DynaPro-MSXTC Operation: An Overview

1. DynaPro-MSXTC Operation:An Overview A step by step guide to operating the DynaPro MSXTC Assumes the operator is familiar with the Start-up Guide, Operator’s Manual, and Data Interpretation Guide. Nov. 12, 2004 中国科学技术大学生命科学学院

2. Outline of Operation • Introduction • Power on DynaPro and launch Dynamics software • Open the preset file Or Select and modify the experimental parameters (solvent, temperature) • Load the sample into the cuvette • Place cuvette into the microsampler • Wait 5 minutes for equilibration to temperature • Launch event scheduler or Acquire data manually • Monitor datalog for indications of acceptable data • Monitor trace view for preliminary indications of homogeneity and heterogeneity • Upon completion of data acquisition, review the ‘goodness’ of data, and take appropriate action. • If the data is ‘good’, accept and save the data into a file • Preliminary analysis of size distributions • If the data is ‘not good’, or if acquisition is interrupted, troubleshoot the problem • When complete, remove the cuvette and recover sample • Clean the cuvette for next sample or for storage • Load the next sample into the cuvette • Decide if include next sample in same data file or if start new data file • Repeat or Exit software and power down DynaPro 中国科学技术大学生命科学学院

3. The purpose of the DynaPro Operation Overview is to provide step-by-step guidance for obtaining ‘good’ data. The manual assumes the reader is familiar with the DynaPro Start-up Guide (e.g. the DynaPro is already installed in your laboratory), the Operator’s Manual (has been reviewed at least one time and have attempted to collect data); and familiar with the Data Interpretation Guide (e.g. some attempt to understand size distributions and their interpretation, and some qualification of the raw data). The manual will not cover the basic theories of light scattering, however it will provide you with references which do cover the theories in detail. Introduction 中国科学技术大学生命科学学院

4. Electrical DynaPro Host Analog/ Digital I/O DynaPro MSXTC Correlator I/O Detector TTL USB Port Optical fibers Laser USB Cable RS232 Serial Personal Computer System Block Diagram 中国科学技术大学生命科学学院

5. Set-up • Refer to the start-up guide provided with your DynaPro. • Remove Host Unit and Plate Reader Optics from Box • Connect optical fibers • Connect Power cords, USB Cable, and RS232 Cable to the back of the Plate Reader • Install Dynamics Software CD and Floppy Disk • Power-on DynaPro • Start software 中国科学技术大学生命科学学院

6. 1. Turn power on to both components 2. Double click the Dynamics V6 Icon Power on and Launch Dynamics 中国科学技术大学生命科学学院

7. 1. Open a Preset file (File, Open Preset) that contains previously defined experiment parameters and software settings. 2. Alternatively, begin a new experiment file (File, New) and manually select and edit the parameters (particularly the Solvent) Open Preset File or Edit Parameters 中国科学技术大学生命科学学院

8. Proterion recommends: • first load the sample unfiltered, with a pipettor or syringe needle; at minimum load 12 microliters into the 12 microliter cuvette. Place the tip onto the bottom of the cuvette and then slowly inject into the sample chamber. • If the data indicate aggregation or presence of large particles, it may be necessary to spin the sample (10,000 rpm for 20-30 minutes Load Sample into Cuvette 中国科学技术大学生命科学学院

9. Insert Cuvette into Microsampler • With the frosty side of the cuvette to the left, insert the cuvette into the optical chamber. • Press the cuvette down until it no longer moves. • Then close the lid of the DynaPro. 中国科学技术大学生命科学学院

10. Wait Five Minutes for equilibration • Proterion recommends waiting approximately five minutes for the sample to reach the temperature of the optical chamber, particularly when the chamber is temperature controlled. • While waiting, make sure the count rates are more than the pure solvent count rate by examining the count rate monitor, as shown to the left. 中国科学技术大学生命科学学院

11. When the cuvette is inserted press the green“Go” button to acquire data. If not green, you must Connect the software to the DynaPro (refer to start up guide or manual) After approximately 100 seconds of data have been acquired, press the “Stop” Button – the same button, which is now the color red. Measure Manually 中国科学技术大学生命科学学院

12. The Event Scheduler is opened by placing the cursor on the ‘Tree’ (e.g. Measurements), right clicking, and choosing Event Scheduler. Measure Automatically • The DynaPro can also be operated in an automatic fashion with the Event Scheduler (access by right clicking mouse on ‘measurement’ tree, left hand side). The Event Scheduler can perform time delays, acquire data, save data, set temperatures and more. 中国科学技术大学生命科学学院

13. Save the Data • Before proceeding, Proterion recommends saving the data to avoid potential data loss due to uncontrollable events such as power failure, PC malfunction, etc. • Press File, Save or the ‘save file’ icon, and insert the name of the file. 中国科学技术大学生命科学学院

14. Good or Bad: Judging the Goodness of the Data • The DynaPro software, Dynamics, does provide basic data analyses that indicate if the data are in ‘acceptable’ ranges. The analyses are based upon simple numerical data filters or qualifiers. Yet these data filters do not always capture or allow for good and bad raw data. • We will explain what is good or bad by commenting on various examples of raw data. The name applied to the raw data is an autocorrelation function. An autocorrelation function is a collection of correlation coefficients – unitless values indicating the level of similarity among sets of data. At this time we will not concern ourselves with the underlying theory and physical meaning of the autocorrelation function. We will examine only how to interpret these functions. • Press the Autocorrelation View at this time: 中国科学技术大学生命科学学院

15. The Autocorrelation Function The DynaPro determines the size of particles in solution by exploiting the physical process of Brownian Motion: the particles are moving in solution as a function of time, and their rate of motion is related to their size. The rate of motion is measured by illuminating the particles with laser light and determining the rate at which light scattered or reflected by the particles changes with time. The technique of autocorrelation determines the rate of these time intensity fluctuations, expressed as an autocorrelation function (shown here). An autocorrelation function is an exponential function comprised of correlation coefficients (y-axis) dependent upon the ‘delay time’ (x-axis), the time-value separating the sets of data. The function can be mathematically described by one or more decays. The rate of decay is related to particle size. A faster decay indicates a smaller particle, a slower decay a larger particle. Autocorrelation functions are determined during each acquisition comprising a measurement, as described earlier. Numerical algorithms are applied to determine the rates of decay or size distributions of the exponential autocorrelation functions. The DynaPro utilizes a proprietary ‘non-negative least squares’ algorithm, a method that finds the size distribution producing the smoothest distribution with the least amount of error. The error is the difference between the measured autocorrelation function and the fitted autocorrelation function. 中国科学技术大学生命科学学院

16. Caution Proceed Increase acquisition time If not pure solvent… Increase acquisition time Increase acquisitions Increase laser power Increase concentration Increase acquisition time Increase acquisitions Increase laser power Increase concentration Spin or filter sample Evaluate Autocorrelation Function Stop 中国科学技术大学生命科学学院

17. Take Action Proceed Stop Caution Before proceeding, attempt to improve the quality of the data by following all or some of the recommended changes to the experiment. Leave the sample in the cuvette and follow these steps. Increase acquisition time (double the current value as a rule of thumb); and/or increase the # of acquisitions (double); Increase laser power (to maximum value of 100%); if none of the above steps lead to functions shown in ‘Proceed’, it may be necessary to increase the concentration of the analyte. Ultimately, one may accept the imperfect data from this category and continue. Do not proceed with data interpretation if the autocorrelation function appears as shown. If the data appear as in the top figure, the sample probably contains large particles, and should be spun (10,000 rpm for 20-30 minutes) or filtered using syringe filters (.1 micron). If the data appear as shown in the lower figure, make sure the cuvette is inserted properly, the lid is closed, and that the sample is not pure solvent. If all of these items check, follow the recommendations under “Caution”. If these steps fail, contact technical support. If the Autocorrelation function is in the Proceed Category, continue with the interpretation of the size distribution analysis, which follows next. 中国科学技术大学生命科学学院

18. Physical Interpretations of Size Distributions Monomodal Monodisperse Monomodal Polydisperse Bimodal Monodisperse Bimodal Monodisperse Polydisperse Radius = R1 Radius = 1.5*R1 The samples contains two types of particles, monomer and trimer. The radius of the trimer is less than twice the radius of monomer so only one peak is resolved, the distribution is monomodal. However the population consists of two species and this increase in size heterogeneity causes an increase in measured polydispersity compared to the samples containing 100% monomer and 100% trimer. Also, the mean radius of the peak will be larger than R1 but smaller than 1.5 R1. The sample contains two types of particles, the monomer and a large aggregate. The large particle is more than twice the radius of the monomer and in sufficient quantities to be measured, so two peaks are resolved by the DynaPro. The mean radius of Peak 1 will be R1 and Peak 2 will be equal to 5*R1. Both species are homogeneous so measured polydispersity is low. The sample contains three types of particles monomer, trimer, and larger aggregate. In this case the DynaPro resolves only two peaks. The monomer and trimer are not resolved from each other and form only one peak, a polydisperse peak. The second peak is formed by the larger particle, which is resolvable from both monomer and trimer. The second peak is monodisperse. Radius = 5*R1 中国科学技术大学生命科学学院

19. Hydrodynamic Radius: The Physical Interpretation of ‘Size’ The DynaPro measures the size distribution of the particles in the sample. The size, previously defined as the radius or diameter of the particle, is represented in this figure as Rh. Rh, or Hydrodynamic Radius, is a particle radius that embodies a ‘hard sphere’ particle which is in fact aspherical and typically surrounded or covered by solvent. Please refer to PSI Books or the article for a more thorough explanation of the physical interpretation of particle size as measured by the DynaPro. 中国科学技术大学生命科学学院

20. Size Distribution Results Results are shown in graphical as well as tabular form. The table located below the size distribution histogram describes the number of peaks and their mean value (Radius), polydispersity (Pd), % polydispersity (%Pd), molecular weight estimated from the measured radius (MW-R), relative amount of light scattered by each population (%Int), and estimated relative amount of mass (concentration) of each peak or species (%Mass). 中国科学技术大学生命科学学院

21. Monomodal Size Distribution Histogram Y-axis Relative amount of light scattered by each bin, % Intensity (% of Total Light Scattered). Represents the probability of existence of the species. This histogram has one peak so we call it a monomodal size distribution. The peak is defined by the mean value and polydispersity. The ‘width’ of the peak is the standard deviation of the weighted bin values, also known as the Polydispersity. X-axis Discrete particle sizes, in nanometers The mean value of the peak is defined by a weighted average of the number of bins comprising the histogram, in this case three. The bins by themselves do not represent real, distinct, physical particles however their mean and standard deviation do. 中国科学技术大学生命科学学院

22. Multimodal Size Distribution What causes Modality? The presence of different and resolvable species in the sample cause modes in the size distribution. To be resolved as a separate peak, a species must have a size (radius) larger than another species by a factor of two or more, and be detectable (produce sufficient scattered light for detection by the DynaPro). Roughly speaking a factor of two in radius is equivalent to a factor of eight (octamer) in MW. When the sizes of the species are below this factor, a separate peak will not be resolved for each species. This histogram has more than one peak so we call it a multimodal size distribution. Specifically this histogram is trimodal. The DynaPro determined three distinct populations exist in this sample. By definition, a multimodal size distribution is heterogeneous: the sample contains distinct populations of particles that are not the same size. The DynaPro can resolve up to four or five modes in a size distribution. For each mode, the DynaPro estimates the relative amount of light scattered and the relative amount of mass based upon one of several possible particle scattering properties. Often times the relative amount of mass of a peak is quite small e.g. less than .1 % and is considered to be negligible. 中国科学技术大学生命科学学院

23. Polydispersity Each peak has a unique mean value and width or Polydispersity. It is useful to normalize Polydispersity to the mean size of the peak, also known as percent polydispersity. Polydispersity refers to the level of homogeneity of the sizes of the particles. When the level of homogeneity is high, the particles can be considered to be virtually identical in their size, or monodisperse. The level of homogeneity is considered high when the percent polydispersity is less than 15%. When the level of homogeneity is low (percent polydispersity greater than 30%), the particle population can be considered to contain significantly different sizes, or ‘polydisperse’. What causes Polydispersity? Heterogeneity is caused by the presence of different species that can not be resolved by the technique of dynamic light scattering (species with sizes less than a factor of two relative to other species exist in solution can not be resolved). A peak containing 100% monomer will have a smaller polydispersity than peak containing a mixture of monomer:octamer. The peaks shown here all have % Polydispersity greater than 30%. Note: refer to appendix for an alternate cause of polydispersity. 中国科学技术大学生命科学学院

24. Monomodal Monodisperse Monomodal ‘Polydisperse’ Multimodal Polydisperse BLGA, 4 mg/ml, PBS, T = 5 C Peaks: 1 Mean Radius: 3.4 nm % Poly: 22.1 % Increasing amounts of Dimer BSA, 2 mg/ml, PBS, T = 25 C Peaks: 2 Peak 1: • Mean Radius: 4.3 nm • % Poly: 32.1 % • Monomer, Dimer, Trimer Peak 2: • Mean Radius: 130.9 • % Poly: 34.5 % • Various non-specific aggregates BLGA, 4 mg/ml, PBS, T = 25 C Peaks: 1 Mean Radius: 2.8 nm % Poly: 13.8 % Majority monomer Size Distribution Interpretations 中国科学技术大学生命科学学院

25. Dynamics V6, the state-of-the-art DynaPro application software, provides several data management and analysis tools specifically designed for large amounts of data. The Trace View is an X-Y based graphical system, with customer selectable X-axis and Y-axis parameters (left and right hand axes, multiple parameters), for data analysis and presentations. The View is easily exported to other MS applications for professional reports and presentations. The Datalog View is a customer configurable Table designed to support database management tools available in Excel or other database programs. Manage and Analyze Data 中国科学技术大学生命科学学院

26. Trace View • Select the Trace View to Present large quantities of data in graphical form, as line or symbols, with customer selectable X-axis and Y-axis parameters. • The control panel provides lists of variables for X, Y axes as well as scaling, zooming, and legend control • Add data to the ‘file’, create a database for your protein or polymer, at any time. 中国科学技术大学生命科学学院

27. Datalog • Select the Datalog View to access customer selected and designed parameters, and export to database management and analysis programs. 中国科学技术大学生命科学学院

28. Excel • Copy and paste from the Datalog View into Excel. With the Excel “AutoFilter” and other data analysis tools sort, organize, search, graph, manipulate the data. The data shown below have been sorted by Radius, increasing value. 中国科学技术大学生命科学学院

29. Remove cuvette, recover sample • Open lid and extract cuvette. • Insert the pipettor tip or syringe needle into the cuvette and recover the sample, if required. • Then rinse thoroughly with water and dry with air or nitrogen. • Insert next sample and continue. • If finished for the day, Proterion recommends placing the cuvette into a detergent solution overnight. 中国科学技术大学生命科学学院

30. Repeat and add data to file • If continuing with experiments, it is possible to continue to add data to the file. • If currently connected, insert cuvette and press the green button. • If not connected, perhaps because it is the next day, press the connect button and then proceed. 中国科学技术大学生命科学学院

31. Ending • If not continuing with experiments, • save the data • Print and summarize results • and then exit the software (File Exit). • Power down the DynaPro 中国科学技术大学生命科学学院