This is an How to....? guide

1). How to acquire a confocal image

2). Technical note on the Line scanning confocal mode

1). How to acquire confocal mode

1. Connect to the microscope
   
2. Mount and focus on your sample
   
3. In the “Optical Control” section, click on “Enable Confocal”:

Confocal Mode active

1. When in confocal imaging mode, the button will turn orange. To exit confocal mode, click the “Enable Confocal” button again.
   
2. While in Confocal imaging mode, the following extra acquisition options are available in the Optical Control panel:
   
1. Enable Navigation Mode
   
2. Line Spacing
   
3. “Enable Navigation Mode” allows you to move faster in your sample. That is because it shows frames and not confocal images (more in Technical note).
   
4. The line spacing can be adjusted by moving the slider or entering a number from 3 to 15. Line spacing also corresponds to the number of frames the software will acquire to generate a confocal image. Hence, the lower the line spacing the faster the confocal acquisition speed. For more information, refer to the technical note.
   
5. Lastly, before acquiring an image the exposure per frame needs to be set. e.g 100 ms. Now, press the acquire button to capture a confocal image.
   
6. Each image will be reconstructed and shown on the acquisition panel.
   
7. Light program and ROI mode are not supported with confocal.
   

Data saving:

Each data set is saved with the raw file, the separate confocal image for each channel and separate widefield reconstructed image in addition to all the data formats saved by Nanoimager software.

Confocal from Multi Acquisition setup (Multi-Acquisition)

Confocal imaging can be done via multi-acquisition and can be combined with Multiposition, Z stack and time-lapse acquisitions.

For activating confocal mode, In the acquisition tab use the dropdown button on Imaging mode and select Confocal. If using multiple acquisitions, this should be done with each step.

Set the exposure time and number of frames to acquire with the lasers with required laser powers.

1. Z stack and time-lapse can be added by select each option from the respective options.
   
2. Press start to acquire. 

Post-processing

1. To generate the desired optical sectioning, in the “Image Display Options” section. The background subtraction factor can be adjusted from 0 to 2. i.e from thickest slice (0) to thinnest slice (2). This can be done during acquisition or post-acquisition.
   
2. Post-processing for changing the subtraction factor can be executed in Analyze tab. Load the data set and select the option to override default confocal settings.
   
3. Now adjust the subtraction factor and click ‘Run Confocal’. The data will be saved in the same folder as the original file.
   

2). Technical note: Line scanning confocal mode

This document provides a technical overview of the laser scanning confocal mode. The Nanoimager's Confocal imaging mode implements an algorithm that helps to remove light from out of focus objects in the acquired images.

Line Spacing

The "Line Spacing" control allows changing the "density" of the of the lines projected onto the sample, which also changes the number of patterns cast on the sample that are then used as part of the algorithm to reduce background light. The optimal line spacing for image quality will depend on the sample, however a lower line spacing results in a faster algorithmic reconstruction.

Navigation Mode

Checking the "Navigate Mode" box puts the system into a mode with minimal processing that can be used to quickly navigate the sample, but has no optical sectioning. This mode is automatically disabled during acquisition.

Note that while in Navigate Mode the sample is fully illuminated as opposed to being pattern illuminated, and so the sample is exposed to more light in this mode than during a Confocal acquisition.

Optical sectioning

The "Optical Section" control allows changing the amount of background light that is removed from the images. Smaller values remove less light and the resulting image represents a thicker slice of the sample, while the larger values do the opposite and represent a thinner slice.

Exposure

Confocal imaging mode has three interdependent exposure properties:

• Exposure per frame (ms)
  

• Total exposure per image (ms)
  

• Frequency (Hz)
  

Exposure per frame (ms) is the number of milliseconds of camera exposure for each individual camera capture. Each individual capture is only one part of the Confocal image. This exposure must be a multiple of 16.66 ms. Note the actual exposure time of the camera is 11 ms less than this value

Total exposure per image (ms) is the number of milliseconds of camera exposure for each Confocal image. Each Confocal image is made up of many individual camera captures which then undergo algorithmic reconstruction. This number is a multiple of the Exposure per frame (ms) (where the multiple is the line spacing) Note the actual summed exposure time of the camera is (11 ms * value of Line Spacing) less than this value

Frequency (Hz) is the number of Confocal images made per second. The actual camera capture rate is faster than this.

Note: These settings are interconnected, changing one will alter the others

The following features are not supported by confocal mode-

1. Light program
   
2. ROI