Version 0.1

This tutorial shows step-by-step, how to acquire emission spectra with an FT250 (equiped with a monochromator) using the Emission Spectrum Wizard in EasyTau 2.

• Switch on the FluoTime 250.
• Put the sample cuvette (here, a solution of Anthracen dissolved in Ethanol) in the sample chamber.
• Make sure that the correct laser (here: LDH-P-C-375) is in place and connected to the laser driver (here: PDL 820).
• Turn on the laser driver and unlock the laser.
• Start the EasyTau 2 software and wait until the status on the bottom right switches from yellow to green:

FT250 Initilization

FT250 Online

• Load a workspace or create a new one by clicking “File“ → „New Workspace“.
• After loading or creating a new workspace, the name of the workspace is displayed in the top line of the window (here, the workspace is called „Demo“).

• Start the „Emission Spectrum Wizard“ by clicking on the according icon:

• The Emission Spectrum Wizard opens up displaying the “Sample Setup“ - Window.

• Enter information about the sample and the solvent in the “Sample”-Tab (in this example: Anthracene solved in Ethanol).
• Additional information about the measurement or the sample may be stored in the “Comment”-Field:

• Select the right excitation source (here an LDH-375 is chosen).
• Set the detection wavelength range (here the range was set from 390 nm to 500 nm with a 1 nm step size).

• Once the measurement parameters are set click the “Optimize Parameters” - Button.

• The “Optimize Parameters” - Window opens.

• Click the “Start Optimization” - Button.

• The system will perform a quick measurement of the emission spectrum and change the attenuator settings for an optimal signal countrate across the chosen wavelength range.
• Once the optimization has finished the “Start Optimization” - Button the count rate displayed in the bar above the optimization-button.

• The bar should lay within the green range to acquire optimal measurement results.

NOTE: On the right side Easytau 2 shows a graphical preview of the emission spectrum. If you want to change the scanning range, you can go back to the “Sample Setup” - Tab by clicking the “Sample Setup” - Button. There, change the measurement parameters as desired and restart the optimization process. You'll have to re-do the optimization if you do this, before you can proceed to the “Acuire Spectrum”-page.

• Click the “Acquire Spectrum” - Button to start the actual measurement of the emission spectrum of the sample.

• The system will then start the acquisition of the emission spectrum.

• The bar on the left indicates the time left until the measurement is done.

• When the measurement is done you can either click the “Save & Exit” - Button to store the acquired data in the workspace or you can - if available - acquire the emission spectrum of a Blank sample to remove any background signal from the data.

• If your signal is relatively low in comparison to the dark counts of the system, a “blank”, i.e. an measurement of the buffer only can be acquired in order to properly subtract the background. This is mainly important with detectors with higher dar count rates, e.g. IR-PMTs. If you don't want to acquire a blank, directly step to the “Save & Exit”-page. To measure a blank, click on the “Acquire Blank” - Button.

• The “Acquire Blank Sample” - Window opens:

• Turn off the laser by turning the key of the laser driver from the “Laser Active” position to the “Locked” position.
• Exchange the Sample in the chamber with the blank sample (here spectral grade Ethanol).
• Close the lid and turn on the laser by turning the key of the laser driver from the “Locked” position to the “Laser Active” position.
• Click the “Start” - Button to start the acquisition of the blank emission spectrum.

• The grey graph overlaying the spectrum shows the normalized spectrum of the blank sample.
• There is also a red graph which is not normalized. In this example the photons counted are so low that this graph can not be displayed.
• When the acquisition is finished click the “Save & Exit” - Button.

• The data will then be stored and highlighted in the workspace on the left.

• Click the “Plot Data” - Button.

• The “Plot” - Window opens up showing the measured data.
• Since the emission spectrum for the Blank Sample was acquired the blue graph represents the already corrected emission spectrum.

• Click on the “Calculation” - Button.

• This opens up a text field below the graph window which shows the formula that was used to calculate the corrected emission spectrum.

• crv[0] is the raw data acquired for the Blank sample
• crv[1] is the raw data curve
• crv[2] is the spectral correction curve for your specific system

• To display the different curves, the display settings for the graph have to be changed first (the measured count rate of the emission spectrum of the Blank sample in our example was so low that it will not be shown with standard settings).
• The display settings for the graph are shown on the top right site of the graph window:

• Click on the checkbox Y-Axis “Min” to activate the manual control of the display settings for this parameter.
• Enter ”-0.01” in the text field behind the checkbox. This lowers the minimum y-value of the shown graph for which data points will be displayed.
• On the right side of the “Plot” - Window you can find all the curves included in the data set under “Raw Data”.

• Click all the three checkboxes next to the three different curves.
• The curves will now also be displayed in the graph window as shown below:

• Click the “Save” - Button to store the plot.

• The data will then be stored in the workspace as shown below:

• Click on the “New Calculation (1).eta file then right click it.
• A context menu opens up where it is possible to rename the file: