ij-macro-programming-workshop

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Exercise 15.1 - Measure objects on all images in a folder

Aim:

• learn how to apply a threshold and use the particle analyzer in a batch macro.

Complete the macro below and run it on the images in the folder 15.

Useful commands:

• run("Set Measurements...", options) sets the features and options that will be measured
• setAutoThreshold(..) sets the threshold using an auto-threshold method, use the macro recorder to find the options
• run("Analyze Particles...", options); runs the particle analyzer with the given options
  • use the macro-recorder to find the options
  • the outlines of the nuclei should be written to an overlay of the image and the objects should be measured
• saveAs("Results", file-path); saves a result table to a tab-separated text file

Your code starts after this line

run("Set Measurements...", "area mean standard modal min centroid center perimeter bounding fit shape feret's integrated median skewness kurtosis area_fraction display add redirect=None decimal=3");
path = getDirectory("Input directory");			// Ask the user for the input directory
print("\\Clear");
print("batch measure nuclei started");
setBatchMode(true);
files = getFileList(path);						// get an array containing the names of all files in the directory path
File.mkdir(path + "out");						// create the output directory
for(i=0; i<files.length; i++) {					// iterate over all files
	image = files[i];							// get the name of the current file
	print("\\Update1: processing file " + (i+1) +"/" + files.length);
	if (indexOf(image, ".tif") != -1) {			// if the current file is a an image...
		open(path + image);
		setAutoThreshold("Default dark");
		run("Analyze Particles...", "size=400-Infinity show=[Overlay Outlines] display exclude in_situ");
		save(path + "out/" + image);
		close();
	}
}
saveAs("Results", path + "out/" + "Results.xls");

> batch measure nuclei started
>  processing file 3/3

Label	Area	Mean	StdDev	Mode	Min	Max	X	Y	XM	YM	Perim.	BX	BY	Width	Height	Major	Minor	Angle	Circ.	Feret	IntDen	Median	Skew	Kurt	%Area	RawIntDen	FeretX	FeretY	FeretAngle	MinFeret	AR	Round	Solidity
A4 dapi 1.tif	6101	126.281	25.558	116	72	222	198.157	86.649	199.334	86.776	353.061	130	50	130	79	140.209	55.403	157.466	0.615	137.786	770438.000	125	0.293	-0.056	100	770438.000	130	60	158.276	56.149	2.531	0.395	0.957
A4 dapi 1.tif	7047	149.474	31.401	157	72	239	428.766	113.529	428.097	113.457	329.120	371	72	111	85	115.781	77.495	158.420	0.818	115.953	1053342.000	151	-0.192	-0.373	100	1053342.000	373	92	156.088	78.673	1.494	0.669	0.973
A4 dapi 1.tif	5455	126.024	26.887	135	72	235	199.684	160.958	200.437	161.086	292.392	149	124	103	71	103.920	66.835	163.869	0.802	104.393	687460.000	126	0.316	0.017	100	687460.000	149	153	170.628	68.839	1.555	0.643	0.964
A4 dapi 1.tif	7524	145.870	32.919	150	72	246	353.094	213.299	353.440	212.639	328.191	305	161	93	104	104.252	91.891	108.347	0.878	105.537	1097527.000	146	0.097	-0.503	100	1097527.000	345	161	102.588	93.000	1.135	0.881	0.980
A4 dapi 1.tif	5653	135.360	23.931	145	72	198	64.660	237.526	65.251	237.117	300.978	11	203	111	69	111.809	64.374	168.139	0.784	112.792	765189.000	138	-0.410	-0.139	100	765189.000	12	222	165.101	64.828	1.737	0.576	0.971
A4 dapi 1.tif	6178	132.127	25.064	132	72	211	552.543	305.316	551.895	304.745	304.392	513	253	78	105	105.948	74.245	102.152	0.838	107.280	816280.000	133	-0.047	-0.373	100	816280.000	540	253	101.834	74.458	1.427	0.701	0.974
A4 dapi 1.tif	4583	137.211	31.462	130	72	224	267.902	300.119	267.431	299.934	296.392	213	272	107	59	108.172	53.944	174.863	0.656	109.385	628839.000	137	0.024	-0.706	100	628839.000	214	283	165.710	58.621	2.005	0.499	0.903
A4 dapi 1.tif	7312	167.040	36.387	167	72	255	360.969	365.884	360.809	365.959	333.120	301	325	115	82	115.715	80.455	175.946	0.828	115.624	1221399.000	169	-0.336	-0.164	100	1221399.000	301	362	174.043	80.706	1.438	0.695	0.978
A4 dapi 1.tif	6820	123.350	25.957	126	72	210	497.701	459.070	499.656	459.478	343.345	451	403	98	109	119.930	72.404	128.003	0.727	121.083	841245.000	123	0.056	-0.814	100	841245.000	462	409	131.987	72.529	1.656	0.604	0.957
nuclei.tif	4656	60.545	12.363	54	41	108	563.192	106.911	562.066	106.638	298.434	513	78	100	59	100.902	58.752	179.535	0.657	100.499	281898.000	59	0.901	0.918	100	281898.000	513	108	5.711	59.000	1.717	0.582	0.955
nuclei.tif	4970	50.532	7.206	41	41	81	1203.534	134.471	1203.957	134.112	440.784	1152	96	107	76	109.552	57.763	156.440	0.321	115.836	251142.000	49	0.969	0.934	100	251142.000	1153	109	152.771	64.374	1.897	0.527	0.861
nuclei.tif	7532	74.958	14.249	80	41	125	93.937	139.042	93.866	138.133	342.090	34	98	119	81	118.247	81.101	3.262	0.809	119.340	564586.000	75	-0.044	-0.006	100	564586.000	34	136	4.325	81.000	1.458	0.686	0.976
nuclei.tif	4775	61.405	11.439	62	41	99	783.236	152.087	783.864	152.005	285.563	733	121	102	63	100.565	60.456	6.408	0.736	102.313	293207.000	61	0.453	-0.278	100	293207.000	733	155	4.485	60.957	1.663	0.601	0.962
nuclei.tif	4833	51.526	8.226	43	41	92	337.884	180.997	338.395	181.737	366.316	300	131	74	100	91.094	67.552	92.418	0.453	100.404	249025.000	50	0.892	0.632	100	249025.000	336	131	95.143	74.000	1.349	0.742	0.845
nuclei.tif	6251	100.673	26.719	100	41	197	608.970	222.146	608.832	223.448	309.605	560	177	98	89	110.489	72.034	143.792	0.819	111.212	629307.000	101	0.248	0.567	100	629307.000	566	188	142.306	71.800	1.534	0.652	0.977
nuclei.tif	5011	53.429	7.981	51	41	84	1025.585	209.996	1025.890	209.275	411.730	968	179	118	62	113.066	56.429	4.474	0.371	118.106	267735.000	53	0.506	-0.253	100	267735.000	968	213	2.426	62.000	2.004	0.499	0.883
nuclei.tif	3849	72.979	20.022	72	41	157	151.210	266.861	151.426	268.349	235.522	116	231	70	71	72.461	67.632	53.765	0.872	74.404	280898.000	70	0.863	0.702	100	280898.000	119	286	36.254	68.333	1.071	0.933	0.970
nuclei.tif	6454	87.154	19.968	80	41	145	822.875	297.016	823.828	297.424	313.706	768	259	109	79	109.872	74.792	168.158	0.824	110.476	562492.000	87	0.051	-0.357	100	562492.000	768	289	170.623	75.671	1.469	0.681	0.976
nuclei.tif	3188	178.801	64.793	255	41	255	271.804	296.490	271.796	295.760	218.108	233	269	76	57	73.426	55.282	172.696	0.842	76.655	570016.000	201	-0.727	-0.727	100	570016.000	233	290	172.504	57.000	1.328	0.753	0.970
nuclei.tif	6234	97.971	24.771	108	41	177	447.669	369.441	448.157	371.505	308.978	412	314	72	113	112.254	70.709	93.223	0.821	113.635	610752.000	100	-0.113	-0.287	100	610752.000	439	314	96.062	71.675	1.588	0.630	0.977
nuclei.tif	4345	69.556	16.028	76	41	123	632.013	349.023	633.947	349.268	268.534	588	316	91	64	91.816	60.253	163.991	0.757	94.921	302221.000	69	0.389	-0.380	100	302221.000	590	331	159.656	63.791	1.524	0.656	0.964
nuclei.tif	6693	90.934	18.041	91	41	161	1115.890	362.511	1115.702	362.864	314.291	1063	318	104	88	109.863	77.567	150.404	0.851	111.893	608620.000	91	0.004	0.799	100	608620.000	1064	343	155.726	78.733	1.416	0.706	0.977
nuclei.tif	3859	66.796	13.888	68	41	108	71.580	364.611	73.106	365.096	283.948	30	329	78	70	76.945	63.856	142.933	0.601	81.025	257765.000	67	0.058	-0.561	100	257765.000	37	343	144.545	67.566	1.205	0.830	0.923
nuclei.tif	4952	61.094	10.969	62	41	112	825.917	442.540	826.619	441.891	288.877	791	394	68	94	92.494	68.167	82.875	0.746	94.895	302539.000	60	0.674	0.716	100	302539.000	817	488	82.126	68.000	1.357	0.737	0.963
nuclei.tif	3580	82.802	23.573	64	41	143	1080.880	425.351	1080.841	426.152	252.936	1037	399	89	54	91.567	49.780	168.472	0.703	90.521	296430.000	81	0.247	-0.803	100	296430.000	1037	411	163.968	49.135	1.839	0.544	0.960
nuclei.tif	4221	70.782	18.245	51	41	132	608.141	453.411	609.522	453.896	278.877	561	423	92	61	91.914	58.471	179.562	0.682	92.779	298770.000	70	0.267	-0.804	100	298770.000	561	450	172.569	59.566	1.572	0.636	0.956
nuclei.tif	4503	64.725	14.720	60	41	116	224.243	484.957	223.192	484.824	272.877	186	444	77	82	88.652	64.673	128.819	0.760	90.802	291456.000	63	0.556	-0.292	100	291456.000	200	448	126.491	65.732	1.371	0.730	0.963
nuclei.tif	3988	72.207	22.197	51	41	138	956.706	510.815	957.509	507.859	261.907	924	468	66	90	90.192	56.299	72.631	0.731	92.655	287961.000	66	0.702	-0.548	100	287961.000	951	558	71.761	59.746	1.602	0.624	0.937
nuclei.tif	3160	92.747	28.132	108	41	171	521.876	517.710	524.533	518.359	251.664	484	487	72	64	78.271	51.404	144.076	0.627	82.662	293080.000	95	-0.008	-0.880	100	293080.000	487	491	145.349	54.113	1.523	0.657	0.919
nuclei.tif	3608	82.563	25.420	57	41	167	654.826	546.803	656.221	547.158	237.664	615	515	80	62	79.561	57.740	163.285	0.803	82.710	297887.000	80	0.427	-0.597	100	297887.000	615	542	165.292	59.032	1.378	0.726	0.961
nuclei.tif	2899	96.265	25.517	112	41	163	806.357	564.048	807.052	563.937	205.279	778	529	57	69	68.987	53.505	111.055	0.865	70.576	279072.000	99	-0.289	-0.568	100	279072.000	795	530	110.746	54.951	1.289	0.776	0.967
nuclei.tif	3231	82.468	23.015	92	41	138	1112.278	564.207	1111.254	564.081	231.907	1075	529	73	67	81.664	50.375	38.165	0.755	83.217	266455.000	82	0.176	-0.738	100	266455.000	1076	586	32.735	50.758	1.621	0.617	0.954
nuclei.tif	3902	74.753	17.624	74	41	145	575.921	621.152	575.602	620.027	264.049	535	582	82	80	96.687	51.384	139.653	0.703	99.358	291686.000	73	0.387	-0.167	100	291686.000	547	584	130.101	54.922	1.882	0.531	0.944
nuclei.tif	4132	69.429	14.281	72	41	121	473.660	639.632	474.657	639.937	248.936	431	608	81	65	81.868	64.262	162.837	0.838	83.726	286881.000	69	0.317	-0.143	100	286881.000	436	622	150.680	64.994	1.274	0.785	0.971
nuclei.tif	4622	56.698	11.250	51	41	97	703.887	654.118	703.850	654.137	355.931	650	617	106	70	105.047	56.022	163.066	0.458	110.164	262058.000	54	0.896	0.398	100	262058.000	650	641	164.197	59.479	1.875	0.533	0.879
nuclei.tif	6132	87.950	20.205	89	41	151	206.769	671.964	207.496	672.254	314.090	155	630	103	85	111.691	69.903	146.088	0.781	112.379	539311.000	89	-0.119	-0.118	100	539311.000	158	644	150.698	71.684	1.598	0.626	0.968
nuclei.tif	4144	61.584	10.329	60	41	93	868.132	768.739	868.806	768.903	267.463	825	731	90	71	96.260	54.813	151.659	0.728	95.000	255205.000	61	0.175	-0.435	100	255205.000	835	737	143.130	56.140	1.756	0.569	0.955
nuclei.tif	4287	54.693	8.618	54	41	91	1175.657	774.702	1176.274	775.718	270.534	1132	741	85	69	83.466	65.397	161.462	0.736	86.608	234471.000	54	1.123	1.849	100	234471.000	1139	751	148.696	67.529	1.276	0.784	0.938
nuclei.tif	4485	55.628	10.563	48	41	106	473.298	788.017	473.738	788.238	318.860	435	743	75	93	101.381	56.327	122.505	0.554	103.465	249492.000	54	0.993	0.763	100	249492.000	443	747	122.768	58.059	1.800	0.556	0.934
nuclei.tif	4005	70.459	14.819	70	41	114	648.668	795.914	649.423	797.291	260.735	610	756	77	77	89.135	57.209	139.386	0.740	92.849	282188.000	70	0.109	-0.673	100	282188.000	617	759	131.070	58.762	1.558	0.642	0.948
nuclei.tif	5464	73.885	14.853	65	41	127	79.575	806.024	80.857	805.305	279.078	39	761	79	91	89.464	77.763	91.851	0.882	91.351	403708.000	73	0.393	0.205	100	403708.000	77	852	84.976	78.433	1.150	0.869	0.974
nuclei.tif	3484	144.319	49.608	180	41	240	317.780	811.305	318.089	811.476	227.622	286	772	62	74	72.268	61.382	80.431	0.845	74.793	502807.000	152	-0.421	-0.795	100	502807.000	293	839	60.350	62.000	1.177	0.849	0.966
nuclei.tif	5205	60.035	10.537	64	41	98	209.712	840.461	208.941	840.475	368.316	174	791	70	98	98.199	67.488	103.616	0.482	99.464	312480.000	60	0.291	-0.314	100	312480.000	201	792	102.779	69.541	1.455	0.687	0.946
nuclei.tif	5110	66.321	15.954	54	41	124	414.876	880.721	415.240	880.828	310.676	379	831	73	97	96.277	67.579	107.220	0.665	99.690	338902.000	63	0.498	-0.594	100	338902.000	403	831	103.339	67.797	1.425	0.702	0.952
nuclei.tif	4174	55.214	7.921	56	41	79	870.033	900.969	870.499	901.293	271.848	832	860	73	85	90.173	58.937	124.413	0.710	90.918	230463.000	55	0.320	-0.521	100	230463.000	849	863	119.667	60.201	1.530	0.654	0.952
nuclei.tif	5001	80.999	19.041	86	41	137	1094.049	925.169	1095.130	926.610	281.848	1053	882	84	86	97.168	65.531	135.264	0.791	97.801	405078.000	81	0.185	-0.148	100	405078.000	1065	885	127.104	65.054	1.483	0.674	0.972
nuclei.tif	3865	57.345	9.737	57	41	91	159.798	955.438	160.242	955.378	252.877	122	919	75	71	75.349	65.310	148.961	0.760	78.772	221640.000	56	0.651	0.102	100	221640.000	123	939	159.955	67.882	1.154	0.867	0.947
nuclei.tif	3863	54.925	10.578	48	41	102	942.878	972.056	942.554	970.288	335.446	907	923	73	103	113.635	43.284	62.089	0.431	113.877	212176.000	53	1.111	1.177	100	212176.000	917	1024	59.381	46.448	2.625	0.381	0.863
nuclei.tif	3957	83.242	25.977	62	41	161	516.635	977.698	517.225	978.815	247.421	481	940	71	74	76.776	65.623	47.568	0.812	79.025	329389.000	79	0.339	-0.833	100	329389.000	488	1005	38.320	66.923	1.170	0.855	0.961
two-nuclei.tif	5880	128.126	24.153	116	83	222	323.421	92.891	324.519	92.926	356.233	256	57	128	78	138.939	53.884	157.267	0.582	136.356	753378.000	127	0.450	-0.001	100	753378.000	256	67	159.837	54.883	2.578	0.388	0.948
two-nuclei.tif	7378	147.230	31.775	150	83	246	327.123	257.133	327.459	256.544	333.990	279	205	93	104	103.090	91.124	109.339	0.831	104.938	1086261.000	147	0.183	-0.548	100	1086261.000	319	205	97.667	92.856	1.131	0.884	0.973

Your code ends before this line

run("Close");
print("batch measure nuclei finished");

> batch measure nuclei finished

The code below is for automatically checking the result. Please ignore it!

ok = (nImages==0);
ok = (ok && File.exists(path+"/out"));
files = getFileList(path+"/out");
ok = (ok && files.length==4);
if (ok)
	showMessage("That's right. Great, you did it!");
else 
	showMessage("Your result is wrong! Please check your macro and try again!");

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