Multi-Dimensional Acquisition

Table of Contents:

1. Getting ready for multi-dimensional acquisition

2. Setting the parameters for acquisition

2.1 General
2.2 Size
2.3 Shutters and Filters
2.4 Z-Steps
2.5 Time Lapse

3. Previewing the specimen

3.1 Size
3.2 Z-Stage

4. Click “OK” and start your experiment!

Multi-Dimensional Acquisition using IPlab on Digital Station #2

1. Getting ready for multi-dimensional acquisition:

• First, you should calculate the best exposure time for your sample with each of the probes that you’ll be using.
• Next, determine the approximate thickness of your sample and the desired step size.
• Press F4 to send light to the camera.

2. Setting the parameters for acquisition:

• To start the Multi-Dimensional Acquisition mode go to the program menu and click “Camera” and then select "Multi Dimensional Acquire".

• The window that opens up has several tabs allowing a user to change several things.
  

2.1 General-

• Preview - Select this to preview the sample before acquisition.
• Experiment Name - Set a name for the experiment to be saved.
• Create Timing Window - This will open a table with the capture time of each image recorded to be saved (may be applicable for time lapse experiments).
• Display After Each Grab - check this to see image after it is collected

2.2. Size-

• Full Frame, ROI or Custom Rect (most users will select “Full Frame”).
• Bin Width / Height - Set as you would to capture single images (Use “#” not “V”).

  2.3. Shutters and Filters-

  

• Shutters 1 and 2
  
  • Shutter X - Select a shutter (Ludl-shutter 1 for fluorescence, 2 for transmitted light.)
  • Open Pos - Open
  • Close Pos - Close
  • Keep open during Z/Preview - Select
    **If you want to capture a z-series of both phase and flourescence, DO NOT SELECT THIS OPTION**
  • Shutter 1 Open Delay(ms) - At least 500 (#) ms if switching filters.
    
• Filter Wheel: Select OlympusIX-FW(1-2)…
  
  • Enable - Select this to choose filter from the list
  • Shutter - Select shutter
  • Exp. Time - Set exposure time.
  • AE - Don’t use AE

  2.4. Z-Steps-

  

• Use Z Step - Select this
• Absolute - Select Absolute
• Start Pos - How far up?
• Stop Pos - How far down? (-)
• Step by - Choose step size (um)
• Reset Current - Select this

  2.5. Time Lapse-

  

• Use Time Lapse - Select this for time lapse imaging
• Set Time Lapse experiment parameters
• Set the file path for saving your files (zseries, time lapse)
• Click 'OK'

***This software currently saves the images with file names based upon the filter. If you take a FITC fluorescence image and then take a transmitted image with the FITC filter in place you will overwrite the data!!

3. Previewing the specimen:

Like the settings window this also has tabs allowing users to change settings. We’ll only cover two of these tabs which are the most common used.

3.1. Size-

This allows you to change the size of the image collected.

3.2. Z-Stage-

• Click "Continue"
• Use the focus knob to move to one end of the sample
• Click "Get" to get the current position
• Click "Abs. Start" / "Abs. End"
• Move to the other end of the sample
• Repeat the last two steps (Get & Abs. Start/End)
• Step by - Another opportunity to change step size
• Calc # Steps - click to see the total number of z slices that will be acquired

4. Click “OK” to start your experiment!

edited by Dustin Grzesik 10/21/2004
updated by Rosana Leonard 03/09/05