In the present study we investigated the association between objective changes in nuclear morphology and cell distribution following induction of caspase-3 in apoptotic cells. We found that caspase-3-positive apoptotic cells show morphological features that can be objectively quantified. A novel morphological indicator, defined as the nuclear circumference divided by form factor, demonstrated the strongest correlation with caspase-3 expression.
Age-related macular degeneration (AMD) is a major cause of blindness in the developed world resulting from a diseased retinal pigment epithelium (RPE) . For the majority of AMD patients (85%), no effective treatment alternative exists. Replacement of the RPE monolayer, however, has been proposed as a future therapy for this group [13–15]. The belief that RPE transplantation holds promise as having therapeutic benefit in the treatment of AMD builds on several studies reporting improved visual function following the restoration of the sub-retinal compartment . Methods for RPE cell replacement include injection of RPE cell suspensions and transplantation of intact RPE cell sheets. Though progress has been made to improve these techniques, the production of RPE transplants is still at an experimental stage. A method for assessing quality of the transplants prior to transplantation would aid in the development of RPE replacement techniques. Quality assessment of transplants pre-operatively can be performed invasively or non-invasively. Invasive techniques may include harvesting and processing of small biopsies from the transplant for analyses of gene and protein content. Although these are powerful techniques, they risk damaging the transplant and necessitate a larger transplant due to loss of tissue during biopsy harvesting. Non-invasive techniques for quality assessment include various microscopy analyses, such as light microscopy and in vivo confocal microscopy (IVCM). The latter has lately been widely employed within the field of ophthalmology, aiding in the identification of putative corneal epithelial stem cell niches embedded in the ocular surface , keratinized surface epithelia in patients suffering from dry eye disease  and in the quantification of corneal endothelium density as part of quality control of corneal transplants . Contrary to invasive techniques involving removal of the analyzed tissue, non-invasive image-based techniques for assessing quality of RPE cell sheets prior to transplantation would allow direct comparison of transplants pre- and post-operatively. As we did not use a non-invasive technique for obtaining images of the cell nuclei in the cultured cell sheets, the current study is a preliminary step towards the development of a method to detect apoptotic cells in RPE transplants based on cytometric measurements. Assessing cell death in RPE transplants prior to surgery could likely aid in quality selection and improve transplantation outcome.
Digital pathology has developed rapidly in recent years. Objective quantification of cell- and tissue- based measures offers the prospect of reducing bias due to subjective interpretation and help meet pathologist workload demand . There are, however, several challenges with objective image quantification that need to be addressed, including image artifacts, such as blurred regions or chromatic aberrations, and batch-to-batch differences . Techniques have been developed for describing images based on pixel, object, and semantic features . Pixel-based image analysis derives information from features such as texture (e.g. sharpness, contrast) and color. The latter has been used to classify breast tumors through data reduction based on diffusion maps . Object-level features encompass higher order features including cellular structures (e.g. nuclei, cytoplasm). Object-level information can be obtained through image segmentation. Semantic level features build on lower level features and make use of preprocessing methods, such as the bag-of-features method . The latter can also include machine-learning technology. Digital pathological techniques are increasingly employed for objective assessment of whole-slide image, the latter of which is gradually becoming common clinical practice .
In our study, staurosporine-incubated cells differed significantly from control cells with regards to their smaller nuclear area and circumference and their higher form factor. Similar results have been reported with several other cell types [6–9, 22]. The decrease in cell nuclei due to DNA loss and the increase in form factor upon initiation of apoptosis form the rationale for using NAF as a morphological indicator of apoptosis.
Both the staurosporine-exposed cultures and the control cultures exhibited a relatively even cell distribution (non-clustered and non-random) in the current study. However, the staurosporine-exposed cultures presented less even cell spacing. Our results are partly in line with a previous study in which blood-derived Jurkat cells temporarily clustered upon initiation of apoptosis . However, the authors did not employ nearest neighbor analysis, but demonstrated cell clustering in photomicrographs. The lack of obvious cell clustering upon apoptosis in the current study may have been due to our use of adherent cells. One third to one sixth of the cell nuclei in our study were co-associated. These numbers are only approximate, however, as they are derived from comparing cell counts with and without the “Watershed” algorithm. Applying “Watershed” in ImageJ could, in theory, inadvertently either 1) removed some cell nuclei by separating them into small fragments that were excluded from the cell count on the basis of area; or 2) increased the number of cells by dividing large single cell nuclei into two or more fragments that were included in the cell count as separate cells. The NND, which was quantified to assess cell nuclei distribution, was defined as the distance between the centroid of each individual nucleus and its closest neighboring nucleus. From this it follows that the NND of associated nuclei would be approximately equal to the sum of the radii of the two nuclei. Thus, this may have affected the computation of cell nuclei distribution, giving a tendency for a somewhat larger NND with larger nuclei.
The use of immunocytochemistry allows for the comparison of protein expression and morphology in single cells. In addition, by employing quantitative immunofluorescence the relative protein expression can be objectively measured. In the current study, we compared single cell caspase-3 expression with the same cell’s morphology measurements. This allowed a more sensitive analysis of the relationship between cell morphology and phenotype than merely comparing the mean phenotype of entire cell cultures/samples [7–9, 22]. Using this method, we found that caspase-3 expression in apoptotic cells showed the highest correlation with a morphologic indicator defined as the nuclear circumference divided by form factor. We also found that the NAF, when computed by its original formula : nuclear area divided by form factor, had greater correlation with caspase-3 expression than when calculating the NAF by the formula: nuclear area multiplied by form factor. In fact, the morphological indicators nuclear circumference, area and form factor alone had greater correlation with caspase-3 expression than nuclear area multiplied by form factor.
We used DAPI as nuclear stain in the current study due to its simple staining protocol, high specificity and availability for quantitative analyses . DAPI can be used with live and fixed cells , and its fluorescence increases 20-fold upon binding to DNA . However, a possible disadvantage of using DAPI is the increase in glare from intensely bright DAPI-stained nuclei. Glare can affect nuclear measures and must be taken into consideration when interpreting the results in the current study. Techniques for avoiding bias due to glare have been described elsewhere . Feulgen stain is considered the gold standard for DNA image cytometry . It has been widely used for objective DNA quantification as the amount of stain seen in the nucleus directly correlates with DNA content . However, the staining protocol is comparatively longer and is more laborious compared to using DAPI. The latter has been shown to yield reliable measurements of staining intensity and nuclear area size . Moreover, DAPI gives similar results as the conventional Feulgen staining , making DAPI a good candidate for use in cytometric analyses.
In this study, we performed both random and stratified sampling [29, 30]. The former was used when 8-bit images were thresholded to binary images, thereby segmenting the images into figure (nuclei) and background (space between nuclei) . Stratified sampling was done when measuring fluorescence intensity of the Cy3-conjugated caspase-3 antibody in the vicinity of each cell nucleus. For closely associated nuclei, we cannot fully exclude the possibility of biased measurements of caspase-3 expression due to overlapping cells. However, this should not have affected the results significantly, as we mainly used monolayer cultures. We employed the default method defined in ImageJ for auto-thresholding each photomicrograph to create binary images. There are, however, at least 16 different thresholding algorithms available in ImageJ, the use of which may have given different results.