Biotek Synergy4 Plate Reader

This manual is meant to complement your training and is not intended to replace on-the-job training.

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Training Contact(s)

Emory Chan

emchan@lbl.gov

67-4114

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Last Updated

10/12/2016

OPERATING PROCEDURE

Overview:

The BioTek Synergy4 Plate Reader is used for acquiring absorption, fluorescence emission, and fluorescence excitation spectra. Biotek’s Gen5 software is used to control the software – this software is not part of the standard Symyx Automation Studio environment. Spectra acquired using the plate reader are uploaded into the Symyx database by saving exported text files tagged with the appropriate deposition experiment ID.

Summary:

Typical applications for this workflow:

1. Measuring fluorescence and absorption spectra for aliquots generated by a WANDA synthesis workflow.

2. Measuring absorption at a particular wavelength.

3. Measuring photoluminescence excitation spectra (PLE)

Specifications:

1. Well volumes 100-300 μl

2. Maximum number of wells per run 96

3. Reaction temperature, range RT – 50 °C

4. Minimum absorption spectrum scan time 15 s

5. Minimum spectral resolution 1 nm

Sample preparation:

1. If you wish to link your data back to the Symyx database, you must use the Automation Studio protocol “Abs/Fluorescence or XRD plate spotting EC8” to deposit your samples.

2. Deposit samples in the 96-well quartz microtiter plate. Organic solvents will dissolve standard disposable polystyrene microtiter plates.

3. If you are manually loading samples

  • Use a 200 μl or 1 ml micropipettor with polypropylene tips

  • Avoid bubbles in wells, as this will scatter incident light

4. For absorption, remember to deposit blanks using the same solvent and volume.

5. IMPORTANT! Gen5 can only acquire groups of wells in the pattern of a rectangle.You should take this into account when you deposit your wells to minimize the time spent acquiring empty wells.

6. IMPORTANT! Because the WANDA glovebox temperatures can affect evaporation rates and quantum yields, be sure to turn the glovebox air conditioning on several hours BEFORE you wish to use the plate reader. From the MBraun touch screen main menu, click to “Functions > Next > Next > Box Cooling” such that the Box Cooling button is green (on). A glovebox temperature of 22 °C will usually equilibrate the plate reader to 25°C.

Data Acquisition

1. Open up the “Gen5 1.05” application on the Windows XP desktop.

2. Create a new experiment (Create New Item > Experiment, or File > New Experiment…)

3. Select a protocol as a starting point for your experiment

  • In this example, we will open “CdSe absorption fluorescence protocol.prt”

  • This protocol measures the fluorescence and absorption spectra for the wavelength ranges typical for CdSe. You can always tweak the parameters later.

4. You should now see the main window that contains your experiment.

  • On the left side of the window, you will see the protocol. Click on the “+” sign next to “Protocol” to view the Procedure and Plate Layout icons.

5. Double-click on the Protocol>Procedure icon6. In the Procedure window, ensure that your plate type is correct.

  • “Hellma 96 quartz flat bottom” for the standard quartz plate

7. Each “Read” line acquires spectral data of a particular type (abs/fluor./PLE) for each well

specified. Double-click on the “Read: (F) Emission Spectrum [430nm to 740nm by 2]”

line.

  • This opens up the “Read Step” window as shown below.

8. REQUIRED: To specify the wells for this particular read step (Emission), click on thebutton in the upper right corner of the Read Step window.

  • Highlight the wells you wish to acquire in blue. You may only acquire a rectangular pattern of wells. Remember to include any blanks!

  • Click “OK” to save changes and go back to the Read Step window.

9. Optional: In the Read Step window, you can change the following parameters:

  • Detection Method: Fluorescence (can be Absorbance)

  • Read Type: Spectrum (can also absorbance at single wavelengths or integrated over a wavelength range)

  • Filter Sets > Sensitivity: A higher sensitivity amplifies the signal in a super-linear manner.

  • The maximum recommended signal is 90,000 counts. If your signal exceeds this, decrease the sensitivity to 90 or below.

  • If you are getting <10,000 counts, you may want to increase the sensitivity in steps of 10.

  • You can turn on the “Automatic Sensitivity Adjustment” by clicking the “Options” button. This doesn’t always work as you wish.

  • Filter Sets > Spectrum Type: Emission (used for fluorescence. Use “Excitation” for PLE)

  • Filter Sets > Excitation, Emission Start, Emission Stop, Emission Step

  • Alter these values to change the excitation and emission parameters for your expected spectrum.

  • Read Speed: Normal (Sweep mode is a faster scan that only uses one acquisition per wavelength step)

  • Next to the “Read Speed: Normal” parameter, click the “…” button to open up the “Measurement Options” window.

  • Measurements per data point: 24 (increase to increase signal to noise)

  • Lamp Energy: High (lower lamp energies have lower signal but allow for faster averaging)

10. Click “OK” in the Read Step window to save your changes and go back to the Procedure

window.

11. Change the Absorbance read step parameters in a similar manner to the Emission read

step by double clicking on the “Read: (A) Spectrum [350nmm to 740nm by 2]” line.

  • REQUIRED: Always remember to specify the wells for each read step by clicking on the button in the upper right corner of the Read Step

  • window

12. Modifying Read Step sequence:

  • Delete a read step by highlighting it in the Procedure window and then Rightclick > Remove.

  • You can add more read steps, shake the plate, or set the temperature by clicking on the proper “Add Step” button.

13. When you are done modifying your procedure, click “OK” to save your changes.

14. In the Protocol sidebar, double-click “Protocol > Plate Layout”

to open up the Plate Layout window

15. REQUIRED: You must specify which wells contain samples and which wells contain blanks. If you do not do this, blank subtraction will not work properly

  • Under “Well Settings > Type”, select “Empty”, then select all of the wells to clear the layout.

  • Under “Well Settings > Type” select “Sample” and then select click on the well positions that correspond to actual samples (not blanks).

  • Under “Well Settings > Type” select “Blank” and then select click on the well positions that correspond to blank wells with pure solvent in them

  • Click “OK” to save your changes and go back to the main experiment window.

16. To acquire spectra, click the “Read Plate” button in the toolbar

REQUIRED: The “Plate Read” annotation window will pop up. In order to link this data back to the Symyx database, you MUST include the Deposition Experiment ID of in the “DepositionID” runtime prompt. This Deposition Experiment ID corresponds the deposition experiment used to spot the quartz 96-well plate. It is not the Plate ID.

  • Optional:

    • Plate ID: Deposition Plate ID

    • Barcode: Library ID

  • If you manually spotted your plate but want to associate your results with a Symyx Library ID, you must run the “Absorption/Fluorescence and XRD spotting” protocol in simulation mode to generate a dummy Deposition

Experiment ID.

17. Viewing your data during acquisition

  • During and after acquisition, you will see the Plate View window, shown below:

  • To view spectra, in the “Data” pull-down menu, choose the “Curves” item that corresponds to a given Read Step (e.g. Curves [Read 2: Emission]). After an acquisition has completed, you can also select the [Blank Read #:Read Type]” curves to see background-subtracted curves (sample - blank).

  • To zoom in on a spectrum, double click on a well position/spectrum to see the spectrum window below:

  • To view multiple spectra, click on the "..." button next to the “Wells” field, then highlight 1-8 well positions.

  • Important: For fluorescence emission or excitation, ensure that the signal is not saturated ( > 100,000 counts). If the signal is saturated, stop acquisition and decrease the sensitivity in the Read Step parameters.

18. When acquisition is completed, save the spectra in the native Gen5 .xpt format.

Exporting Data (for Symyx Database, etc.)

1. Gen5 can export spectral data in user-customizable text files. To ensure compatibility with the Symyx data loaders, follow the instructions below.

2. In the Protocol side bar, double-click “Protocol > File Export Builder” to open the File Export Builder window.

3. The right-hand “Export Content” list contains the data and parameters that will be output, in order, in the exported text file.

4. The third item in the “Export Content” list is the “Well Data” item. “Well Data” specifies that spectral data be output in columnar format in which each column corresponds to a given well position, and each well corresponds to a given wavelength.

5. To output a data for a given read type, ensure that the 3rd Export Content item is the “Blank Read” Well Data for a given Read Type (e.g. Blank Read 2: EM Spectrum” for

background-subtracted fluorescence). If the wrong read type already occupies this position, select it and then click the “Remove” button. Add the proper well data by selecting it from the left-hand “Available Data Views” list on the left of the File Export Builder window and clicking the “Add” button. This will bring up the “Edit” well data window, shown below:

6. In the “Wells” tab in the Edit well data window, type “A1-H12” in the “Wells” field, even if you didn’t use all the wells (unused wells will be ignored by the Symyx data loaders).

7. Important: For absorption, the default precision is usually too low for the absorbance values. To increase the number of significant digits, click the “Data” tab in the Edit window, then click on the "..." button to the right of the <Decimal,3> text in the “Format” column. This will bring up the “Numeric Format” window:

  • In the “Decimal places” field, type “5”.

8. Click “OK” in all of the windows that you opened to save changes and return to the main experiment window. This preceding actions will NOT export the file – they just configure the export file format.

9. To actually export a text file, click the “File Export” button in the toolbar.

10. IMPORTANT: if you want the Symyx data file loaders to automatically parse and upload your plate reader data, you must save your files in the correct folders, as listed below:

  • Absorption spectra: C:\PlateReaderData\Fluorescence

  • Fluorescence spectra: C:\PlateReaderData\UV-Vis

  • Note: after the Symyx file loaders process your exported file, they will move these files to the “processed” directory inside the text file’s current directory. Sometimes the Renaissance file loader will throw errors while your file is saving, but many times, the file is processed fine. Ensure that your files were uploaded to the database by checking that the corresponding data appears in Symyx Data.

Browser.

11. IMPORTANT: You cannot export both Fluorescence and UV-Vis data in the same text file if you wish to import them into the Symyx database. You must modify the File Export Builder and export a text file once for each Read Type (absorption, emission). You may only have ONE “Well Data” item in your Export Content list.

12. To import these text files into Igor Pro, use the WANDAworks Igor Pro library, which will be described elsewhere.

More information

  • For more information about the Gen5 software, use the online help files located in the “Help” menu in Gen5, or go to the Biotek website at

    • Original file path for this manual: Macintosh HD:Users:emory:Documents:LBL postdoc:robot:symyx:manual:manual - biotek plate reader