TITLE: Geographic Information Systems (GIS)-based spatial analysis of cell distribution
AUTHORS: Adalberto Merighi, Laura Lossi

[DESCRIPTION]
This protocol describes how to perform a Geographic Information Systems (GIS)-based spatial analysis of cerebellar images. It can be used for any biological images to study cellular or molecular spatial distributions, or, more generally, the distribution of any biological feature of interest.
The procedures described here can be employed singularly or in combination to analyze clustering/dispersion by GIS spatially. It is based on the use of ESRI ArcMap to calculate the Average Nearest Neighbor, the High/Low Clustering (G tool), the Multi-distance Spatial Cluster Analysis (Ripley’s K Function), and the Spatial Autocorrelation (Global Moran’s I). It is also shown how to represent the features' distribution graphically.

[BEFORE_START]
Familiarize yourself with the basics of spatial statistics.

[GUIDELINES]
It may be useful to read articles in which ArcGIS has been applied to the study of biological samples, e.g. https://www.frontiersin.org/articles/10.3389/fevo.2021.642255/full

[STEPS]
SECTION: Calculation of cells&#x27; X-Y coordinates
33. 
SECTION: Calculation of cells&#x27; X-Y coordinates
1. Open the image to be analyzed using Fiji: File → Open     
 
                                                                                                                                                                                                                                                                                                                                              
SECTION: Calculation of cells&#x27; X-Y coordinates
2. Set the appropriate scale for the image using Analyze → Set Scale. In the pop-up window report the distance in pixels related to the known distance using the correct unit of length (μM). Leave the pixel aspect ratio at 1.0.
 

SECTION: Calculation of cells&#x27; X-Y coordinates
3. Use the Multipoint tool and click with the mouse on the center of each labeled cell. The tool should be configured so that clicked cells are visualized directly on the image. To do so double-click with the mouse on the tool icon and tick the Label points box.


SECTION: Calculation of cells&#x27; X-Y coordinates
4. Set the measurements to be computed using Analyze → Set Measurements. In the pop-up window verify that all boxes are not ticked. Set the Decimal places box to 3. Use Analyze → Measure to calculate the X-Y coordinates. A new window pops up where the results are shown in tabular form.
SECTION: Calculation of cells&#x27; X-Y coordinates
5. Save data as a .csv file.
SECTION: Image elaboration with ArcMap
6. Load all .csv files to an ad hoc folder in ArcMap.
SECTION: Image elaboration with ArcMap
7. Open the program and create a new map document: File → New → New Maps → My Templates → Blank Map.
SECTION: Image elaboration with ArcMap
8. On the ribbon click View → Data Frame Properties. In the pop-up window click Coordinate System. Click on the world icon and select New → Projected Coordinate System. For name type the name of the new coordinate system e.g. Microscope Coordinate System. For Linear Unit Name choose Millimeter. Leave all other parameters unchanged. Save the new coordinate system: the program creates a new folder named Custom that contains the file Microscope_Coordinate_System.
 

SECTION: Image elaboration with ArcMap
9. • Save the Blank Map file and name it with the name of the image under investigation, e.g. Image1. The program saves a file named Image1.mdx
SECTION: Image elaboration with ArcMap
10. Add the X-Y coordinates of the cells to the map. On the toolbar click the Add data icon then → Add Data. Choose the .csv file with the X-Y coordinates of the cells and upload it. The program creates a new layer on the map with the same name as the .csv file and an attribute table containing the cell coordinates. Right-click with the mouse on the new layer and choose Display XY Data. In the pop-up window, be sure that the X and Y fields for the layer correspond to the fields of X and Y coordinates in your .csv file, and press the OK button. A window appears with the warning Table Does Not Have Object-ID Field. This is because the layer created so far is an X-Y event layer that must be converted into a feature layer for further analysis. Press the OK button and the positions of the cells will be displayed as in the figure below. 
 


  
SECTION: Image elaboration with ArcMap
11. Convert the X-Y event layer into a feature layer. On the layer right click → Data → Export Data → All features. Select Use the same coordinate system as this layer source data and press OK. The program generates a new layer named Export_Output_#. By double-clicking with the mouse on the layer name, the Layer Properties window opens and it is possible to customize the data by e.g. changing the layer name and using a different symbology to display the cells.  


SECTION: Analysis of cell distribution with spatial statistics
12. Average Nearest Neighbor

The Average Nearest Neighbor tool calculates the nearest neighbor index based on the average distance from each feature (i.e. a labeled Purkinje neuron) to its nearest neighboring feature. The Average Nearest Neighbor tool returns five values: Observed Mean Distance, Expected Mean Distance, Nearest Neighbor Index, z-score, and p-value. The values are written as messages at the bottom of the Geoprocessing pane during tool execution and passed as derived output values. With the Generate Report box ticked the tool produces an HTML report file with a graphical summary of the results (see figure below).
 
                                                                                                                                                                                                                                                                                                                                                                                                                                                   
SECTION: Analysis of cell distribution with spatial statistics
15.                Graphical visualization of cell counts 

By this approach, it is possible to display graphically on the map a series of polygons (tessellation) over the labeled cells and to join the cell counts to each polygon so that a graphic display of the cell distribution is obtained.                                         
SECTION: Analysis of cell distribution with spatial statistics
12.1. On the toolbar select the Arc Toolbox icon then→ Spatial Statistics Tools → Analyzing Patterns → Average Nearest Neighbor
SECTION: Analysis of cell distribution with spatial statistics
12.2. In the pop-up windows for Input Feature Class select Export Output #, for Distance Method, select EUCLIDEAN DISTANCE, and tick the box Generate Report.
  

SECTION: Analysis of cell distribution with spatial statistics
14. Multi-distance Spatial Cluster Analysis (Ripley’s K Function)

The Multi-Distance Spatial Cluster Analysis (Ripley's K Function) determines whether features or the values associated with features exhibit statistically significant clustering or dispersion over a range of distances. The tool output is a table with two fields: ExpectedK and ObservedK containing the expected and observed K values, respectively. Because the L(d) transformation is applied, the ExpectedK values will always match the Distance value. A field named DiffK contains the Observed K values minus the Expected K values. As a confidence interval option is specified, two additional fields named LwConfEnv and HiConfEnv will be included in the Output Table. The output graph is shown in the figure below.                                                                       
SECTION: Analysis of cell distribution with spatial statistics
13. High/Low Clustering (G tool)       

The High/Low Clustering tool measures the degree of clustering for either high or low values using the Getis-Ord General G statistic. The High/Low Clustering tool returns four values: Observed General G, Expected General G, z-score, and p-value. The values are written as messages at the bottom of the Geoprocessing pane during tool execution and passed as derived output values. With the Generate Report box ticked the tool generates an HTML report file with a graphical summary of results (see figure below).          
 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
SECTION: Analysis of cell distribution with spatial statistics
13.1. On the toolbar select the Arc Toolbox icon then→ Spatial Statistics Tools → Analyzing Patterns → High/Low Clustering (Getis-Ord General G)
SECTION: Analysis of cell distribution with spatial statistics
13.2. In the pop-up windows for Input Feature Class select Export Output #, for Input Field select XM, for Conceptualization of Spatial Relationship select INVERSE DISTANCE, for Distance Method, select EUCLIDEAN DISTANCE, for standardization select NONE, and tick the box Generate Report.

  

SECTION: Analysis of cell distribution with spatial statistics
14.1. On the toolbar select the Arc Toolbox icon then→ Spatial Statistics Tools → Analyzing Patterns → Multi-distance spatial cluster analysis (Ripley’s K function)
SECTION: Analysis of cell distribution with spatial statistics
14.2. In the pop-up windows for Input Feature Class select Export Output # for Output Table leave the program generated name (Export Output # MultiDistan), for Compute Confidence Envelope (optional) choose 99_PERMUTATIONS, tick the box Display Results Graphically
 
  
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
SECTION: Analysis of cell distribution with spatial statistics
15.1. Generate tessellation. With the mouse select the Export_Output # layer. On the toolbar select the Arc Toolbox icon then → DataManagementTools → Sampling → GenerateTessellation. This tool generates a polygon feature class of a tessellated grid of regular polygons which will entirely cover a given extent. In the pop-up window leave unchanged the path of the Output Feature Class. For Extent click on the folder icon and choose Same as layer Export Output #. Selection of the shape type is optional. Check that HEXAGON is selected by the program. The program creates a new layer named Generate Tessellation # and the tessellation appears above the cells (see figure below).
 


  

SECTION: Analysis of cell distribution with spatial statistics
15.2. Select hexagons with cells. From the ribbon click Selection → Selectbylocation. In the pop-up window, for Target layer(s) select Generate tessellation #, for the Source layer select Export_Output #, and for the Spatial selection method for target layer feature(s) choose Intersect the source layer feature. Click OK. The hexagons containing cells are highlighted (see the left figure below).
SECTION: Analysis of cell distribution with spatial statistics
15.3. Join cell positions to selected hexagons. Select the Arc Toolbox icon on the toolbar→ AnalysisTools → Overlay → SpatialJoin. In the pop-up window, for Target features select Generate Tessellation #, for Join Features select Export_Output #. The program creates a new layer named Export_Output # SpatialJoin# with the hexagons containing cells visualized in a different color than those with no cells (see the right figure below).

  

SECTION: Analysis of cell distribution with spatial statistics
15.4. Graphical visualization of the cell counts on the map. Remove the layer Generate Tessellation # from the map (right-click with the mouse) and turn off the visibility of the layer Export Output #. Only the hexagons with cells remain visible (see the left figure below). With the mouse right-click on layer → Properties → Symbology → Show quantities → Select color ramp (e.g. cyan-to-purple) → Fields: Value = Join-Count; Normalization = none. Hexagons on the map are displayed in different colors according to the number of cells that they contain (see the right figure below).

  

SECTION: Analysis of cell distribution with spatial statistics
16. Spatial Autocorrelation (Global Moran’s I)

The Spatial Autocorrelation (Global Moran’s I) tool measures spatial autocorrelation based on feature locations and attribute values using the Global Moran's I statistic. 
The tool returns five values: Moran's I Index, Expected Index, Variance, z-score, and p-value. The values are written as messages at the bottom of the Geoprocessing pane during tool execution and passed as derived output values. With the Generate Report box ticked the tool produces an HTML report file with a graphical summary of results (see figure below). 
SECTION: Analysis of cell distribution with spatial statistics
16.1. On the toolbar select the Arc Toolbox icon then → Spatial Statistics Tools → Analyzing Patterns → Spatial Autocorrelation (Global Moran’s I).
SECTION: Analysis of cell distribution with spatial statistics
16.2. In the pop-up windows for Input Feature Class select GenerateTessellation#Spati#, for Input Field select Join-Count, for Conceptualization of Spatial Relationship select INVERSE_DISTANCE, for Distance Method select EUCLIDEAN DISTANCE, for STANDARDIZATION select ROW. Tick Generate Report and click OK.
SECTION: Analysis of cell distribution with spatial statistics
16.3. After the tool has run, no layer is added to the map but a report is generated. To view the report in the ribbon, click Geoprocessing → Results.
SECTION: Analysis of cell distribution with spatial statistics
16.4. In the results list, expand the Spatial Autocorrelation (Moran's I) folder and click on the Report File to view the report in the browser window (see figure below).  The report file is automatically saved as a .html file in the ArcGIS folder of the computer.