Journal: Open Research Europe

Article Title: Methodological approaches in aggregate formation and microscopic analysis to assess pseudoislet morphology and cellular interactions

doi: 10.12688/openreseurope.14894.2

Figure Lengend Snippet: ( A – D ) Post-processed image of the aggregated INS1E cells (green) and nuclei (blue) with applied background noise reduction and immunofluorescence signal normalisation by each software. ( E – H ) The threshold segmentation of individual nuclei ID represents the thresholding success for all four software, Fiji, CellProfiler, GA3 and Imaris, respectively. ( I – L ) Focus on cell segmentation between single cells in the aggregate and how well each of the four different software could identify single cells. ( M ) The software was compared to manual counting for their performance to quantify nuclei and correct for multiple counts. The data represent an over– or underestimated count of nuclei in the different images containing aggregates of cells. The software GA3 and Imaris overestimated the count of nuclei with an average of 13% and 10%, respectively. Fiji and CellProfiler underestimated the count of nuclei with an average of 69% and 50%, respectively. ( N ) A comparison between the software for their success segmenting single cells in the cell clusters. GA3 and Imaris had close quantifications to the manual cell count with a slight underestimation of 8% and 6%, respectively. CellProfiler showed results close to GA3 and Imaris along with larger variation in over– and underestimations of the cell count with an average of 12% overestimation. Fiji showed a consistent underestimation of the cell count with an average value of 83% from the manual count. Results are calculated by the relative change, and the data comprised nine z-stack data sets.

Article Snippet: Cell quantification was completed on digital images using four different software packages: Fiji , CellProfiler (Broad Institute, Cambridge, MA, USA), NIS Elements GA3 (Nikon Instruments), Imaris 9.5.0 (Bitplane, South Windsor, CT, USA).

Techniques: Immunofluorescence, Software, Comparison, Cell Counting

Journal: Open Research Europe

Article Title: Methodological approaches in aggregate formation and microscopic analysis to assess pseudoislet morphology and cellular interactions

doi: 10.12688/openreseurope.14894.2

Figure Lengend Snippet: ( A ) GA3 and Imaris produced equal quantification of the nuclei except in image four, where GA3 slightly overestimated the count compared to Imaris. In most z-stacks, CellProfiler was closer to the manual count than Fiji except in dataset one and eight. However, overall, they underestimated the number of nuclei. ( B ) For the quantification of single cells, both GA3 and Imaris, resulted in equal counts as the manual count except in dataset four and seven. However, CellProfiler showed equal accuracy as GA3 and Imaris in many z-stacks, except in dataset two, three, four and eight. Fiji had a consistent underestimation of ~83% in all datasets. ( C ) Comparing the distinction between core and mantle in the aggregates. The manual counting had a lesser distinction between the core and mantle distribution of the INS1E cells compared to both GA3 and Imaris that used a percentage area distribution mask to quantify the distribution of the cells in each area. This resulted in an 89% and 17% core versus mantle distribution in GA3 and 83% and 17% in Imaris. ( D ) Another method was to quantify overlapping signals between two immunofluorescence channels within a 0 µm distance. The GA3 and Imaris software resulted in equal count except in datasets nine and seven. However, in most situations, the manual quantification had a higher count than GA3 and Imaris. Only dataset three, five, six and eight had comparable results as the software. Results are calculated by the relative change, and the data set included nine z-stack data sets.

Article Snippet: Cell quantification was completed on digital images using four different software packages: Fiji , CellProfiler (Broad Institute, Cambridge, MA, USA), NIS Elements GA3 (Nikon Instruments), Imaris 9.5.0 (Bitplane, South Windsor, CT, USA).

Techniques: Produced, Immunofluorescence, Software