The input czi file has the following channels
The corresponding segmentions from Dotdotdot
Download the toolbox directory and assign the paths of input czi file and toolbox to Matlab variables as shown below. The toolbox should be added to the Matlab’s current working directory to run the code.
>> filename = '/dcl01/lieber/ajaffe/Maddy/RNAscope/dotdot_vignette/dotdot_vignette/Mouse2.czi';
>> toolbox = '/dcl01/lieber/ajaffe/Maddy/RNAscope/dotdot_vignette/dotdot_vignette/toolbox';
>> addpath(genpath(toolbox)) %adding toolbox path to current working directoryThe command rnascope_mouse takes in the filename and toolbox as inputs. This command uses segmct function from the toolbox CellSegm for nuclei segmentatation.
The CellSegm toolbox provides the user with several input options for smoothing (coherence enhancing diffusion, edge enhancing diffusion, gaussian) and thresholding (iterative thresholding, adaptive thresholding, gradient thresholding, ridge enhancement). The inputs for segmct function are the raw DAPI channel, estimated nucleus size (minimum and maximum in cubic micron) and a matlab structured array called prm which takes in the above mentioned options.
The options given to prm suitable for the current mouse data are shown below.
prm.method = 'adth'; % adaptive thresholding
prm.adth.filtrad = 13; % Filter size for thresholding
prm.adth.th = 0.01; % threshold
prm.split = 1; % 1 = split the segmented nuclei, 0 = donot split.
prm.splitth = 1; % The amount of splitting is controlled by PRM.SPLITTH where lower values gives stronger splitting.
prm.splitplane = round(Z/2); % Start splitting from the middle plane
prm.h=[voxelSizeX,voxelSizeY,voxelSizeZ]; % x,y,z pixel to um
minN = 0.038; % minimum nucleus volume in cubic micron given as volume of one voxel for current data
maxN = ((4/3)*(pi)*(Z*0.4/2)^3); % maximum nucleus volume in cubic micron given as volume of sphere with nucleus radius "Z*0.4/2" (the images are taken to approximately cover the depth of a nucleus (Z), so the nucleus radius in cubic micron is (Z/2)*(pixel resolution in Z))
[cellbw1,~,~,~] = cellsegm.segmct(DAPIchannel,minZ,maxZ,'prm',prm);The above prm structure is default for the rnascope_mouse function. When the rnascope_mouse is run it produces the following output on command line explaining the process.
The channel/dye names in the sample mouse data were wiped out when we cropped the images (since they were too big to version control on GitHub). Line #20 in rnascope_mouse adds default channel names to these sample data, which should be commented out when using the code with real data.
>> rnascope_mouse(filename, toolbox)
Reading Images: 1 of 72 Frames
Reading Images: 2 of 72 Frames
Reading Images: 3 of 72 Frames
...
Reading Images: 69 of 72 Frames
Reading Images: 70 of 72 Frames
Reading Images: 71 of 72 Frames
Reading Images: 72 of 72 Frames
Elapsed time is 6.512189 seconds.
"extracted" "Cy5" "DsRed" "EGFP" "DAPI"
segmenting DAPI
This is SEGMCT for segmentation of cytoplasmically stained cells
This is SEGMCT using settings % describing what values and options are used in 'prm'
method : adth
illum : 0 % correct for any illumination in images (default = 0 (yes), = 1 (no))
illumdiameter : 25
splitth : 1
split : 1
h : 0.313 0.313 0.396
just : 0.9
splitplane : 9
voxelvol : 0.038796
minvol : 38
minvolvox : 979
maxvol : 195432.1958
maxvolvox : 5037467
minvolfull : 38
maxvolfull : 195432.1958
splitvolvox : 979
This is SMOOTHIM using settings % using default "gaussian" smoothening filter
planewise : 1
gpu : 0
h : 0.313 0.313 0.396
method : gaussian
Using Gaussian smoothing
Using settings
diameter : 5 5 3
stdev : 2
Using segmentation method ADTH in SEGMCT % using "adaptive" thresholding
This is SEGMADTH using settings
th : 0.01
filtrad : 13
h : 0.313 0.313 0.396
minvolvox : 979
maxvolvox : 5037467
Splitting cells using distance function
This is SPLITCELLS splitting objects
Number of objects due to splitting: 8
This is SMOOTHIM using settings
planewise : 1
gpu : 0
h : 0.313 0.313 0.396
Using Gaussian smoothing
Using settings
diameter : 9 9
stdev : 3
Number of objects afters splitting: 20
Remove small parts that are not cells
Removed 8 regions due to small size
Removed 0 regions due to large size
Number of objects left: 12
Elapsed time is 52.015566 seconds.
no.of nuclei detected: 14
CHANNEL img.Cy5: 426 dots detected
1 cells finished in time 0.044959s
2 cells finished in time 0.083727s
3 cells finished in time 0.11191s
4 cells finished in time 0.14625s
5 cells finished in time 0.18092s
6 cells finished in time 0.20822s
7 cells finished in time 0.24323s
8 cells finished in time 0.2756s
9 cells finished in time 0.30331s
10 cells finished in time 0.33273s
11 cells finished in time 0.35857s
12 cells finished in time 0.38855s
13 cells finished in time 0.41298s
14 cells finished in time 0.43719s
completed Cy5
CHANNEL img.DsRed: 781 dots detected
1 cells finished in time 0.051917s
2 cells finished in time 0.11335s
3 cells finished in time 0.15307s
4 cells finished in time 0.2112s
5 cells finished in time 0.26617s
6 cells finished in time 0.30689s
7 cells finished in time 0.35975s
8 cells finished in time 0.41578s
9 cells finished in time 0.45942s
10 cells finished in time 0.50974s
11 cells finished in time 0.55287s
12 cells finished in time 0.60081s
13 cells finished in time 0.63754s
14 cells finished in time 0.67165s
completed DsRed
CHANNEL img.EGFP: 284 dots detected
1 cells finished in time 0.027005s
2 cells finished in time 0.051664s
3 cells finished in time 0.072558s
4 cells finished in time 0.098938s
5 cells finished in time 0.12521s
6 cells finished in time 0.14503s
7 cells finished in time 0.16891s
8 cells finished in time 0.19495s
9 cells finished in time 0.21682s
10 cells finished in time 0.23933s
11 cells finished in time 0.25986s
12 cells finished in time 0.29001s
13 cells finished in time 0.31296s
14 cells finished in time 0.33317s
completed EGFP
Elapsed time is 0.334633 seconds.The rnascope_mouse function outputs the following four .mat files that are saved where the input .czi file is located.
These matfiles are matlab structures with each field being a channel from the .czi file.
>> load('~/Mouse2_img.mat')
>> img
img =
struct with fields:
Cy5: [201×201×18 double]
DsRed: [201×201×18 double]
EGFP: [201×201×18 double]
DAPI: [201×201×18 double]segmented images ~/Mouse2_segmentation.mat
>> load('~/Mouse2_segmentation.mat')
>> Segmentations
Segmentations =
struct with fields:
DAPI: [201×201×18 double]
Cy5: [201×201×18 logical]
DsRed: [201×201×18 logical]
EGFP: [201×201×18 logical]
imshow(max(Segmentations.DAPI,[],3)) %displays the following image
total segmented dots in each channel ~/Mouse2_totaldots.mat
>> load('~/Mouse2_totaldots.mat')
>> excel_totaldots
>> excel_totaldots
excel_totaldots =
struct with fields:
DAPI: [14×9 table]
Cy5: [426×8 table]
DsRed: [781×8 table]
EGFP: [284×8 table]
>> head(excel_totaldots.DAPI) %display 1st few rows of this object
ans =
8×9 table
Volume Centroid BoundingBox VoxelIdxList VoxelList VoxelValues MeanIntensity MinIntensity MaxIntensity
______ __________________________ ____________ ________________ ________________ ________________ _____________ ____________ ____________
8565 62.444 143.6 9.809 [1x6 double] [ 8565×1 double] [ 8565×3 double] [ 8565×1 double] 10628 466 44982
11057 162.73 156.14 8.6432 [1x6 double] [11057×1 double] [11057×3 double] [11057×1 double] 9519.9 215 45876
2408 5.2022 154.21 8.961 [1x6 double] [ 2408×1 double] [ 2408×3 double] [ 2408×1 double] 7397.1 573 27740
12556 108.64 139.28 9.579 [1x6 double] [12556×1 double] [12556×3 double] [12556×1 double] 8743.5 734 50481
11766 46.25 106.34 10.424 [1x6 double] [11766×1 double] [11766×3 double] [11766×1 double] 18578 993 65535
2829 51.367 196.84 10.746 [1x6 double] [ 2829×1 double] [ 2829×3 double] [ 2829×1 double] 9791.3 718 34668
10231 90.073 70.494 9.7296 [1x6 double] [10231×1 double] [10231×3 double] [10231×1 double] 11861 929 57322
11066 42.672 26.197 11.052 [1x6 double] [11066×1 double] [11066×3 double] [11066×1 double] 12175 165 64592
Dots in nuclei per channel ~/Mouse2_dots_of_ROI.mat
>> load('~/Mouse2_dots_of_ROI.mat')
>> excel_dots_of_ROI
excel_dots_of_ROI =
struct with fields:
Cy5: [14×6 table]
DsRed: [14×6 table]
EGFP: [14×6 table]
>> head(excel_dots_of_ROI.Cy5) %display 1st few rows of this object
ans =
8×6 table
ROI dotname count Volume Location Intensity
______ _____________ _____ _____________ ___________ ___________
'ROI1' [ 6×1 double] 6 [ 6×1 double] { 6×1 cell} { 6×1 cell}
'ROI2' [35×1 double] 35 [35×1 double] {35×1 cell} {35×1 cell}
'ROI3' [ 7×1 double] 7 [ 7×1 double] { 7×1 cell} { 7×1 cell}
'ROI4' [59×1 double] 59 [59×1 double] {59×1 cell} {59×1 cell}
'ROI5' [16×1 double] 16 [16×1 double] {16×1 cell} {16×1 cell}
'ROI6' [ 7×1 double] 7 [ 7×1 double] { 7×1 cell} { 7×1 cell}
'ROI7' [37×1 double] 37 [37×1 double] {37×1 cell} {37×1 cell}
'ROI8' [ 4×1 double] 4 [ 4×1 double] { 4×1 cell} { 4×1 cell}
The above matlab tables for all the images are consolidated into excel files (long_data - ROI level, man - Image level) saved in the path path1, using final_table_mouse matlab function.
path1 = '/dcl01/lieber/ajaffe/Maddy/RNAscope/dotdotdot/dotdotdot_repo/dotdotdot/output';
ext = 'Mouse*.czi';
toolbox = '/dcl01/lieber/ajaffe/Maddy/RNAscope/dotdotdot/dotdotdot_repo/dotdotdot/toolbox';
channels = {'Cy5','DsRed','EGFP','DAPI'};
final_table_mouse(path1,ext,toolbox,channels)
/dcl01/lieber/ajaffe/Maddy/RNAscope/dotdotdot/dotdotdot_repo/dotdotdot/output : 3
file 1 completed
file 2 completed
file 3 completedThese csv files long_data, man are extracted into R objects like below.
The output files have the following columns:
man.csv represents image-level summary statistics for each channel of input data (represented as *)
- var1: image name,
- PO_* - total C1 pixels in the image
- DO_* - total C1 dots in the image
- IO_* - Mean Intensity of C1 dots in the image
- MI_* - Mean Intensity of the C1 channel in the image
longdat.csv represents ROI-level (or nuclei-level) summary statistics for each channel of input data (represented as *)
- names: image name in that image
- Volume: ROI volume
- P_* : total C1 pixels in that ROI
- MI_P_* : Mean intensity of C1 pixels in that ROI
- D_* : total C1 dots in that ROI
- V_* : mean volume/size (in pixels) of C1 dots in that ROI
- MI_D_* = Mean intensity of C1 dots in that ROI
These two CSVs can be read into R (or python, or Matlab, or your favorite statistical sofware tool).
One potential analysis involved assigning ROIs to qualitative/categorial expression groups. Foe example, ROIs with more than 0 transcripts are recruited for each channel and k-means is run on each channel data like below. ROIs with 0 transcripts are assigned directly to "Low" group.
# the following dataframe is made from the above long_data for channel1 (c1 - BdnfEx4) excluding ROIs that have less than 1 transcript
head(c1)
no_of_dots_c1 avg_dot_size_c1 avg_dot_Intensity_c1 treatment Image_no
ROI2_1 10 2.300000 5263.275 ECS 1
ROI4_1 5 6.600000 6167.333 ECS 1
ROI5_1 16 6.750000 4759.467 ECS 1
ROI7_1 3 3.333333 5746.333 ECS 1
ROI9_1 3 5.000000 6338.833 ECS 1
ROI10_1 10 3.600000 5313.039 ECS 1
results_c1 = kmeans(subset(c1, select = c("no_of_dots_c1","avg_dot_size_c1")),3) #this clusters all the ROIs in c1 into 1,2,3 groups based on number of dots per ROI and average dot size per ROIThe results_c1 object has the output from k-means. The cluster component of the results object shows which group each ROI is assigned to and the centers component shows where each of the cluster's centers lie. ROIs assigned to group 1 are labelled as "Low" since the centers lie at the lowest end and ROIs assigned to group 3 are labelled as "High" since the centers lie at the highest end, the rest of the ROIs that are group 2 are labelled as "Medium".
head(results_avgdotsize_c11$cluster)
ROI2_1 ROI4_1 ROI5_1 ROI7_1 ROI9_1 ROI10_1
1 1 2 1 1 2
results_avgdotsize_c11$centers
no_of_dots_c1 avg_dot_size_c1
1 3.49784 3.005068
2 15.40827 6.598141
3 36.03497 10.029988
The k-means output when plotted
Phenotype data can be merged with the ROI-level dotdotdot output (longdat.csv) and differential expression analyses can be performed with linear mixed effects modeling, treating the image file as a random intercept.