscosity
    1. Procedure
      • Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)
    2. Data
      • Include a snapshot of the 0.84 μm fluorescent beads monitored.
      • Plot two or more example bead trajectories for each of the glycerin samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)
    3. Analysis and Results
      • Plot the average MSD vs τ results for all glycerin samples (A, B, C, and D); use log-log axes. Use the minimum number of axes that can convey your results clearly.
      • Include a table of the diffusion coefficient, viscosity and glycerin/water ratio for each of the samples (A, B, C, and D).
      • Provide a bullet point outline of all calculations and data processing steps.
    4. Discussion
      • How do your viscosity calculations compare to your expectations? (This chart is a useful reference.)
      • Include a thorough discussion of error sources and the approaches to minimize them. It may be helpful to list out the error sources in a table, including a category for the error source, type of error (random, systematic, fundamental, technical, etc.), the magnitude of the error, and a description and way to minimize each one.
  1. Particle Tracking in Cells
    1. Procedure
      • Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)
    2. Data
      • Include a snapshot of the 0.84 μm fluorescent beads monitored.
      • Plot two or more example bead trajectories for each of the samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)
    3. Analysis and Results
      • Combine your data with others from the class to increase your sample size.
      • Plot the average MSD for untreated and Cyto D treated cells on a single set of log-log axes.
    4. Discussion
      • What kind of motion do you see described by your MSD vs τ results?
      • What differences do you see between the untreated and Cyto D treated MSD curves?
      • Please suggest an interpretation of the behavior of your cells based on your data.
      • Include a discussion of your error sources.

Optical microscopy lab

Code examples and simulations

Background reading

References

  1. ↑ A. Einstein, On the Motion of Small Particles Suspended in Liquids at Rest Required by the Molecular-Kinetic Theory of Heat, Annalen der Physik (1905).
  2. ↑ E. Frey and K. Kroy, Brownian motion: a paradigm of soft matter and biological physics, Ann. Phys. (2005). Published on the 100th anniversary of Einstein’s paper, this reference chronicles the history of Brownian motion from 1905 to the present.
  3. ↑ R. Newburgh, Einstein, Perrin, and the reality of atoms: 1905 revisited, Am. J. Phys. (2006). A modern replication of Perrin's experiment. Has a good, concise appendix with both the Einstein and Langevin derivations.
  4. ↑ M. Haw, Colloidal suspensions, Brownian motion, molecular reality: a short history, J. Phys. Condens. Matter (2002).
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