Confocal Microscopy

Confocal Microscopy enables imaging of thin optical section in optically thick specimens (living biological tissue).

Many students (such as pictured above at Confocal Day, 6-24-09) are becoming interested in confocal as an interesting and powerful tool.

The two matlab codes below calculate the optical power of the confocal optical signal falling on a detector as a function of the power of the laser used and the molar concentration of the fluorescent chemical species in the focal region.

Matlab instructions:

  1. Download the two Matlab files
  2. Check out the comments in the code
  3. Adapt the code for your experimental conditions
  4. Run the program

Source code in Matlab:

Master Code

clear all
close all

%The molar extinction coefficient of Eosin Y dissolved in basic ethanol 
%based on the absorbance measurements made by Junzhong Li on 12/11/97 using
%a Cary 3 [H. Du, R. A. Fuh, J. Li, A. Corkan, J. S. Lindsey, 
%"PhotochemCAD: A computer-aided design and research tool in photochemistry
%," Photochemistry and Photobiology, 68, 141-142, 1998]. The absorbance data
%was normalized to have an extinction coefficient of 112000 at 524.8 nm
%[P. G. Seybold, M. Gouterman, and J. Callis, "Calorimetric, photometric 
%and lifetime determinations of fluorescence yields of fluorescein dyes," 
%Photochem. Photobiol., 9, 229-242, 1969]. 
%To convert this data to absorbance A=-log10(T), multiply by the molar 
%concentration and the pathlength in centimeters. To convert this data to 
%absorption coefficient in (cm-1), multiply by the molar concentration and 2.303. 

EoData % Loads Eosin data data[nm extcoef]

figure(1)
plot(data(:,1),data(:,2),'k-')
xlabel('wavelength [nm]')
ylabel('Molar extinction coefficient [cm^-1/M]')

% Optical sectioning
title('pick wavelength (click, hit enter)')
samp = ginput;
lambda = samp(1)*1e-9;      % [m] wavelength
NA = 0.9;                   % [-]
dZ = 2*0.63*1.34*lambda/NA^2;    % [um]


QY = 0.5;                   % [-] quantum yield of fluorescence
data(:,2) = data(:,2)*100;  % Ext. Coef. [cm-1/M] --> [m-1/M]

n = 100;            % [-] sample points
Pmax = 50e-3;       % [W] laser power
dP = Pmax/n;        % [W] laser power
cMAX = 1;           % [mM]
dc = cMAX/n; % [mM]

T_optics = 0.5; % [-] transmission to the sample
T_tissue = exp(-60*0.0100); % [-] = exp(-[cm-1][cm])
T = T_optics*T_tissue;  % [-] Total Transmission

ix = 0;


for Plas = dP:dP:Pmax   
    ix = ix +1;
    Po(ix) = Plas*T;
    iy = 0;
    for cMM = dc:dc:cMAX
        iy = iy +1;
        c(iy) = cMM / 1000;   % [mM-1] --> [M-1]
        mua(iy) = c(iy)*samp(2);  % [m-1] = [M-1]*[m-1/M] 
        Pabs = Po(ix)*(1-exp(-mua(iy)*dZ)); % [W] absorbed in confocal vol
        Pemit = Pabs*QY; % [W] emitted from confocal vol
        Pdet(ix,iy) = Pemit*T ;% [W] detected
    end
end



figure(1)
surf(Po/T,c*1000,Pdet)
xlabel('laser power [W]')
ylabel('concentration [mM]')
zlabel('detected signal [W]')
% break
Lp=input('laser power (in Watts)?');
Ceo=input('concentration (mM)');
Ceo=Ceo/1000;
P10=Lp*T;
mua=Ceo*samp(2);
P1abs=P10*(1-exp(-mua*dZ));
P1emit=P1abs*QY;
P1det=P1emit*T;
figure(1); 
title(sprintf('The light at detector=%0.3e(W)',P1det),'fontsize',14)

Eosin Data

data = [
    219.75	   32895
 220.00	   31973
 220.25	   32689
 220.50	   31935
 220.75	   31286
 221.00	   30902
 221.25	   31456
 221.50	   31019
 221.75	   31351
 222.00	   29886
 222.25	   29802
 222.50	   30350
 222.75	   30233
 223.00	   29301
 223.25	   29811
 223.50	   28066
 223.75	   29315
 224.00	   28876
 224.25	   28053
 224.50	   28296
 224.75	   28752
 225.00	   27995
 225.25	   28393
 225.50	   28243
 225.75	   28182
 226.00	   27857
 226.25	   27796
 226.50	   26833
 226.75	   27795
 227.00	   27159
 227.25	   27040
 227.50	   27707
 227.75	   26195
 228.00	   27684
 228.25	   27338
 228.50	   27681
 228.75	   26818
 229.00	   25920
 229.25	   26701
 229.50	   26389
 229.75	   26460
 230.00	   27359
 230.25	   27089
 230.50	   26482
 230.75	   26348
 231.00	   26392
 231.25	   26574
 231.50	   26352
 231.75	   26049
 232.00	   26149
 232.25	   26884
 232.50	   26749
 232.75	   27129
 233.00	   26155
 233.25	   26877
 233.50	   26285
 233.75	   26522
 234.00	   26406
 234.25	   26570
 234.50	   27457
 234.75	   26276
 235.00	   27505
 235.25	   26902
 235.50	   27004
 235.75	   27786
 236.00	   26780
 236.25	   27154
 236.50	   27074
 236.75	   26286
 237.00	   27552
 237.25	   27188
 237.50	   27719
 237.75	   27404
 238.00	   26989
 238.25	   27754
 238.50	   27703
 238.75	   28055
 239.00	   27952
 239.25	   27360
 239.50	   27659
 239.75	   28481
 240.00	   27878
 240.25	   28418
 240.50	   28517
 240.75	   27913
 241.00	   28231
 241.25	   27960
 241.50	   28570
 241.75	   28630
 242.00	   29269
 242.25	   29095
 242.50	   28778
 242.75	   28249
 243.00	   28271
 243.25	   28735
 243.50	   28856
 243.75	   28430
 244.00	   28495
 244.25	   28046
 244.50	   28446
 244.75	   29283
 245.00	   28294
 245.25	   28489
 245.50	   27937
 245.75	   27884
 246.00	   27894
 246.25	   27187
 246.50	   27607
 246.75	   27742
 247.00	   27002
 247.25	   27478
 247.50	   27347
 247.75	   27296
 248.00	   27211
 248.25	   27901
 248.50	   27883
 248.75	   27919
 249.00	   27840
 249.25	   27199
 249.50	   28084
 249.75	   27739
 250.00	   27779
 250.25	   28124
 250.50	   27791
 250.75	   28303
 251.00	   28267
 251.25	   27984
 251.50	   27825
 251.75	   27380
 252.00	   27853
 252.25	   27951
 252.50	   28274
 252.75	   27909
 253.00	   27720
 253.25	   28169
 253.50	   27876
 253.75	   28432
 254.00	   28022
 254.25	   28083
 254.50	   27913
 254.75	   27764
 255.00	   27959
 255.25	   28766
 255.50	   28007
 255.75	   28119
 256.00	   27233
 256.25	   27298
 256.50	   27435
 256.75	   28078
 257.00	   27602
 257.25	   28503
 257.50	   28340
 257.75	   28505
 258.00	   28292
 258.25	   28568
 258.50	   27781
 258.75	   28450
 259.00	   28154
 259.25	   28376
 259.50	   28699
 259.75	   28440
 260.00	   28562
 260.25	   27932
 260.50	   28230
 260.75	   28363
 261.00	   26915
 261.25	   27754
 261.50	   27634
 261.75	   27453
 262.00	   26051
 262.25	   26893
 262.50	   26419
 262.75	   25686
 263.00	   24660
 263.25	   24115
 263.50	   23535
 263.75	   22438
 264.00	   22414
 264.25	   21936
 264.50	   20895
 264.75	   20355
 265.00	   19227
 265.25	   18662
 265.50	   17662
 265.75	   17831
 266.00	   16906
 266.25	   16283
 266.50	   15240
 266.75	   15075
 267.00	   14331
 267.25	   13554
 267.50	   12955
 267.75	   12383
 268.00	   11671
 268.25	   11453
 268.50	   10724
 268.75	   10813
 269.00	   10045
 269.25	    9738
 269.50	    9017
 269.75	    9081
 270.00	    8412
 270.25	    8244
 270.50	    7546
 270.75	    7116
 271.00	    7388
 271.25	    7288
 271.50	    6844
 271.75	    7020
 272.00	    5994
 272.25	    5559
 272.50	    6094
 272.75	    6155
 273.00	    5678
 273.25	    5144
 273.50	    5751
 273.75	    5299
 274.00	    5140
 274.25	    4591
 274.50	    4398
 274.75	    4503
 275.00	    4386
 275.25	    4730
 275.50	    3924
 275.75	    4839
 276.00	    4564
 276.25	    4818
 276.50	    4686
 276.75	    4442
 277.00	    4502
 277.25	    3764
 277.50	    3912
 277.75	    4122
 278.00	    3833
 278.25	    4145
 278.50	    3257
 278.75	    3302
 279.00	    3989
 279.25	    3658
 279.50	    3677
 279.75	    3291
 280.00	    3169
 280.25	    3351
 280.50	    3507
 280.75	    3031
 281.00	    3568
 281.25	    3280
 281.50	    2693
 281.75	    3498
 282.00	    3539
 282.25	    2813
 282.50	    3337
 282.75	    3629
 283.00	    3671
 283.25	    3397
 283.50	    3265
 283.75	    3447
 284.00	    3723
 284.25	    3667
 284.50	    3363
 284.75	    3207
 285.00	    3599
 285.25	    3929
 285.50	    3088
 285.75	    3854
 286.00	    3111
 286.25	    4637
 286.50	    3481
 286.75	    3626
 287.00	    3999
 287.25	    4003
 287.50	    4016
 287.75	    4103
 288.00	    3841
 288.25	    4472
 288.50	    3984
 288.75	    3967
 289.00	    4236
 289.25	    4826
 289.50	    3935
 289.75	    4793
 290.00	    4948
 290.25	    5014
 290.50	    4990
 290.75	    4681
 291.00	    4941
 291.25	    5036
 291.50	    5673
 291.75	    5631
 292.00	    5257
 292.25	    5173
 292.50	    5120
 292.75	    5925
 293.00	    5874
 293.25	    5739
 293.50	    6418
 293.75	    6049
 294.00	    6819
 294.25	    6428
 294.50	    6965
 294.75	    6539
 295.00	    7223
 295.25	    7193
 295.50	    7289
 295.75	    7145
 296.00	    7860
 296.25	    7034
 296.50	    7947
 296.75	    7855
 297.00	    7826
 297.25	    8343
 297.50	    8045
 297.75	    8262
 298.00	    8214
 298.25	    8898
 298.50	    8869
 298.75	    9036
 299.00	    9058
 299.25	    9273
 299.50	    9470
 299.75	    9372
 300.00	   10123
 300.25	   10232
 300.50	   10588
 300.75	   10476
 301.00	   10421
 301.25	   10899
 301.50	   10675
 301.75	   11553
 302.00	   12226
 302.25	   12373
 302.50	   11669
 302.75	   11799
 303.00	   11768
 303.25	   12763
 303.50	   12499
 303.75	   12947
 304.00	   12455
 304.25	   13205
 304.50	   13304
 304.75	   13810
 305.00	   13765
 305.25	   14177
 305.50	   13727
 305.75	   14537
 306.00	   14907
 306.25	   14667
 306.50	   13791
 306.75	   14322
 307.00	   14974
 307.25	   15269
 307.50	   14801
 307.75	   14271
 308.00	   14585
 308.25	   14814
 308.50	   15501
 308.75	   15280
 309.00	   15065
 309.25	   15487
 309.50	   16007
 309.75	   15223
 310.00	   15317
 310.25	   15393
 310.50	   15001
 310.75	   15171
 311.00	   15865
 311.25	   15232
 311.50	   15691
 311.75	   15966
 312.00	   15178
 312.25	   15020
 312.50	   15261
 312.75	   15330
 313.00	   14969
 313.25	   14180
 313.50	   14313
 313.75	   14747
 314.00	   15029
 314.25	   14042
 314.50	   14005
 314.75	   14401
 315.00	   14527
 315.25	   14465
 315.50	   14569
 315.75	   13380
 316.00	   13779
 316.25	   13442
 316.50	   12755
 316.75	   13274
 317.00	   13217
 317.25	   12888
 317.50	   12656
 317.75	   12907
 318.00	   11747
 318.25	   11622
 318.50	   11107
 318.75	   12657
 319.00	   11435
 319.25	   11048
 319.50	   11277
 319.75	   11376
 320.00	   11352
 320.25	   10804
 320.50	   10115
 320.75	   10940
 321.00	   10708
 321.25	    9218
 321.50	    9199
 321.75	    9361
 322.00	   10483
 322.25	    9808
 322.50	    9237
 322.75	    9127
 323.00	    8536
 323.25	    8345
 323.50	    8869
 323.75	    8996
 324.00	    8674
 324.25	    7960
 324.50	    6887
 324.75	    7767
 325.00	    7403
 325.25	    7124
 325.50	    7100
 325.75	    7226
 326.00	    7963
 326.25	    7138
 326.50	    7450
 326.75	    7966
 327.00	    6804
 327.25	    7265
 327.50	    6958
 327.75	    6892
 328.00	    6125
 328.25	    6925
 328.50	    6225
 328.75	    5874
 329.00	    6435
 329.25	    5699
 329.50	    6238
 329.75	    6081
 330.00	    6214
 330.25	    6288
 330.50	    6524
 330.75	    5963
 331.00	    6309
 331.25	    5022
 331.50	    5653
 331.75	    5558
 332.00	    4839
 332.25	    4195
 332.50	    5042
 332.75	    4847
 333.00	    5598
 333.25	    5160
 333.50	    5348
 333.75	    5505
 334.00	    5079
 334.25	    5064
 334.50	    5206
 334.75	    5552
 335.00	    5344
 335.25	    6107
 335.50	    5144
 335.75	    5811
 336.00	    5558
 336.25	    4856
 336.50	    5630
 336.75	    4853
 337.00	    4757
 337.25	    5334
 337.50	    6131
 337.75	    5576
 338.00	    6005
 338.25	    5875
 338.50	    6029
 338.75	    6049
 339.00	    5440
 339.25	    6507
 339.50	    5764
 339.75	    5854
 340.00	    6422
 340.25	    6256
 340.50	    5712
 340.75	    5088
 341.00	    6609
 341.25	    5809
 341.50	    5687
 341.75	    5084
 342.00	    6450
 342.25	    6396
 342.50	    6161
 342.75	    6330
 343.00	    5875
 343.25	    5980
 343.50	    5821
 343.75	    6163
 344.00	    6060
 344.25	    6057
 344.50	    6029
 344.75	    6521
 345.00	    6258
 345.25	    5890
 345.50	    6329
 345.75	    5620
 346.00	    6275
 346.25	    5102
 346.50	    5641
 346.75	    5414
 347.00	    5347
 347.25	    4979
 347.50	    5688
 347.75	    5449
 348.00	    4640
 348.25	    5006
 348.50	    5004
 348.75	    4533
 349.00	    4231
 349.25	    6025
 349.50	    4685
 349.75	    3959
 350.00	    4212
 350.25	    4309
 350.50	    3600
 350.75	    3983
 351.00	    3823
 351.25	    3739
 351.50	    3855
 351.75	    3252
 352.00	    3630
 352.25	    3322
 352.50	    3826
 352.75	    3766
 353.00	    3259
 353.25	    3680
 353.50	    3229
 353.75	    3795
 354.00	    3080
 354.25	    2687
 354.50	    3047
 354.75	    2526
 355.00	    2853
 355.25	    2876
 355.50	    2526
 355.75	    2458
 356.00	    2430
 356.25	    2389
 356.50	    2323
 356.75	    2566
 357.00	    1905
 357.25	    2144
 357.50	    2414
 357.75	    2267
 358.00	    2510
 358.25	    2231
 358.50	    2307
 358.75	    1627
 359.00	    2008
 359.25	    2341
 359.50	    2120
 359.75	    1984
 360.00	    1981
 360.25	    1766
 360.50	    2014
 360.75	    1894
 361.00	    1608
 361.25	    1330
 361.50	    1772
 361.75	    1954
 362.00	    1534
 362.25	    1813
 362.50	    2317
 362.75	    1824
 363.00	    1853
 363.25	    1647
 363.50	    1450
 363.75	    1506
 364.00	    1239
 364.25	    1115
 364.50	    1609
 364.75	    1716
 365.00	    1414
 365.25	    2188
 365.50	    1337
 365.75	    1505
 366.00	    1327
 366.25	    1465
 366.50	    1375
 366.75	    1578
 367.00	    1455
 367.25	    1736
 367.50	    1549
 367.75	    1141
 368.00	    1540
 368.25	    1286
 368.50	    1629
 368.75	    1430
 369.00	     986
 369.25	    1971
 369.50	    1561
 369.75	    1652
 370.00	    1552
 370.25	    1478
 370.50	    1128
 370.75	    1454
 371.00	    1544
 371.25	    1100
 371.50	    1563
 371.75	    1547
 372.00	    1228
 372.25	    1687
 372.50	    1432
 372.75	    1442
 373.00	    1572
 373.25	    1705
 373.50	    1980
 373.75	    1379
 374.00	    1077
 374.25	    1272
 374.50	    1627
 374.75	    1238
 375.00	    1703
 375.25	    1619
 375.50	    1696
 375.75	    1234
 376.00	    1803
 376.25	    1549
 376.50	    1708
 376.75	    1269
 377.00	    1875
 377.25	    1509
 377.50	    1203
 377.75	    1880
 378.00	    1691
 378.25	    1627
 378.50	    1337
 378.75	    1374
 379.00	    1797
 379.25	    1690
 379.50	    1719
 379.75	    1823
 380.00	    1238
 380.25	    1535
 380.50	    1622
 380.75	    1342
 381.00	    2069
 381.25	    1201
 381.50	    1597
 381.75	    1892
 382.00	    1707
 382.25	    1555
 382.50	    2030
 382.75	    1438
 383.00	    1795
 383.25	    1862
 383.50	    1298
 383.75	    1283
 384.00	    1791
 384.25	    1811
 384.50	    1808
 384.75	    2012
 385.00	    1691
 385.25	    1419
 385.50	    1628
 385.75	    1701
 386.00	    1748
 386.25	    1643
 386.50	    1811
 386.75	    1937
 387.00	    1804
 387.25	    1545
 387.50	    2033
 387.75	    2033
 388.00	    1765
 388.25	    1874
 388.50	    2167
 388.75	    1899
 389.00	    1742
 389.25	    2070
 389.50	    2113
 389.75	    1852
 390.00	    1818
 390.25	    2238
 390.50	    2251
 390.75	    1560
 391.00	    1830
 391.25	    1906
 391.50	    2063
 391.75	    1944
 392.00	    2197
 392.25	    2062
 392.50	    2108
 392.75	    1921
 393.00	    1959
 393.25	    1762
 393.50	    2127
 393.75	    2080
 394.00	    1769
 394.25	    1954
 394.50	    2164
 394.75	    2027
 395.00	    2036
 395.25	    1958
 395.50	    2010
 395.75	    1839
 396.00	    1923
 396.25	    1881
 396.50	    2111
 396.75	    1943
 397.00	    2185
 397.25	    2018
 397.50	    1994
 397.75	    2208
 398.00	    1913
 398.25	    1900
 398.50	    2079
 398.75	    2080
 399.00	    2192
 399.25	    1892
 399.50	    1920
 399.75	    2165
 400.00	    1969
 400.25	    1828
 400.50	    1834
 400.75	    2291
 401.00	    2194
 401.25	    2056
 401.50	    2262
 401.75	    1963
 402.00	    2118
 402.25	    2052
 402.50	    1703
 402.75	    1523
 403.00	    1868
 403.25	    1833
 403.50	    1790
 403.75	    1954
 404.00	    2067
 404.25	    1800
 404.50	    1925
 404.75	    1857
 405.00	    1899
 405.25	    1999
 405.50	    1617
 405.75	    1705
 406.00	    1720
 406.25	    1790
 406.50	    1878
 406.75	    1860
 407.00	    1666
 407.25	    1526
 407.50	    1735
 407.75	    1748
 408.00	    1923
 408.25	    1903
 408.50	    1703
 408.75	    1620
 409.00	    1480
 409.25	    1597
 409.50	    1496
 409.75	    1518
 410.00	    1733
 410.25	    1657
 410.50	    1445
 410.75	    1643
 411.00	    1226
 411.25	    1427
 411.50	    1568
 411.75	    1442
 412.00	    1672
 412.25	    1632
 412.50	    1695
 412.75	    1568
 413.00	    1869
 413.25	    1779
 413.50	    1305
 413.75	    1716
 414.00	    1301
 414.25	    1366
 414.50	    1484
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 645.25	       0
 645.50	       0
 645.75	       4
 646.00	       0
 646.25	       0
 646.50	       0
 646.75	     107
 647.00	     248
 647.25	       0
 647.50	       0
 647.75	       0
 648.00	     168
 648.25	       0
 648.50	       0
 648.75	       0
 649.00	       0
 649.25	       0
 649.50	      87
 649.75	      77
 650.00	       0
 650.25	       0
 650.50	       0
 650.75	       0
 651.00	       0
 651.25	       4
 651.50	     118
 651.75	      76
 652.00	      52
 652.25	       0
 652.50	     262
 652.75	       0
 653.00	       0
 653.25	       0
 653.50	       0
 653.75	     146
 654.00	     109
 654.25	       0
 654.50	      85
 654.75	       0
 655.00	       0
 655.25	       0
 655.50	       0
 655.75	       0
 656.00	       0
 656.25	       0
 656.50	       0
 656.75	       0
 657.00	       0
 657.25	       0
 657.50	       0
 657.75	       0
 658.00	      96
 658.25	     211
 658.50	       0
 658.75	       0
 659.00	       0
 659.25	       0
 659.50	       0
 659.75	     243
 660.00	       0
 660.25	       0
 660.50	       0
 660.75	       0
 661.00	       0
 661.25	       0
 661.50	       0
 661.75	     423
 662.00	       0
 662.25	       0
 662.50	       0
 662.75	      18
 663.00	       0
 663.25	       0
 663.50	       0
 663.75	       0
 664.00	       0
 664.25	      38
 664.50	       0
 664.75	       0
 665.00	       0
 665.25	       0
 665.50	     102
 665.75	     173
 666.00	       0
 666.25	       0
 666.50	       0
 666.75	       0
 667.00	     303
 667.25	       0
 667.50	     194
 667.75	      37
 668.00	       0
 668.25	       0
 668.50	       0
 668.75	       0
 669.00	       0
 669.25	       0
 669.50	       0
 669.75	     296
 670.00	       0
 670.25	       0
 670.50	      32
 670.75	       0
 671.00	       0
 671.25	       6
 671.50	       0
 671.75	       0
 672.00	       0
 672.25	       0
 672.50	      85
 672.75	      37
 673.00	       0
 673.25	       0
 673.50	       0
 673.75	       0
 674.00	     137
 674.25	       0
 674.50	      32
 674.75	      28
 675.00	       0
 675.25	       0
 675.50	      58
 675.75	       0
 676.00	       0
 676.25	      14
 676.50	       0
 676.75	      91
 677.00	       0
 677.25	     146
 677.50	       0
 677.75	       0
 678.00	       0
 678.25	      47
 678.50	       0
 678.75	      55
 679.00	       0
 679.25	       0
 679.50	       0
 679.75	       0
 680.00	       0
 680.25	       0
 680.50	      93
 680.75	       0
 681.00	      74
 681.25	       0
 681.50	      54
 681.75	      27
 682.00	       0
 682.25	      53
 682.50	       0
 682.75	     148
 683.00	       0
 683.25	       0
 683.50	       0
 683.75	      49
 684.00	       0
 684.25	      65
 684.50	       0
 684.75	       0
 685.00	     166
 685.25	       0
 685.50	       0
 685.75	       0
 686.00	       0
 686.25	      20
 686.50	       0
 686.75	       0
 687.00	      45
 687.25	       0
 687.50	       0
 687.75	       0
 688.00	       0
 688.25	     150
 688.50	       0
 688.75	       0
 689.00	      33
 689.25	      10
 689.50	     126
 689.75	       0
 690.00	       0
 690.25	      11
 690.50	       0
 690.75	       0
 691.00	       0
 691.25	       0
 691.50	       0
 691.75	       0
 692.00	       0
 692.25	     270
 692.50	       0
 692.75	       0
 693.00	       0
 693.25	       0
 693.50	     179
 693.75	       0
 694.00	       0
 694.25	       0
 694.50	      20
 694.75	       0
 695.00	       0
 695.25	     138
 695.50	       0
 695.75	       0
 696.00	       0
 696.25	       0
 696.50	     152
 696.75	       0
 697.00	      15
 697.25	      62
 697.50	       0
 697.75	       0
 698.00	       0
 698.25	       0
 698.50	       0
 698.75	      15
 699.00	       0
 699.25	       0
 699.50	      84
 699.75	       0
 700.00	      19
];

Once you are comfortable calculating the amount of light you'll detect learn about detection in the powerpoint below. This will help you let the optics be the limiting factor and not poor detection technique.

Powerpoint on Detection of Optical Confocal Signal

Additional files:

Noise analysis for PMT Matlab Code

%%%%NEP_PMT.m
%%%%%%%%%%Calculates NEP from PMT data
G=1e6; %%gain of the PMT
Ra=5e4; %%radiance responsivity of anode (A/W)
d=6; %%%secondary emission ratio
F=d/(d-1);
Ida=1e-9; %%anode dark current (A)
logB=[-2:1:8]; %%log of bandwidth in Hz
B=10.^(logB); %%bandwidth in Hz
e=1.6e-19; %%electronic charge (C)
NEP=e*G*F*B/(Ra)+((e*G*F*B).^2+4*e*Ida*G*F*B).^(0.5)/(Ra);
figure(2);clf
loglog(B,NEP,'linewidth',3.0)
hold on
plot(B,P1det+zeros(1,length(B)),'k--','linewidth',3.0)
xlabel('Bandwidth(Hz)','fontsize',14)
ylabel('NEP(W)','fontsize',14)
break
Gtia=1e5; %%TIA gain (Ohms=V/A);
NEP_I=NEP*Ra;
ENI=0.2e-9;
figure(3);clf
loglog(B,NEP_I,'linewidth',3.0)
hold on
plot(B,ENI+zeros(1,length(B)),'k--','linewidth',3.0)
xlabel('Bandwidth(Hz)','fontsize',14)
ylabel('current (A)','fontsize',14)

S=P1det*Ra*Gtia; %%%signal at the output of TIA
figure(4);clf
semilogx(B,S+zeros(1,length(B)),'k--','linewidth',3.0)

Noise analysis for DAQ Matlab Code

%%%DAQ_Noise.m
%%%%%%%%%%%%%%%%%%
bits=16; %%% number of bits
Vmax=0.2; %%%maximum voltage
% lsb=Vmax/(2^bits-1); %%%least significant bit
lsb=5.2e-6;
NEP_V=NEP*Ra*Gtia; %%%minimum input at detector S/N=1
figure(5); 
loglog(B,NEP_V,'linewidth',3.0)
hold on
plot(B,lsb+zeros(1,length(B)),'k--','linewidth',3.0)
xlabel('Bandwidth(Hz)','fontsize',14)
ylabel('NEP at Daq(V)','fontsize',14)

Hamamatsu PMT info 1 PDF

Hamamatsu PMT info 2 PDF

 

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