McClean: Plotting Stacked Histograms

Summary

This explains the basics of plotting histograms stacked vertically (this allows you to see the shift in, for instance, fluorescence in a population of cells analyzed by flow cytometry).

Example

Your data could be anything. In this example, the variable "Data" (stored in MakeStackHistData.mat) contains five (5) rows, each of which contain 9000 fluorescence readings from a flow cytometry experiment. Each row represents a time point, with induction of GFP increasing with time.

%% Preliminaries:

close all; clear all;
load('MakeStackHistData.mat')

Chose bins (you probably want to use the same bin for every plot, since you will be stacking them along one y-axis) and then bin your data using the Matlab "hist" command. We also keep track of the distributions' means since we use this to color the histograms later.

%Set up bins (we are making histograms of flow cytometry data so we chose logarithmically spaced bins):
bins=logspace(0,4,60);
x=bins;

%Bin the data using "hist" and keep track of the number of elements "n" in each bin "x" for each row in "Data".  Also keep track of the mean of each row of "Data":

HistData=[];  
Means=[];

for i=1:5
    [n,x]=hist(Data(i,:),x);
    HistData=[HistData; n./sum(n)];
    Means=[Means mean(Data(i,:))];
end

We set up a colormap so that our histograms change in color as the mean of their distribution increases:

%% Define a colormap for the histograms that will make the histograms brighter as the mean of the distribution increases

% In this case we chose to make the histograms brighter green at higher
% mean values since the flow cytometry data is of GFP.
 
%Define a color map
MMColorMap=zeros(5,3);

%Define colors so that they scale with the difference between the mean
%fluorescence at a given timepoint and the mean at time 0


MM=sort(Means);
MMdiff=Means-Means(1);
MMdiff=MMdiff./(max(MMdiff));


MMColorMap(1:end,2)=MMdiff;  %The colormap is RGB, so changing the second column changes the green values.

%Set up the figure and axis properties:
h=figure; hold; colors=colormap;

set(gca,'XScale','log')
set(gca,'XLim',[10,2000])
set(gca,'PlotBoxAspectRatioMode','manual')
set(gca,'PlotBoxAspectRatio',[1 3 1])
set(gca,'FontSize',12)
set(gca,'XTick',[100 1000 10000 100000])
set(gca,'YTick',[0 1])
ylabel('Fraction of Cell Population','FontSize',14)
xlabel('Fluorescence [a.u.]','FontSize',14)

Plot the histograms along the y-axis. We choose the spacing variable empirically so that the plot "looks good":

spacing=.15;  %Spacing along the y-axis chosen empirically 

for i=1:5
    fill([x(1);x'; x'],[i*spacing; (HistData(i,:)+i*spacing)'; ones(1,length(x))'*i*spacing],MMColorMap(i,:),'LineStyle','none')
    semilogx(x,HistData(i,:)+i*spacing,'LineWidth',3,'Color','k');
end

Save your figure in a variety of formats for later use (recall that we made h our figure handle):

saveas(h,'ExampleStackedHistograms','fig')
saveas(h,'ExampleStackedHistograms','png')
saveas(h,'ExampleStackedHistograms','ai')
saveas(h,'ExampleStackedHistograms','pdf')
 

Code

You can copy and paste the code below into a Matlab m-file to run all of the examples shown above. You will also the "Data.mat" example data:

%% Preliminaries:

close all; clear all;
load('Data.mat')

%% Define the bins to use for our data (you will need to adjust this depending on your data):

%In this case we are using the same bins for each data set.  You probably
%want to do this when you are plotting stacked histograms.

bins=logspace(0,4,60);
x=bins;

%% Bin your data using Matlabs "hist" function.  

%The variable "n" will be the number in each bin described by the variable
%"x".  HistData will become a matrix of the normalized bins (normalized to
%the total number of elements).  Means will become a vector of the mean
%value for each distribution, which we will use when coloring our
%histograms (so that colors roughly correspond to the mean of the
%distribution).

HistData=[];  
Means=[];

for i=1:5
    [n,x]=hist(Data(i,:),x);
    HistData=[HistData; n./sum(n)];
    Means=[Means mean(Data(i,:))];
end



%% Define a colormap for the histograms that will make the histograms brighter as the mean of the distribution increases

% In this case we chose to make the histograms brighter green at higher
% mean values since the flow cytometry data is of GFP.
 
%Define a color map
MMColorMap=zeros(5,3);

%Define colors so that they scale with the difference between the mean
%fluorescence at a given timepoint and the mean at time 0


MM=sort(Means);
MMdiff=Means-Means(1);
MMdiff=MMdiff./(max(MMdiff));


MMColorMap(1:end,2)=MMdiff;

%Set up the figure and axis properties:
h=figure; hold; colors=colormap;

set(gca,'XScale','log')
set(gca,'XLim',[10,2000])
set(gca,'PlotBoxAspectRatioMode','manual')
set(gca,'PlotBoxAspectRatio',[1 3 1])
set(gca,'FontSize',12)
set(gca,'XTick',[100 1000 10000 100000])
set(gca,'YTick',[0 1])
ylabel('Fraction of Cell Population','FontSize',14)
xlabel('Fluorescence [a.u.]','FontSize',14)

%% Plot the histograms along the y-axis

spacing=.15;  %Spacing along the y-axis chosen empirically 

for i=1:5
    fill([x(1);x'; x'],[i*spacing; (HistData(i,:)+i*spacing)'; ones(1,length(x))'*i*spacing],MMColorMap(i,:),'LineStyle','none')
    semilogx(x,HistData(i,:)+i*spacing,'LineWidth',3,'Color','k');
end


%% Save the histogram figure
saveas(h,'ExampleStackedHistograms','fig')
saveas(h,'ExampleStackedHistograms','png')
saveas(h,'ExampleStackedHistograms','ai')
saveas(h,'ExampleStackedHistograms','pdf')

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

• Megan N McClean 17:27, 17 July 2013(EDT): This ought to get you started. There are many improvements that could be made. For instance, a more sophisticated/attractive color scheme or automatic selection of the spacing along the y-axis. Knock yourselves out!

Files

Example with pMM165 (pGAL1-GFP)

Contact

• Megan N McClean 14:01, 11 June 2012 (EDT)

or instead, discuss this protocol.