A new multichannel method quantitating TUNEL in detached photoreceptor nuclei

Abstract

Nuclear co-localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer-based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co-localization. We established a new ImageJ-based automated multichannel thresholding (MCT) method to quantitate nuclear co-localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL-positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross-sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as "typical" or high density "hotspot" TUNEL regions (n = 10/group). Images were analyzed and compared between the MCT method and the published techniques including both "standard" and "high" settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built-in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland-Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R² = 0.8972, hotspot: R² = 0.9000) with minor to non-significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R² = 0.8036, standard/hotspot: R² = 0.4309, high/typical: R² = 0.7895, high/hotspot: R² = 0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer-based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells.

Keywords: Apoptosis; Cell death; Image analysis; ImageJ; Retina; Retinal detachment; TUNEL; Thresholding.

Fig. 1

Immunohistochemistry image capture of an example hotspot region showing the TUNEL (red) and DAPI (blue) staining. The RGB cropped ONL region (A) is processed by the ‘Color Threshold’ function in ImageJ by adjusting the red and blue channels to create a black & white image (B) that shows black as a positive region and white as negative space. The black & white image is run through a binary watershed segmentation along with manual watershed adjustment before it is processed by the ‘Analyze Particles’ function in ImageJ to count the positive cells. This creates an outline map of the positive, detected cells (C). The final image (D) is created by overlaying the outline (C) onto the original image (A) to provide a visual assessment of counted TUNEL and DAPI+ cells.

Fig. 2

Immunofluorescence image (A) of a typical TUNEL (red, B) and DAPI (blue, C) region prior to cropping. The white box shows the region of analysis used in (B–H). The merged cropped region (D) was processed with the primary four methods: the ImagePro method (E), the RA Toolkit standard setting (F), the RA Toolkit high setting (G), and the MCT method (H). The corresponding individual total cell counts acquired with each method are shown in (I) where ‘E’ refers to ImagePro, ‘F’ refers to the RA Toolkit standard setting, ‘G’ refers to the RA Toolkit high setting, and ‘H’ refers to the MCT method. Scale bar denotes 50μm. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, GCL – ganglion cell layer.

Fig. 3

Immunofluorescence image (A) of a hotspot TUNEL (red, B) and DAPI (blue, C) region prior to cropping. The white box shows the region of analysis used in (B–H). The merged cropped region (D) was processed with the primary four methods: the ImagePro method (E), the RA Toolkit standard setting (F), the RA Toolkit high setting (G), and the MCT method (H). The corresponding individual total cell counts acquired with each method are shown in (I) where ‘E’ refers to ImagePro, ‘F’ refers to the RA Toolkit standard setting, ‘G’ refers to the RA Toolkit high setting, and ‘H’ refers to the MCT method. Scale bar denotes 50μm. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, GCL – ganglion cell layer.

Fig. 4

Immunofluorescence image (A) of the hotspot TUNEL region shown previously. The white box indicates the magnified region (B–H). The merged (B), TUNEL (C), and DAPI (D) channels are shown with no analysis overlays. The following analysis methods are shown with the counted cells overlaid with outlines: the manual MCT method (E), the ImagePro method (F), the RA Toolkit standard setting (G), and the RA Toolkit high setting (H). White arrowheads indicated regions where there exists positive TUNEL signal but no positive DAPI signal. White arrows indicate a regions of conjoined TUNEL signal but with two distinct nuclei. Scale bar denotes 50μm.

Fig. 5

Bland-Altman mean-difference plots for TUNEL+ cells//mm² show agreement between the manually thresholded MCT method, the ImagePro method, and the RA Toolkit standard and high settings in both typical and hotspot image types (A). Solid lines represent the mean difference. Dashed lines represent the 95% limit of agreement (± 1.96 SD). Regression analysis of TUNEL+ cells/mm² detected by the MCT method versus the ImagePro method and the RA Toolkit standard and high threshold settings in both typical and hotspot image types (B). Dotted lines represent the linear regression trendline. Dashed lines represent a perfect one-to-one correlation for comparison.

Fig. 6

Immunofluorescence images of typical TUNEL+ regions thresholded by manual and automated MCT methods (A). Immunofluorescence images of hotspot TUNEL+ regions thresholded by manual and automated MCT methods (B). For both A and B, the first column shows the cropped region with no analysis outlines (top) and the outlines of the manual MCT method (bottom). On subsequent columns the top row is thresholded with the MaxEntropy algorithm while the bottom row is thresholded with the RenyiEntropy algorithm. The second column uses the IsoData algorithm to threshold the DAPI+ cells, the third column uses the Li algorithm, the fourth column uses the Mean algorithm, and the fifth column uses the Otsu algorithm. Scale bar denotes 50μm.

Fig. 7

TUNEL+ cell counts for all analysis methods. Images 1 through 10 represent hotspot regions while images 11 through 20 represent typical regions. Hotspot cell count scale to the left y-axis from 0 to 600 while typical cell counts scale to the right y-axis from 0 to 300. The primary analysis methods are represented by dots and full lines (RA Toolkit standard – open red circle, RA Toolkit high – filled red circle, ImagePro – open black circle, MCT: Manual – filled black circle). All methods utilizing the MaxEntropy algorithm are represented by triangles while methods utilizing the RenyiEntropy algorithm are represented by squares. The count using MaxEntropy without the MCT method is represented by a black triangle with a dotted line while the count using RenyiEntropy without the MCT method is represented by a black square with a dashed line. The subsequent automated MCT methods are colored according to the DAPI thresholding algorithm used (IsoData – Red, Li – Green, Mean – Purple, Otsu – Blue).