Software Workflow - Optical Unfolding
This tutorial serves as a guide for an Optical Unfolding experimental workflow on Dianthus α, starting from creating a new experiment in DI.Control, running the acquisition, and exporting data, through to analyzing results in the NanoTemper Hub.
Note: Optical Unfolding is a measurement mode that can be unlocked for Dianthus instruments equipped with Spectral Shift optics. NT.23 instruments are not capable of performing Optical Unfolding measurements, but can be physically upgraded to enable this functionality.
1. Create a New Experiment in DI.Control
View: New Experiment Interface (Figure 1)
Purpose: Start a new Optical Unfolding experiment and define the well layout for the plate.
When you open DI.Control, the New Experiment screen is displayed. You have two options to populate the well information:
Quick Start: Click the Quick Start button to configure the wells manually, one by one.
Excel Import: Click the Excel Import button to load a pre-filled spreadsheet that automatically populates all well information. This option is recommended when you have many conditions to set up.
Once you have selected your preferred method and filled in the well information, proceed to the main acquisition setup screen.
2. Configure the Optical Unfolding Acquisition
View: Main Display Window - Optical Unfolding Setup (Figure 2)
Purpose: Define the laser on-time and an optional delay before starting the measurement.
On this screen, configure the following parameters:
Laser On-Time: Set the duration the laser will be active. The recommended value is 60 seconds, which achieves approximately 80°C in the well.
Delay Before Start: If you need to wait before the acquisition begins (for example, to allow sample equilibration), enter a delay time. Leave this at 0 if no delay is required.
Verify that the laser on-time is set to the recommended 60 seconds unless your experimental protocol specifies otherwise.
Note: This measurement will denature your protein samples and should therefore be conducted last in your experimental sequence.
Once you are satisfied with the settings, add the experiment to the queue and click the Start button to begin the acquisition.
3. Export the Data
View: Plate Mapping View, after acquisition is complete (Figure 2, circled in red)
Purpose: Export the acquired data in JSON format for analysis in the App Hub.
When the acquisition is finished, navigate to the Plate Mapping view. Locate the Export JSON button in the top right corner of the screen (circled in Figure 2). Click it to save your data as a JSON file.
Make a note of where the file is saved, as you will need it in the following steps.
4. Log into the App Hub
View: NanoTemper App Hub - Login and Application Selection (Figure 3)
Purpose: Access the online analysis platform and select the Optical Unfolding Analysis application.
Open your web browser and go to https://hub.nanotempertech.com/apps.
Log in with your credentials.
From the list of available applications, select Optical Unfolding Analysis.
5. Load the JSON Data File
View: File Upload Screen (Figure 4)
Purpose: Import the JSON file exported from DI.Control into the App Hub for analysis.
You can load your file in one of two ways:
Drag and drop the JSON file directly into the upload area.
Click the upload area to browse and locate the file on your computer.
Wait for the upload to complete before proceeding to the next step.
6. Review the Results
View: Main Results Window (Figure 5)
Purpose: Examine the unfolding data displayed as ratio versus time, and identify the time of inflection.
The Results screen is organized into the following panels:
Center panel: Displays the unfolding conditions plotted as ratio versus time.
Top tabs: Allow you to switch between the graph view and a table view containing numerical data, including the automatically identified time of inflection.
Right panel: Allows you to select specific plot requirements and customize the display.
Left panel: Provides navigation. Click Overview to see all previous analyses, or create a new analysis from here.
You can rename an analysis at any time by clicking on its title. This helps you stay organized when working with multiple experiments.