Pasquale Chieco

Basic strategies for valid cytometry using image analysis

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetics in difficult imaging situations, such as low fluorescence signals and high absorbance signals.

Effect of administrative vehicle on oral 1,1-dichloroethylene toxicity

Abstract The relationship between the acute toxicity and biologic fate of 1,1-dichloroethylene (1,1-DCE) was examined in fasted and fed male Sprague-Dawley rats given 200 mg 1,1-DCE/kg orally in a mineral oil, a corn oil, or an aqueous Tween-80 vehicle. Exhalation of unchanged 1,1-DCE by individual rats was monitored at selected 15-min intervals for 5 hr and all rats were sacrificed at 6 hr. The administrative vehicle affected the magnitude of liver injury in fasted rats; with mineral oil or corn oil, injury was massive [> 100-fold elevation of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)] whereas with the aqueous Tween-80 vehicle, injury was moderate t 15-fold elevation of GOT and GPT). In contrast, in all fed groups liver injury was slight (two- to threefold elevation of GOT and GPT). Rats with massive liver injury also had an ∼50-fold increase in plasma free hemoglobin and their kidneys exhibited numerous granular “heme” casts in Henles loop without apparent degenerative changes in either glomeruli or tubular epithelium. No pathologic changes were observed in heart, lungs, spleen, adrenals, or duodenum. The administrative vehicle did not affect the total amount of 1,1-DCE exhaled, which was approximately half the given dose, nor did the vehicle affect the initial rapid phase of 1,1-DCE exhalation, which lasted ∼1 hr and had t 1 2 values ranging from 15 to 21 min for the fasted groups and from 10 to 13 min for the fed groups. In contrast, the later slow phase of 1,1-DCE exhalation was predictably affected by the administrative vehicle; t 1 2 values for the fasted and fed groups, respectively, were most prolonged with the poorly absorbed mineral oil vehicle (257 and 280 min), intermediate with the more digestable corn oil vehicle (73 and 103 min), and briefest with the more absorbable aqueous Tween vehicle (22 and 42 min). Further studies are needed to determine if the relative resistance of the fed animals to hepatic injury is due to the capacity of these animals to detoxify 1,1-DCE for a longer duration than the fasted animals.

Image Cytometry: Protocols for 2D and 3D Quantification in Microscopic Images

Microscopy-based imaging is booming and the need for tools to retrieve quantitative data from images is urgent. This book provides simple but reliable tools to generate valid quantitative gene expression data, at the mRNA, protein and activity level, from microscopic images in relation to structures in cells, tissues and organs in 2D and 3D. Volumes, areas, lengths and numbers of cells and tissues can be calculated and related to these gene expression data while preserving the 2D and 3D morphology. Image cytometry thus provides a comprehensive toolkit to study molecular processes and structural changes at the level of cells and tissues.

Improvement in soluble dehydrogenase histochemistry by nitroblue tetrazolium preuptake in sections: a qualitative and quantitative study.

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.

Image Cytometry Protocols

In the May 2013 issue of the Journal of Histochemistry & Cytochemistry, two papers were published based on quantification in light microscopic images or image cytometry (Garrido et al. 2013; Ruiz-Rosado et al. 2013). As Garrido and co-workers stated, valid quantification is essential to obtain meaningful numbers. However, the authors of this letter to the editor have serious questions regarding both publications and would like to outline these here. Our criticism is focused on different issues in the two publications, so we discuss them separately. The article by Garrido et al. (2013) describes quantitative histological assessment of xenobiotic effects on rat liver, thyroid gland, and the pars distalis of the pituitary gland using whole-slide automated image analysis. We consider it as an important publication that allows preclinical screening of compounds for safety evaluation. This methodology combines quantitative and structural information on the effects of xenobiotics on tissues and we hope that it will have a considerable impact on drug screening research. However, the major flaw of this article is the lack of information on how the image cytometry was exactly performed. On the basis of the procedures described in the Materials & Methods section of the article, it is impossible to reproduce the study. Information that is lacking concerns issues such as, (1) What were the exact fixation and staining procedures? (2) How was the staining [polymerized diaminobenzidine (polyDAB)] intensity quantified? (3) Hematoxylin and polyDAB have overlapping absorbance spectra, and thus, what is the light that has been used to illuminate the sections—white light or light of a specific wavelength? If the latter is the case, what wavelength? (4) How was the threshold set to distinguish between “dark staining” and “weak staining”? (5) The same questions concern the quantification in H&E stained sections. How did the authors segment the hematoxylin-stained nuclei from the eosinstained cytoplasm? (6) What type of microscope, what magnifications, and what type of camera were used, including numbers of pixels of whole-section images and higher magnifications? (7) Digital cameras are very sensitive to infrared radiation while our eyes are not, and infrared blocking filters have to be used to reduce noise in the digital images. Therefore, the question arises whether infrared radiation was blocked or not. In summary, essential details of the image cytometry procedures and, in particular, the image capturing procedures are lacking. We have recently published protocols for valid image cytometry in 2D and 3D that include all these essential aspects of the methodology (Chieco et al. 2013). We hope that image cytometry and quantification in microscopic images will acquire the status that is predicted in the July 2012 issue of Nature Methods (see Editorial 2012) and in Yuan et al. (2012), which describe an essential role for image cytometry technology in large-scale cancer studies. We hope that the protocols for the retrieval of meaningful quantitative data from microscopic images that we described (Chieco et al. 2013) will help to bring image cytometry into that position and help authors to provide details on methodology in publications that are necessary to check whether 500497 JHCXXX10.1369/0022155413500497Journal of Histochemistry & CytochemistryNoorden and Chieco research-article2013