The multi-dimensional acquisition window

From Endrov

This window provides the quickest way of acquiring Z-stacks, with different channels, at different positions, over time. The window is found in "Windows . Recording. Acquire: Multi-dim", and has many settings:

Multidimacq.png

To acquire a new recording, first do "File . New" to create an empty file. Select it bottom-right "Store in" and optionally change the name of the object. Once all other settings are ok you can press the red acquire button.

Contents

Channels

Channels are fundamental - You should always have channels set up to store proper metadata about your images. Use the "+" button to add additional channels (for example, simultaneous DIC and GFP). For each channel you can set:

  • The exposure time (in ms)
  • The stepping in Z (Z++). For example, by having one channel 1 and the other 2, the second channel will have only half the number of slices of the first
  • Offset in Z (First Z). Use this if you want one channel to not include the first number of slices
  • The stepping in time (t++). In analogy to stepping in Z, it can be used to skip time frames.
  • Frame averaging. By taking more than 1 image and averaging them, some types of noise can be removed

Slices

Enable slices to do 3D stacks. You need to tell the program the lower and upper limits of the stack, in um. The number of slices can be entered directly, or by calculating them through the slice distance.

Time

Enable this to record over time. You have to specify the rate at which images are taken, in Hz or letting the camera decide. You also need to specify how many frames you want; either through number of frames (#f) or by total time.

Positions

If you wish to do multi-position (or multi-well imaging), you can specify those positions here. The positions are first created in the Positions window, see separate section.

When imaging over large areas, you will find that the slides are warped, that the agarose has different thickness etc, causing the 0-point of the surface to be different. Using an autofocus every time the stage is moved can alleviate this problem. Tick the box to enable it. You need to have configured a software autofocus, or have a microscope equipped with the hardware.

Order

When mixing dimensions, the order of dimensions is crucial. For example, a 2-channel 3D stack can be created in two ways:

  • Channel 1 and 2 are captured for a first slice. Stage moves to next Z position and the procedure is repeated.
  • Channel 1 is selected, all images for this channel are captured, Channel 2 is selected, and all those images are captured too.

The most suitable order depends on wear-and-tear of the components, the speed of moving mechanical components and the required repeatability in moving the stage.