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EMBL Hamburg Biological
Small Angle Scattering
BioSAXS

DLS Basic Training

1) Turning on the Instrument

From a “cold start”, the DLS Nanostar requires at least 30 minutes of warm up time before starting a measurement. The on-switch is located on the front of the DLS unit at the bottom right hand corner. Make sure that the software on the instrument has loaded, as indicated on the front touch-panel display of the DLS unit.

 

2) Communicating/controlling the DLS instrument from the DLS instrument

Limited control of the DLS instrument can be performed by command operations listed on the front touch panel of the DLS instrument. There are several tabs to choose from including:

Main tab – shows a basic display, by default cell temperature and static light scattering  recorded in real time. The axes of the display can be altered using the drop down menu options. For example, it is possible to display ‘count rate’ vs temperature, etc. The time axis can also be adjusted. Importantly, the cell temperature can be adjusted from the Main display. Touch the ‘Cell Temp Set’ box to bring up the cursor in the box, then use the arrows and numeric key pad to adjust the cell temperature to the desired value:

With Nitrogen flow = 4-80 °C (**currently there is NO N2 option!!)

Without Nitrogen flow = 20-80 °C ** (current default working range).

 

When the temperature is equilibrated, the recorded temperature changes colour from yellow to green.

The Main display also indicates whether the laser is on or off. The laser is automatically controlled. However, if you would like to leave the instrument on overnight ENSURE THE LASER IS SWITCHED OFF before you leave. Simply tap “Laser” and the display will toggle the laser to off.

NOTE: by default laser attenuation and laser power are auto-enabled to reduce the chances of flooding the detector with photons and damaging it.

QELS tab – This tab shows the Quasi-Elastic Light Scattering output being recorded in real time, i.e., the DLS time correlation function data (DLS data). This can be useful to assess how your sample scattering intensity behaves prior to measurement (i.e., assess whether he sample is garbage).

 

Alarm tab – If a problem is detected by the instrument, an alarm will sound. This can get annoying. The audible alarm can be disabled by un-ticking the ‘Audio alarm’ box. A list of possible alarm options are listed in the tab and will be indicated by a yellow (no need to panic, just be patient) or red (panic) box. Record which alarm has sounded. The Vapour alarm will sound if too much condensation builds up in the unit OR if some sort of vapour is detected (for example an aerosol from a spray can). Condensation or vapor can be removed by heating the cell with the top of the sample holder left open. The alarm should stop.

 

Sysetm tab – there is no need to go into this tab. You can adjust the temperature from this tab if absolutely required. DO NOT ALTER THE LASER AUTO ATTENUATION!

 

Comm tab – this is for Wyatt service personnel and troubleshooting purposes only. Users have no need to go into this tab. The PIN must remain set at 5595.

 

3) Setting up the experimental folder environment and copying DLS presets

The password for the computer is currently set to password. This will likely change and the updated password be posted on the computer. Before opening the software that controls the DLS instrument, make sure you set up a dedicated User folder. This folder can be written to:

C:\Documents and Settings\jekyll_admin\My Documents\Dynamics\PreSets\Experiments Dynamics 7.1.7.16\Internal Users\Username

 

 

As it is boring to remember this pathway, simply click on the  “Shortcut to DLS Experiments” icon on the desktop to open the folder.

 

In the experiments folder there is a folder called Presets for DLS. Copy the appropriate preset required to your experimental folder. The preset options are, as the name suggests, preset scripts to drive the DLS instrument. There are two main preset options:

 

a)      Batch mode analysis. Single temperature.

b)      Temperature screen.

After you have set up the folder environment, open the Dynamics 7.1.7 software on the computer.

 

4) Starting DYNAMICS Software for DLS data recording and event scheduling

The Dynamics software is used to control the DLS instrument, record data and analyse the results. The opening screen to the Dynamics software looks like this:

To open a preset click on the ‘open preset’ button. Make sure to navigate to your Experimental/User folder (in the Experiments Dynamics 7.1.7.16 folder!)

 

An open preset is shown below. There are a number of options listed in the left hand column that can be expanded or minimized using the + or – check buttons.

 

 

 

 

Of particular interest are:

Parameters–Fixed–Instrument

Describes the instrument ‘resting mode’ when not in use. NEVER disengage the auto attenuation (it should always be True). The default temperature is 25 °C. To change the temperature simply enter the desired default temperature in the screen:

 

 

Parameters–Sample

Describes the sample, including the sample name, as well as the type of cuvette to be used for the analysis. For SLS measurements it is important to include the sample concentration! To add a sample, simply click on ‘Add’. A dialogue will appear and type in the sample name.

 

To delete a sample name simply go to the sample name drop down menu, highlight the sample name you do NOT want and then click Delete.

Remember to define the protein concentration and Cuvette! The cuvette can be selected from the dropdown options. There are three options:

i) Quartz Cuvette = JC-282 (1 microlitre)

ii) Wyatt Disposable 4ml

iii) Eppendorf 50 ml.

NOTE – external users are limited to the disposable cuvettes (3 EUR each)! The disposable cuvettes are as easy and reliable as the quartz cuvette. Also note that the Eppendorf cuvettes (or UVettes) require a specialised cell holder, with the long path length oriented to the primary beam (top-to-bottom).

Finally in the sample tab define what the next measurement to be measured from the drop-down menu otherwise the sample name and conditions will NOT be copied over to the experiment!

 

 

Parameters–sample–solvent

Define the solvent. Unfortunately these are preset. If you are really keen it is possible to define your own solvent, but this requires measuring the viscosity of the sample (possible using this instrument.)

Parameters–sample–cuvette

Define the cuvette from the drop-down menu. For the quartz cuvette do please do not re-calibrate the cuvette. The disposable cuvettes do not require calibration.

 

Parameters–sample–names

Define the naming convention of the experiments by clicking Apply. By default each measurement will be labeled:

{Number} {Value: Sample} in {Value: Solvent Name} at {Value: Temp}

 

5) Setting up an event schedule

Parameters–Event Schedule

If you employ a preset, there is not much to do in the event schedule. The event schedule is a list of commands that are performed one by one in series by the software and instructs the DLS instrument what to do at each step. The default preset more-than-likely is sufficient for most users. However, to change a parameter (for example the temperature or “save data as”) simply click in the corresponding box next to the event name and change the parameter.

 

You can add events to the schedule by first clicking what event you would like to add from the drop-down menu, in this case an additional Wait command.

click the Add button….

 

Then shift the new event using the move up or move down buttons to insert it into the appropriate place in the schedule.

 

Below is a temperature ramping experiment. Note the loop command; the software drives the DLS instrument to repeat the commands between the loop until a fixed parameter is met, in this instance when the temperature reaches 25 °C.

 

 

 

Connect the software/computer to the Nanostar instrument

Click the square panel button at the top left hand corner of the experiment window. A green light will come on indicating that the instrument is connected. This is the start button. Click the start button to begin the experiment. Note that the experiments will not begin until the lid of the cell holder of the DLS instrument has been closed (door interlock).

 

6) Loading cuvettes into the instrument

Quartz (1 ml):

The quartz cuvette has a beveled corner. Load it into the instrument so that the beveled corner is oriented on the bottom left hand corner of the sample holder. Handle the quartz cuvette with powder-free gloves (simply because it is really boring removing fingerprints between each sample.) To clean the cuvette use the provided cleaning solution (approximately 5 ml in the cuvette for 20 min) followed by copious amounts of water. Do not use ethanol from the squirt bottles – it is grubby. AVOID HIGH pH SOLUTIONS AND NaOH over extended time periods – this will eat away the cuvette! Use the lens cleaning cloth to remove dust from the outside of the cuvette.

For high temperature measurements, place the lid on the cuvette.

 

Wyatt 4 ml.

The Wyatt 4 ml has a ‘nose’ on one of its ‘feet.’ The nose on the foot goes into the instrument at the TOP left hand corner of the sample holder. Use kimwipes to remove dust from the outside of the cuvette.

For high temperature measurements load 4-10 microlitres of sample into the inner compartment of the cuvette then add a few drops of Paraffin oil on top of the sample and quarter-fill the outer compartment of the cuvette with buffer.  Place the stopper onto the cuvette.

Eppendorf 50 ml.

The SAMPLE HOLDER for the Eppendorf UVette also has a nose (sharp spiky protrusion) that is placed into the instrument at the TOP left hand corner of the sample holder. Remember to place the UVette into the sample holder so that the long path length is oriented in the direction of the primary beam (top-to-bottom). Use kimwipes to remove dust from the outside of the cuvette.  For high temperature measurements, add a few drops of Paraffin oil on top of the sample.

 

Hints and tips: NEVER USE A SHARPE NEEDLE TO LOAD A SMPLE INTO A CUVETTE. Use disposable plastic gel-loading or small plastic pipette tips. Filter your samples or spin at high speed to remove dust and other contaminants. Dust will ruin the DLS experiment.

 

Results

The DLS data are recorded in real time. The final results and result/summary views can be selected from the following tabs during or at the completion of a run:

Tabular format:

 

Correlation coefficient:

 

 

Peak heights - % Mass distribution vs Rh

 

 

All plots and data plots can be saved as .csv or tables as .xml files by right clicking on the area to be saved and choosing “Export”. Plots can also be saved as JPG and EMF files. The plots can also be edited (styles), again via the right click option. Of course the experiment can be saved in total and opened in a copy of Dynamics 7.1.7 on another computer.

Renaming measurements?

Forgot to change the sample name in the Sample set up? Simply highlight the measurement to be renamed and rename it!


  Last modified: August 6, 2013

© BioSAXS group 2013