Array Advice - Scanning of Arrays

View tips and solutions to common problems for the scanning of arrays:

Tips

  • If possible, place the scanner in a box with ozone scrubbers to reduce ozone levels to <5ppb.
  • Ensure correct side of slide is placed in slide holder/scanner according to manufacturer’s instructions.
  • Always scan slides immediately after washing to avoid degradation of signal due to environmental ozone.

Solutions to Common Problems

Problem 1 - Low Cy5 signal gradient across slide

Cause: “Dry” ozone- this occurs when the slide is being scanned. Most evident when using a slide holder where a Cy5 degradation gradient can be caused by higher Cy5 degradation at the open end of the slide holder.

Fig 1. Low Cy5 signal gradient across slideFig 1. Low Cy5 signal gradient across slide

Solution:

  1. Ensure that the slide is scanned immediately after washing
  2. Place the scanner in an enclosed area with ozone scrubbers

 

Problem 2 - Low Cy5 signal towards the edges of a feature

Fig 2. Low Cy5 signal towards edges of a featureFig 2. Low Cy5 signal towards edges of a feature

Cause: “Wet” ozone- after washing, the outer edges of each spot will dry before the centre of the spot, thus the edges of the spot will be exposed to the atmosphere (and hence ozone) before the centre

Solution:

  1. Ensure that the slide is scanned immediately after washing
  2. Place the scanner in an enclosed area with ozone scrubbers

 

Problem 3 - Scanner jitters (observed as rough edges on the array spots)

Fig. 3. Example of scanner jittersFigure 3. Example of scanner jitters

Cause: Auto-focus issue where the laser beam does not respond quick enough when the auto-focus stage moves backward or forward.

Solution: Contact the scanner manufacturer

 

Problem 4 - Bright red spot on array

Fig. 4. Example of bright red spot on array

Fig. 4. Example of bright red spot on array 

Cause: Scanner slide holder may have fluorescent residue.

Solution:  Clean slide holder with isopropanol. If spot remains, place scotch tape on the spot on the slide holder and then remove it.

 

CytoSure™ products are for research use only; not for use in diagnostic procedures