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- Click the User login button in the top left of the user interface.
- In the dialogue box that appears, type a Username and Password and click Create user.
- Whenever you use the machine in future, simply click User login, type in your username and password, and click Login. Your images will be saved in a folder in this location: C:\Data\your_username.
Single colour dSTORM
Setting
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acquisition conditions (for single colour 2D dSTORM)
The instructions below assume that the sample is stained following instructions are appropriate for single colour 2D dSTORM using a sample labelled with a far-red dye like Alexa 647.
- Click View in the Acquisition Control area to get a live feed from the acquisition camera.
- There will be two live images, which correspond to two simultaneous colour channels. For dSTORM using a far-red dye you only need Channel 1, so untick Channel 0 in the Image Display Options on the left. For dyes other than far-red you will need Channel 0.
- The live camera image will only show noise until you illuminate the sample with a laser. Change the percentage power in the Light Control area by either clicking on the spin buttons or typing in a value and pressing the Return key. The laser power in the image below is set to 100% but it is best to use a lower power like 10% to avoid photobleaching while setting up your specimen. Click on the button above the laser (e.g. red 640 button) to switch on the illumination.
- Focus on your specimen. If you are using Z lock you may need to adjust the Z offset as explained in the section on focusing.
- Turn off the laser when not imaging so you don’t photobleach the specimen.
Acquiring images
Acquisition Control
| Note |
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Acquisition Control is the most obvious way to start an acquisition because it is easily visible in the user interface. However, the best way I've found to acquire images is to use Multiple Acquisition Setup, which is explained later. |
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| Warning |
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The lasers are not automatically turned on when acquisition is initiated this way, so you must turn them on yourself in the Light Control area, or the images will all be captured with the lasers off. This is one reason why Multiple Acquisition setup is better. |
Multiple Acquisition Setup
- The best way start acquiring data is to use multidimensional acquisition. Click the Multiple Acquisition Setup button under the focus camera view on the left.
- In the Acquisition tab, click Single, set imaging mode to Normal, adjust the laser power and make sure the button above the percentage slider is clicked (text is white, colour is bright). Set the exposure and number of images.
- Ignore the Priority and AutoFocus and Z Reference tabs. They are not important for conventional 2D dSTORM at a single stage position.
- In Saving, specify the Dataset Tag and the Acquisition Tag (see Acquisition Control above for an explanation). If you are logged into your user login all files will be automatically saved in your folder.
- Click the Start button at the bottom to begin the acquisition.
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