Using PYME Acquire

The camera & preview

PYMEAquire will start with the camera running in a live preview mode. The mapping of the (high bit depth) camera data to the display is controlled by the display widget. By default this auto-scales the available dynamic range (max value - min value) in the image to 8 bits for display. This auto-scaling behaviour can be disabled by unchecking the Auto option. When auto-scaling is disabled, the scaling can be adjusted by dragging the red reference lines on the histogram, or by clicking Optimise to achieve a once off min-max scaling. For historical reasons this display assumes a maximum camera dynamic range of 12 bits and with cameras possesing a higher dynamic range it is possible to saturate the display before the camera saturates. The display widget also allows the display magnification to be changed.

Integration time

The camera integration time can be set using the integration time slider or the accompanying combobox.

Region of interest

A region of interest can be selected by using the mouse to select an area in the preview window and then selecting Controls > Camera > Set ROI from the menu, or by using the ascociated keyboard shortcut (F8). The ROI can then be cleared through the menu, or by pressing F8 again.

Advanced camera properties

Some cameras (eg the Andor EMCCDs) will cause an additional pane of advanced settings to be shown which allow, for example, the EM gain end TE cooling temperature to be set. A not completely obvious aspect of the acquisition software is that it will run the camera in continuous mode (ie letting the camera acquire frames one after the other and stream them into a buffer). This has the advantage of allowing the higest possible frame rates, but means that the camera frames and any hardware actions are essentially asynchronous. If syncronised motion is required, the camera can be placed into single shot mode whereby control returns to the software after each frame. For the Andor camera, this is achieved in the Advanced section of the EMCCD controls. This is probably advisable when doing, for example, conventional z-stacks. Z-stacks in protocols avoid the syncronisation problem by recording a timestamp of when the movement occured and calculating, post-hoc, which frame it corresponds to.

Multiple camera support

PYMEAcquire has some basic support for multiple cameras. If multiple cameras are defined in the initialisation file, a control will be shown permitting switching between the cameras.

Acquiring data

Acquiring single images

Single images (snapshots) may be acquired by either selecting Acquire > 1 pic or by pressing F5. These can be saved as .tif, .h5, or .npy (see PYME Data Formats)

Z-Stacks

3D image stacks such as used for conventional widefield microscopy and, for example PSF measurements can be obtained using the control at top right. Again, the options are .tif, .h5, or .npy

Streaming/Spooling

Streaming functionality (the Spooling panel) is what is typically used for localisation microscopy, and permits data to be streamed either to disk in .h5 format or to a server process (default) for real time analysis. If you want to save directly to file and analyse later, the Save to Queue option needs to be unchecked. Filenames are automatically generated from the current date and user name (although can be changed manually). To access some of the controls (eg ‘Spool Directory’ and ‘Save to Queue’ you might need to either drag the ‘Hardware’ controls down, or unpin the Z-Stack controls.

Before starting streaming you can choose a protocol, which defines actions to be performed during the sequence. You can then choose to acquire a simple sequence (by clicking on Series), or one in which the focus is stepped at regular intervals (Z-Series). The Z-Series uses the z-stepping regime defined using the Z-stack control.