Z stack confocal software#
Z stack confocal free#
Z stack confocal windows#

In the example above, an xyz scan of a GFP-expressing mushroom body in an adult Drosophila brain, there are 291 xy frames in the stack. The “Crop” function in the “Edit” portion of the “Process Tools” allow you to copy and isolate a subset of the xy frames and create a new file. Your start/end points can be cleared by clicking the trash can icon, or you can turn the focus knobs and click the set buttons to set new start/end points. You also have the option to specify the number of z steps.Īfter you do your scan, you can click the “Maximum Projection” button to merge the z stacks: This will change the total number of z steps.

Z stack confocal free#

You are free to disregard the recommended z-step size and type in any compatible number. Clicking the “+” bar shows you the formula and give you to option to apply if it isn’t already active.

Z stack confocal software#

When “System Optimized” is on, the software will select a “recommended” optical section thickness, based on the objective and laser line in use. Once you have set your begin/end points, the panel will tell you many optical sections there are, and their thickness: Anything that does not have much useful signal can always be edited out later, but if you set the start/end points too short you will have to redo the scan. It’s also a good idea to err on the side of extra stacks. Always check every channel you can re-click on a set button to add more z layers if needed. For example, if you set your start/end points based on a nuclear dye imaged in the first detection window, but you also are detecting a membrane marker with a different fluor, basing your stack limits on the nuclear stain might cut out some of the membrane stain. You should check these positions to see whether your stack settings will collect all the data from these channels. The “Move to Begin” “Move to Center” and “Move to End” buttons are very useful here. If you are scanning with more than one detection window you will need to check the levels for each one, at different points of the z-stack.

Z stack confocal windows#

Two new windows (marked with yellow *), with values for the beginning and ending z-coordinates, will appear to the right.īe aware that the optimal exposure levels you set at the beginning may turn out to be not so optimal as you focus up and down through the sample, so adjust them as needed. Now turn the knob in the opposite direction until you have moved past your sample from the other direction. Turn a little further and click “Begin” to set your start point. Next you will need to turn the focus knob (You can use either the USB control panel or the focus knobs on the STP6000 box) in one direction (it doesn’t matter which) until the useful signal from your specimen starts to disappear. Click the “Live” button to scan, then adjust the gain and offset. To set up a z-stack, find a region of interest via the eyepieces and bring it into focus. The buttons you will use most frequently set the start and end of the z-stack, and are located on the top left of the panel: Our system is not equipped to use the “z-Galvo” mode, so select “z-Wide”. The first thing you should check is whether the drop down is set to “z-Wide”. The Z-stack panel allows you to set the Z-parameters: If you select the “xyz” mode without defining any start/end points for the z-axis, you can still collect data, but it will just be in the current optical plane (essentially an “xy” scan). Z-stacks can be merged to create a detailed 3 dimensional representation of the sample. The use of a pinhole in collecting the light from the specimen prevents the out-of-focus light from obscuring the in-focus image. Scan modes with a “z” dimension allow the user to collect images in multiple optical planes.