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• When your specimen is in focus, close the shutter and click the "Acquisition" tab.

Imaging Setup

Smart Setup

The easiest way to set up imaging conditions is to use Smart Setup.

Click on the Smart Setup button and add the dyes you want to image.

Choose from Fastest, Best Signal or Smartest proposals. Basically, Fastest will be simultaneous (one Track), Best Signal will capture all channels separately, moving the filters each time to optimise signal (which is slow). Smartest optimises the filter positions so that they don't have to move, but still captures the channels sequentially.

The applicable lasers will automatically turn on and the filters will automatically move into the positions programmed for that dye combination and configuration.

Manual Setup

• Expand Imaging Setup
• Choose LSM and Channel
• (Non Descanned is for multiphoton only)
• (Lambda Mode and Online Fingerprinting are for spectral imaging)

Smart Setup

• Use Smart Setup if you want to easily set up your experimental settings (e.g. filter and detector combinations) depending on your dyes.

• Pick Click the + button and pick dyes from the list, and Smart Setup will arrange the light path to optimally capture those dyes with three combinationspossible options:
  • Fastest (simultaneous acquisition). Fast but with a risk of crosstalk).
  • Best Signal (Sequential acquisition with the spectral windows optimised for each dye in turn to maximise signal, but may be slow)
  • Smartest (Line sequential with the spectral windows balanced to get the best signal with the best speed)

Image Removed

• You can manually setup or adjust the light paths in imaging Setup

Image Removed

• • will capture all channels separately moving the filters each time to optimise signal (which is slow).
  • Smartest optimises the filter positions so that they don't have to move, but still captures the channels sequentially. This reduces crosstalk and is faster than Best Signal.

Image Added

• In the example image above, Fastest shows all three colours being excited and captured simultaneously in one Track (Track 1). Sequential acquisitions would require more than one Track.
• Best Signal creates three tracks for the three selected dyes, one for each channel so they are all captured separately but switching . Switching between the tracks takes time because the components (e.g. dichroic mirrors) movephysical components must move.
• Smartest creates two tracks, with spectrally separated dyes in the same track as each other. Also, the physical components don't move to save time, which saves time.
• Note that the optimal settings for other dye combinations may look different.
• NB lasers are always set to 2% in Smart Setup - but you can change them afterwards
• Click Live to get a live scan.

If you have multiple tracks then you can define whether you switch them every Line (only suitable when the light path is set up in the Smartest manner), Frame or Frame Fast (only suitable when the light path is set up in the Smartest manner).

You can edit the imaging setup after loading the Smart Setup configuration, or alternatively you can create an imaging setup manually from scratch without using Smart Setup.

Acquisition Mode

• Expand Acquisition Mode. In here you can change a variety of parameters but the main ones are the scan settings:

• • Scan Mode: Whether the scan switches channels in Frame or Line mode (frame by frame or line by line)laser scans 2D frames, lines or spots. Most of the time you will have this set to Frame.
  • Frame Size = Scan Format or the number of spots that the laser scans on the sample (e.g., 512 x 512, 1024 x 1024, etc.).
  • Line Step: Increases scan speed but lowers the resolution in one dimension by skipping lines in the scan.
  • Scan Speed: the line frequency. Expressed in pixel dwell time as opposed to in Hz.
  • Averaging, including number of scans and whether it should be done line by line or frame by frame. A method by which photon noise can be reduced in images.
  • Method: Mean = Average, Sum = Accumulate.
  • Bit Depth: 8 bit is default.
  • Direction: unidirectional or bidirectional (Stick to unidirectional).
  • Scan Area: Zoom and Scan Field Rotation.

Channels

• Expand Channels
  

• In here you can determine which channels are actually scanned by unchecking the tracks and you can define laser powers and detector settings such as detector gain
• For most purposes you will use Integration Mode, which is the normal mode of using a PMT, but photon counting can be used to quantify signals so that each intensity level corresponds to a detected photoelectron.
• Range Indicator at the bottom of the screen will indicate saturation and black pixels

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