Table 2 The compatibility of ImmunoRatio with variable staining and image acquisition settings

   too dilute (1:400)

too low labeling index (Figure 6a)

   optimal (1:100)

best match with visual counting (Figure 6b)

   too strong (1:25)

cytoplasmic background causing overly high labeling indexes (Figure 6c)

Hematoxylin counterstaining

   weak

insufficient nuclear segmentation (Figure 6d)

   optimal

best match with visual counting (Figure 6e)

   strong

false segmentation of cytoplasmic structures (Figure 6f)

Microscope objective magnification

(using 1× phototube)

   10×

non-carcinomatous cells often included*

   20×

for accurate result, an average of three images per sample is recommended

   40×

for accurate result, averaging several images per sample is recommended

Image brightness

   underexposed (too dim)

mean gray intensity of the blankfield image <200

   in optimal range

as guided by the Camera Adjustment Wizard of ImmunoRatio

   overexposed (too bright)

mean gray intensity of the blankfield image >250

Image contrast

   too low

foreground mean gray intensity over 85% of the background mean gray intensity

   in optimal range

as guided by the Camera Adjustment Wizard of ImmunoRatio

   too high

foreground mean gray intensity under 50% of the background mean gray intensity

Image compression

   uncompressed (lossless)

slow network transmission (slower overall analysis time)

   JPEG, quality factor 50 to 100 (lossy)

optimal for ImmunoRatio

   JPEG, quality factor <50 (lossy)

visible image artifacts