|
|
| (One intermediate revision by one other user not shown) |
| Line 1: |
Line 1: |
| '''Tethered particle motion (TPM)''' is a biophysical method that is used for studying various [[polymers]] such as [[DNA]] and their interaction with other entities such as [[proteins]].
| | e - Shop Word - Press is a excellent cart for your on the web shopping organization. Affilo - Theme is the guaranteed mixing of wordpress theme that Mark Ling use for his internet marketing career. PSD files are incompatible to browsers and are suppose to be converted into wordpress compatible files so that it opens up in browser. After confirming the account, login with your username and password at Ad - Mob. In the most current edition you can customize your retailer layout and display hues and fonts similar to your site or blog. <br><br>These websites can be easily customized and can appear in the top rankings of the major search engines. Infertility can cause a major setback to the couples due to the inability to conceive. A Wordpress plugin is a software that you can install into your Wordpress site. They provide many such popular products which you can buy for your baby. For a Wordpress website, you don't need a powerful web hosting account to host your site. <br><br>Digital photography is a innovative effort, if you removethe stress to catch every position and viewpoint of a place, you free yourself up to be more innovative and your outcomes will be much better. When a business benefits from its own domain name and a tailor-made blog, the odds of ranking higher in the search engines and being visible to a greater number of people is more likely. After age 35, 18% of pregnancies will end in miscarriage. Nonetheless, with stylish Facebook themes obtainable on the Globe Broad Internet, half of your enterprise is done previously. Article Source: Stevens works in Internet and Network Marketing. <br><br>Digg Digg Social Sharing - This plugin that is accountable for the floating social icon located at the left aspect corner of just about every submit. Quttera - Quttera describes itself as a 'Saa - S [Software as a Service] web-malware monitoring and alerting solution for websites of any size and complexity. re creating a Word - Press design yourself, the good news is there are tons of Word - Press themes to choose from. IVF ,fertility,infertility expert,surrogacy specialist in India at Rotundaivf. If your blog employs the permalink function, This gives your SEO efforts a boost, and your visitors will know firsthand what's in the post when seeing the URL. <br><br>You will know which of your Word - Press blog posts are attracting more unique visitors which in turn will help you develop better products and services for your customers. In fact portfolio Word - Press themes is a smooth and attractive but considerably flawed Word - Press theme in creating simpler to the photographers or designers to develop a specific internet site showcasing their most current perform since it appear modern-day and has fantastic typography and large photographs which would develop an attractive wanting portfolio internet site. It can be concluded that white label SEO comprise of a third party who resells a contract involving IT expert or consultant, SEO professional and end user. If you adored this article and also you would like to get more info relating to [http://GET7.pw/wordpress_dropbox_backup_818488 wordpress backup plugin] nicely visit our own web-page. And, it is better that you leave it on for the duration you are writing plugin code. Verify whether your company has a team of developers or programmers having hands-on experience and knowledge about all Word - Press concepts. |
| [[Image:Tethered particle motion sketch.JPG|right|thumb|250px|'''TPM''' - the bead diffuses in the solution, but because of the tethering, it can move only in restricted volume.]]
| |
| The method allows observers to measure various physical properties on the substances, as well as to measure the properties of biochemical interactions with other substances such as proteins and enzymes.
| |
| TPM is a [[single molecule experiment]] method.
| |
| | |
| ==History==
| |
| TPM was first introduced by Schafer, Gelles, Sheetz and Landick in 1991.<ref>[http://www.nature.com/nature/journal/v352/n6334/pdf/352444a0.pdf Schafer, D.A., et al., ''Transcription by single molecules of RNA polymerase observed by light microscopy.'' Nature, 1991. '''352''': p. 444-448.]</ref> In their research, they attached [[RNA polymerase]] to the surface, and gold beads were attached to one end of the DNA molecules. In the beginning, the RNA polymerase "captures" the DNA near the gold bead. During the [[Transcription (genetics)|transcription]], the DNA "slides" on the RNA polymerase so the distance between the RNA polymerase and the gold bead (the tether length)is increased. Using an [[optical microscope]] the area that the bead moves in was detected. The transcription rate was extracted from data.<br>
| |
| Since then, a lot of TPM experiments have been done, and the method was improved in many ways such as beads types, biochemstry techniques, imaging (faster cameras, different microscopy methods etc.) data analysis and combination with other single-molecule techniques (e.g. optical or magnetical tweezers).
| |
| | |
| == Principle of the method ==
| |
| One end of a polymer is attached to a small bead (tens to hundreds of nanometer), while the other end is attached to a surface.
| |
| Both the polymer and the bead stay in an aqueous environment, so the bead moves in [[Brownian motion]]. Because of the tether, the motion is restricted. Using an [[optical microscope]] and [[CCD camera]], one can track the bead position in a time series. Although the bead is usually smaller than the [[diffraction limit]], so the image is a spot which is larger than the bead itself ([[point spread function]]), the center of the spot represents the projection on the X-Y plane of the end of the polymer ([[end-to-end vector]]). Analyzing the distribution of the bead position can tell us a lot of information about the polymer.
| |
| [[Image:New TPM simulation.gif|right|thumb|250px|'''TPM simulation'''. The simulation done by [[MATLAB]], using [[Freely Jointed Chain]] model of [[polymer]]. The green points represent the projection of the end-to-end vector of the polymer on the XY plane.]]
| |
| ===Excursion number===
| |
| In order that the motion would be polymer dominated, and not bead dominated, one should notice that the excursion number<ref>[http://dx.doi.org/10.1103/PhysRevLett.96.088306 Segall, D.E., et al., ''Volume-exclusion effects in tethered-particle experiments: bead size matters.'' Physical Review Letters, 2006. '''96''': p. 088306-1-4.]</ref> will be less than 1:<br>
| |
| <center><math>N_R\equiv\frac{r}{\sqrt{L\cdot{l_p/3}}}<1</math></center>
| |
| where <math>r</math> is the bead radius, <math>L</math> is the [[contour length]] of the polymer and <math>l_p</math> is the [[persistence length]] (50 nm in physiological conditions) of the polymer. (It is possible to work also when <math>N_R>1</math>, but it should be treated carefully.)
| |
| | |
| ===Bead types===
| |
| * '''Metal beads:''' The metallic beads (usually gold) scatter light with high intensity, so one can use very small beads (~40 nm diameter), and still have a good picture. From the other hand, metallic beads are not the appropriate tool for [[optical tweezers]] experiments.
| |
| * '''Polystyrene beads:''' The [[polystyrene]] bead scatter light weaker than metallic (in order to get the same intensity as getting from 40 nm gold bead, the polystyrene bead should be ~125 nm!<ref>Paul R. Selvin, Taekjip Ha, ''Single Molecule Techniques (Chapter 19)'', Cold Spring Harbor Laboratory Press, 2008</ref>), but it has the advantage that it can be combined with optical tweezers experiments.
| |
| * '''Fluorospheres:''' The major advantage of fluorospheres is that the excitation [[wavelength]] and the emission wavelength are not the same, so [[dichroic filter]] can be used to give a cleaner signal. The disadvantage of the fluorospheres is [[photobleaching]].
| |
| | |
| All of the bead types and diameters (with the biochemistry marker, look at the tether assembly section) are manufactured by commercial companies, and can purchased easily.
| |
| | |
| ==Chip and Tether Assembly==
| |
| ===Chip assembly===
| |
| A chip can be made of two coverslips. One of them should be drilled to make two hole, allowing the reagents to be injected into the flowcell. The slides should be cleaned to remove dirt. A bath sonicator is a good tool for that, 15 minutes in Isopropanol should do the trick. Next, the a channel should be made. One way of doing so is to cut parafilm in the center, leaving a frame of parafilm that would be used as a spacer between the slides. The slides should be assembled one on the other with the cut parafilm between them. The final step is to heat the chip so that the parafilm will melt and glue the slides together.
| |
| | |
| ===Tether Assembly===
| |
| First, the chip has to be passivated so that the polymer won't stick to the glass, there are plenty of blocking reagents available (BSA, alpha-casein, etc.) and one should find what works best for the specific situation
| |
| Next, the surface should be coated with an antibody or other reactive molecule (such as anti-[[digoxigenin]]) that will bind to an antigen ([[digoxigenin]]) at one end of the polymer. After an incubation of about 45min, the excess antibody has to be washed away.
| |
| After washing the excess antibody, the polymer should be injected into the chip and incubated for about the same time. The polymer had been modified before at the ends. One end has a biotin tail and the other has a digoxigenin tail. After incubation, unbound polymer has to be washed out from the cell. Then, anti-[[biotin]] coated beads should be injected to the flowcell and incubate for about 30-45min. Excess beads should be washed out.
| |
| | |
| == Data Analysis ==
| |
| [[Image:TPM Experiment video.ogv|right|thumb|250px|Video taken from TPM experiment, using darkfield microscope. The green framed beads are tethered beads, and the red framed are immobilized beads.]]
| |
| | |
| ===Tracking===
| |
| As mentioned above, the image doesn't show the bead itself but a larger spot according to its '''PSF''' ([[Point spread function]]). In addition, the [[pixel]] size on the camera may reduce the resolution of the measure.
| |
| In order to extract the exact bead's position (that corresponds to the end-to-end vector), the center of the spot should be found as accurate as possible. It can be done with good resolution using two different techniques, both based on spot characteristics. The light intensity in the [[focal plane]] distributed as [[airy disk]], and has circular symmetry.<br>
| |
| '''Gaussian fitting:''' A 2-dimensional [[Gaussian function]] is a good approximation for [[airy disk]]. By fitting this function to the spot one can find the parameters <math>x_0</math> and <math>y_0</math> that are the coordinates of the center of the spot, and of the end-to-end vector.<br>
| |
| '''"Center of mass":''' The second technique is to find the center of intensity,<ref>[http://www.biophysj.org/cgi/reprint/89/2/1272.pdf Blumberg, S., et al., ''Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy.'' Biophysical Journal, 2005. '''89''': p. 1272-1281.]</ref> using the definition of [[center of mass]]:
| |
| <center><math>\vec{R}_{cm}=\frac{1}{I_{tot}}\cdot\sum_{k=1}^{N}I_k\cdot\vec{r}_k</math></center>
| |
| where <math>\vec{R}_{cm}</math> is the center of mass coordinate, <math>\textstyle I_{tot}</math> is the total intensity of the spot, and <math>\textstyle I_k</math> and <math>\textstyle\vec{r}_k</math> are the intensity and coordinate of the ''k''-th pixel. Because of the circular symmetry, the coordinate of the center of intensity is the coordinate of the center of the bead.<br>
| |
| Both techniques give us the coordinate of the end-to-end vector in a resolution better than pixel size.
| |
| | |
| [[Image:TPM drift correction.jpg|left|thumb|250px|'''TPM drift:'''<br> '''top:''' The green graph is the data of the TPM experiment, the black curve is smoothing of the data (the drift).<br>'''bottom:''' Subtraction of the drift from the data.]]
| |
| | |
| ===Drift correction===
| |
| Usually, the whole system drifts during the measuring. There are several methods to correct the drift, generally these can divided into 3 groups:<br>
| |
| '''Frequency:''' The Brownian motion frequency is much larger than the drift frequency, so one can use [[high-pass filter]] in order to remove the drift. Similar effect can achieve by smoothing the data, and subtraction of the smoothed from the data (see figure).<br>
| |
| '''Averaging over few beads:''' If few beads are shown in the frame, because every bead moving randomly, averaging over the position of them for every frame should give us the drift (it should subtracted from the data for having clean data).<br>
| |
| '''Immobilized bead:''' If an immobilized bead is shown in the frame, we can take its position as a reference, and correct the data by the immobilized bead's position. (Another advantage of looking at immobilized bead, is the fact that the motion of it can tell us about the accuracy of the measure.)<br>
| |
| Of course one can use more than one method.
| |
| | |
| ===Polymer characterization===
| |
| It is common to fit [[random walk]] statistics to the end-to-end vector of the polymer.<ref>Rubinstein, M. & Colby, R.H., ''Polymer Physics (chapter 2.5 – Distribution of end-to-end Vector)'', OXFORD University Press (2003).</ref> For 1-dimensional we'll get the [[Normal distribution]], and for 2-dimensional the [[Rayleigh distribution]]:
| |
| | |
| <center><math>
| |
| P_{1D}(x)dx=\sqrt{\frac{3}{4{\pi}Ll_p}}\exp{\left(-\frac{3x^2}{4Ll_p}\right)}dx \,\,\,;\,\,\,\,\,\,
| |
| P_{2D}(R)dR=2{\pi}R\frac{3}{4{\pi}Ll_p}\exp{\left(-\frac{3R^2}{4Ll_p}\right)}dR
| |
| </math></center><br>
| |
| where <math>L</math> is the contour length and <math>l_p</math> is the persistence length.<br>
| |
| After collecting the data of time series, one should fitting the [[histogram]] of the data to the distribution function (one or two dimensional). If the contour length of the polymer is known, the only fitting parameter is the persistence length.
| |
| | |
| ===Spring constant===
| |
| Due to [[entropic force]], the polymer acts like [[Statistical_mechanics#Random walks in space|Hookian spring.]] According to [[Boltzmann distribution]], the distribution is proprtional to exponent of the ratio between the [[elastic energy]] and the [[thermal energy]]:
| |
| <center><math> P(x)\propto \exp{\left(-\frac{\frac{1}{2}kx^2}{K_BT}\right)} </math></center>
| |
| where <math>k</math> is the [[spring constant]], <math>K_B</math> is [[Boltzmann constant]] and <math>T</math> is the temperature. By taking the [[logarithm]] of the distribution <math>P(x)</math> and fitting it to a [[parabola]] shape, one can get the spring constant of the polymer:<ref>[http://dx.doi.org/10.1117/12.699040 Dietrich, H.R.C., et al., ''A new optical method for characterizing single molecule interactions based on dark field microscopy.'' Proceedings of the SPIE, 2007.]</ref>
| |
| <center><math>\displaystyle k=2{\alpha}K_BT</math></center>
| |
| where <math>\alpha</math> is the coefficient of <math>x^2</math> from the parabola fit.
| |
| | |
| ==Advantage and disadvantage==
| |
| ===Advantage===
| |
| * Simple setup system.
| |
| * Not expensive.
| |
| * Observations are made in the polymer's natural environment (no external forces are being used).
| |
| * Suitable to various microscopy method (e.g. [[TIRFM]], [[Dark field]], [[Differential interference contrast microscopy]], etc.).
| |
| * Can be combined and manipulated using other methods.
| |
| * High variety of applications.
| |
| | |
| ===Disadvantage===
| |
| * Low spatial resolution (~30 nm).
| |
| * Fits to [[in vitro]] experiments only.
| |
| | |
| ==References==
| |
| {{reflist}}
| |
| | |
| [[Category:Biophysics]]
| |
e - Shop Word - Press is a excellent cart for your on the web shopping organization. Affilo - Theme is the guaranteed mixing of wordpress theme that Mark Ling use for his internet marketing career. PSD files are incompatible to browsers and are suppose to be converted into wordpress compatible files so that it opens up in browser. After confirming the account, login with your username and password at Ad - Mob. In the most current edition you can customize your retailer layout and display hues and fonts similar to your site or blog.
These websites can be easily customized and can appear in the top rankings of the major search engines. Infertility can cause a major setback to the couples due to the inability to conceive. A Wordpress plugin is a software that you can install into your Wordpress site. They provide many such popular products which you can buy for your baby. For a Wordpress website, you don't need a powerful web hosting account to host your site.
Digital photography is a innovative effort, if you removethe stress to catch every position and viewpoint of a place, you free yourself up to be more innovative and your outcomes will be much better. When a business benefits from its own domain name and a tailor-made blog, the odds of ranking higher in the search engines and being visible to a greater number of people is more likely. After age 35, 18% of pregnancies will end in miscarriage. Nonetheless, with stylish Facebook themes obtainable on the Globe Broad Internet, half of your enterprise is done previously. Article Source: Stevens works in Internet and Network Marketing.
Digg Digg Social Sharing - This plugin that is accountable for the floating social icon located at the left aspect corner of just about every submit. Quttera - Quttera describes itself as a 'Saa - S [Software as a Service] web-malware monitoring and alerting solution for websites of any size and complexity. re creating a Word - Press design yourself, the good news is there are tons of Word - Press themes to choose from. IVF ,fertility,infertility expert,surrogacy specialist in India at Rotundaivf. If your blog employs the permalink function, This gives your SEO efforts a boost, and your visitors will know firsthand what's in the post when seeing the URL.
You will know which of your Word - Press blog posts are attracting more unique visitors which in turn will help you develop better products and services for your customers. In fact portfolio Word - Press themes is a smooth and attractive but considerably flawed Word - Press theme in creating simpler to the photographers or designers to develop a specific internet site showcasing their most current perform since it appear modern-day and has fantastic typography and large photographs which would develop an attractive wanting portfolio internet site. It can be concluded that white label SEO comprise of a third party who resells a contract involving IT expert or consultant, SEO professional and end user. If you adored this article and also you would like to get more info relating to wordpress backup plugin nicely visit our own web-page. And, it is better that you leave it on for the duration you are writing plugin code. Verify whether your company has a team of developers or programmers having hands-on experience and knowledge about all Word - Press concepts.