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

Abstract

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.

Glucose-6-phosphate dehydrogenase activity in spinach as measured by image analysis: A new approach for plant enzyme histochemistry

“…For many years, scanning and integrating microdensitometers have been widely considered to be the best instruments for such in situ studies. However, automated instruments using video methods have also been employed successfully during the past few years in quantitative enzyme histochemistry and cytochemistry in animal tissue sections (Hildebrand & Schleicher, 1986;Blanco et al, 1988;Lawrence et al, 1989;Van Noorden, 1989;Laborde et al, 1990;Van Noorden & Butcher, 1991;Markovic et al, 1994). Despite the important development of image analysis systems and the various advantages they show over microdensitometers, they do not seem to have been used for quantification of enzyme reactions in plant tissue sections.…”

“…It is now possible to collect equivalent data more quickly and more easily with image analysers. Such equipment has been used successfully in the past decade for quantifying the activity of various enzymes in animal tissues (Lawrence et al, 1989;Van Noorden, 1989;Chieco et al, 1994). However, to our knowledge, this approach does not yet appear to have been exploited in plant histochemistry.…”