Low magnification spinning disk confocal:

considerations on camera choice

One of the advantages of having 2 camera ports on a spinning disk confocal is the possibility of combining 2 cameras with very different specifications.

The most common camera types (large field and 11um pixel size) can be paired with a camera with small pixels (4.25um) to better match the sampling rate and consequent resolution that can be achieved with low magnification objectives.

In this specific test, we tested side by side an Andor Sona and a Teledyne Photometrics IRIS15 with 4X, 10X and 20X objectives (Plan Apo Lambda, Nikon) on the X-LIGHT V3 confocal, using a large specimen of fluorescently labelled late stage mouse embryo.

The first difference between the two cameras tested is the field of view (FOV). The IRIS15 has a rectangular chip (5056x2560pixels) with a diagonal of 25mm, while the Sona has a square chip (2048×2048 pixels) with a diagonal of 32mm.

Fig. 1_Diagram showing FOV of microscope, IRIS15 and Sona.

With the Sona, it is necessary to apply a crop to fit the achievable field of view of the microscope and the confocal. The achievable FOV on the sample is shown in Fig. 2.

Fig. 2_ Achievable full field acquisition on Nikon Ti2 microscope (25mm FOV) with a Plan Apo Lambda 4x objective. IRIS15 on the left, Sona on the right. Scale bar 1mm

Differences in achievable FOV are essential in acquisition speed, especially when large specimens need to be acquired. Sampling rate/Resolution also play a predominant role, on a camera-based acquisition, this parameter is determined by pixel size (4.25um vs 11um). Differences in resolution are more difficult to appreciate when looking at the stitched image of a large specimen, but can easily be appreciated when cropping or zooming in.

Cropped regions of identical sizes are compared for the two cameras at three magnifications:

Objective 4X Plan Apo Lambda (NA 0.2)

Fig. 3_ Detail of acquisition (800x800um ROI) with IRIS15 (left) and Sona (right). Objective Plan Apo Lambda 4X, Nikon. Scale bar 0.1mm

Objective 10X Plan Apo Lambda (NA 0.45)

Fig. 4_ Detail of acquisition (400x400um ROI) with IRIS15 (left) and Sona (right). Objective Plan Apo Lambda 10X, Nikon. Scale bar 0.1mm

Objective 20X Plan Apo Lambda (NA 0.75)

Fig. 5_ Detail of acquisition (200x200um ROI) with IRIS15 (left) and Sona (right). Objective Plan Apo Lambda 20X, Nikon. Scale bar 0.1mm

The last key factor to consider is the sensitivity, or quantum efficiency (QE) of the different cameras. In this specific case, the IRIS15 has a lower QE compared to the Sona. This difference can be compensated by using a longer exposure time or by selecting a higher excitation power.

To conclude, when choosing your camera, it is essential to consider the following questions:

  1. Which objectives are required for the key applications?
  2. Is it possible to have two cameras?
  3. What is most important between speed and resolution?
  4. How photo-sensitive and photo-stable is the sample?

The application note has been prepared in collaboration with BergmanLabora.

 

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