Fig 1.
Outline of the FluoroCellTrack algorithm.
The starting and ending points are depicted by ovals, the input and output commands are depicted by parallelograms, the processing steps are rectangles, and the decision steps are diamonds. The green arrows denote the steps in droplet detection, the blue arrows show contour detection, and the purple arrows represent information extracted from combining droplet and contour outputs. The dotted lines represent extended applications of the algorithm.
Fig 2.
Comparison of edge detector and CHT in detecting droplets.
Left: The edge detector function involved manual thresholding resulted in incorrect detection: low T led to false positive values while high T led to data destruction, as highlighted in dashed white boxes. Right: CHT resulted in precise detection of droplets with exact requirement of 25<R<50 microns, as highlighted in white boxes. CHT outlined the droplets with green boundaries and orange centers which were finally counted for further analysis. Scale bar represents 70 μm.
Fig 3.
Steps involved in contour detection.
A mask was generated from the input image (A) through definition of color boundaries. This mask was convolved with the input image, smoothened and converted to grayscale as shown in (B). Edge detection, dilation and erosion was performed on (B) to yield (C) which was then subjected to random walker segmentation to map the contour centers as shown in (D). The detected contours were finally checked for area where cellular area equal to or greater than 130 pixels were outlined and labeled (E). Scale bar represents 70 μm.
Fig 4.
Quantification of encapsulation information.
(A) The process of cell detection involved setting of color boundaries, edge detection, segmentation to finally generate ROIs around cells including live cells (green), dead cells (red) and overlapping cells (yellow) outlined in red, blue and purple. Differently colored ROIs are counted for respective type of cells (as shown in output). (B) Detection of droplets through CHT mark them with green outlines and orange centers. The overlapping droplet center map with cell center map and defining droplet axis centers and radius boundaries resulted in generation of encapsulation information in output data. Scale bar represents 70 μm.
Fig 5.
Comparison of FluoroCellTrack to manual inspection to quantify single OPM-2 cell viability.
Cells were treated with different dozes of BTZ for 24 h (A), 48 h (B), and 72 h (C). An average of ~99% similarity was observed between the outputs of FluoroCellTrack and manual inspection in terms of single cell and multiple cell encapsulation (SCE and MCE) and assessment of cell viability. A minimum of 275 cells and n = 787 droplets were analyzed in each case.
Fig 6.
Identification of co-encapsulated single cells and NPs.
A) Representative images of (i) GFP-HeLa cells and (ii) RFP-MDA MB 231 cells tracked by Eu3+-doped NPs and (iii) GFP-HeLa cells and (iv) RFP-MDA-MB-231 cells tracked by Tb3+-doped NPs. White boxes denote droplets co-encapsulated with cells and NPs. Eu3+-doped NPs and Tb3+-doped NPs are colored magenta and blue in (ii) and (iii) to distinguish them from RFP- and GFP-expressing cells. B) Approach to quantify droplet tracking data using an image from RFP-MDA-MB-231 cells co-encapsulated with Eu3+-doped NPs. Eu3+-doped NPs are outlined in yellow while RFP-MDA-MB-231 cells are outlined in blue. The single droplet represents the RFP-MDA-MB-231 cell being tracked by Eu3+-doped NPs. Scale bar represents 70 μm.
Fig 7.
Comparison of FluoroCellTrack to manual inspection of co-encapsulation droplet tracking data.
Droplet subpopulation identification was performed for RFP-expressing MDA-MB-231 cells tracked by (A) Eu3+-doped NPs and (B) Tb3+-doped NPs. SC-single cells, MC-multiple cells. n = 787 droplets were analyzed for each experiment.
Fig 8.
Quantification of intracellular fluorescence using FluoroCellTrack for the uptake of 50 μM ARG.
The input image (A) of single HeLa cells containing the ARG peptide was processed to detect droplets (B). (C) Cell detection required area thresholding to filter out debris (dashed white box) from cells (white box) trapped in droplets. (D) Overlapping of droplet center and contour maps produced output data consisting of encapsulation and intracellular fluorescence information.
Fig 9.
Quantification of intracellular parameters to understand heterogeneous peptide uptake.
(A) The outlined fluorescent cell from the fluorescent channel of the input image was traced for its center map and the center was detected in red shown in (B). The 8-bit histogram plot was generated to observe the frequency of grayscale intensity values in (C) leading to the generation of maximum, minimum peak values of intensity along with variance in intensity values which is finally mapped into a 16-bit scale as shown in the output data (D). Scale bar represents 10 μm.