The use of continuous monitoring and computer-assisted image analysis for the histochemical quantification of hexokinase activity

The effect of endogenous 6-phosphogluconate dehydrogenase (6PGDH) on the histochemical quantification of low-Km hexokinase activity in rat submandibular salivary glands has been investigated using a Seescan Solitaire Plus image analysis system and a modified black and white Newvicon TV camera. Absorbance readings of neutral density filters were close to their known absorbance values. A significant correlation was found between the absorbance of Nitro BT reduction products within sections, with and without the use of a 588 nm interference filter. Furthermore, absorbance readings obtained from 8 microns-thick sections were 1.92 and 2.22 times greater than values obtained from 4 microns-thick sections using 'white' and 588 nm light respectively. The level of background illumination was not critical for absorbance measurements provided it was below the level that saturated the Newvicon camera and was not changed between background and image capture. The greatest variations in absorbance readings on tissue sections were associated with changes in zoom and objective combinations. Our studies indicate that this relatively low cost image analysis system can give reproducible absorbance readings from various structures defined in digitized, captured images of tissue sections. Results from continuous monitoring studies indicated that the high levels of 6PGDH activity in excretory ducts caused a 1.67-fold overestimation of hexokinase activity as assessed by absorbance readings performed throughout a 22-minute reaction period. By contrast, overestimation of hexokinase activity in salivary gland acini only became apparent after 8 min incubation with a 1.4-fold overestimation being seen at 22 min. This difference appears to reflect the relatively low hexokinase and 6PGDH activities present in acini compared with excretory ducts.