This report estimates the expected fluorescence overlap between Opal fluorophores when using the designated fluorescence filter sets in fluorescence microscopy applications. These are rough estimates only, based on fluorescence spectra of the Opal fluors and these specific Chroma filter sets. Variables such as light sources and the influence of sample chemistry on fluorescence ex. & emission spectra will affect the spectral overlap observed in real samples.

1. Opal
fluorophore
Excitation
maxima
Emission
maxima
Filter Set (See following pages for lot data)
green = catalog part, red = non-catalog part
Opal 520 494 525
Recommended: ET485/25x, T505lpxr, ET520/20m
Modified #1: ET480/40x, T505lpxr, ET520/20m
Opal 540 523 536 Recommended: ZET520/20x, T535lpxr, ET550/20x
Opal 570 550 570 Recommended: ET546/22x, T560lpxr, ET585/30m
Opal 620 588 616 Recommended: ET580/20x, T600lpxr, ET615/30m
Opal 650 627 650 Recommended: CT620/20x, T640lpxr, ET660/30m
Opal 690 676 694 Recommended: ET665/30bp, T690lpxr, CT725/60m
* data for Modified #1 Filter set for Opal 520
Estimated overlap values for custom Chroma filter sets with Opal fluors
Values describe expected brightness relative to intended Opal fluor
Fluorophore
Filter set
Opal 520 Opal 540 Opal 570 Opal 620 Opal 650 Opal 690
520 Filter set 100 15.5 (15.5)* — — — —
540 Filter set 12.1 100 18.0 — — —
570 Filter set — 4.71 100 11.3 — —
620 Filter set — — 1.53 100 3.18 —
650 Filter set — — — 4.42 100 2.20
690 Filter set — — — — 2.82 100

2. The data in the table above are my estimates of expected fluorescence overlap when using the
custom Chroma filter sets with the six listed Opal fluorophores (fluors). All supporting spectral data
and calculations are in the following pages. Overlap values >10% are indicated in red.
I do not have access to authentic Opal fluor spectra. All fluorescence ex. & em. spectra are proxies.
Care was used to choose existing fluorescent spectra proxies for each Opal fluor by matching the
shape of spectra in published excitation and emission graphs. The FWHM, excitation maxima &
emission maxima of these spectra were then adjusted to match that of the published Opal fluor data.
Differences are minimal and resulting spectra should provide reasonably accurate estimates.
However, these are estimates, and actual values may vary significantly depending on several factors
such as:
 brightness of each Opal fluor relative to others
 abundance/concentration of each Opal fluor relative to others
 variation of illumination intensity across the spectral range of an excitation filter passband
 amount and spectral variation of tissue autofluorescence
 spectral variation of fluorescence ex. & emission spectra due to pH, local chemistry, etc.
Values were obtained by plotting the integrals of an ex. or em. filter passband with the appropriate ex.
or em. fluorescence spectra and quantifying this “area under the curve”.
For example, one finds the integral of the excitation filter intended for use with Opal 540 and the
fluorescence excitation spectra of Opal 540.
Then one finds the integral of the emission filter intended for Opal 540 and its associated fluorescence
emission spectra. These are expressed in dimensionless units.
Next, these two values are multiplied to calculate the estimated "brightness" (or specific fluorescence
signal intensity) of a given filter with a given fluor.
To calculate overlap, one multiplies the excitation crosstalk and emisson bleedthrough integrals of
"contaminating" fluors with a given filter set's spectra, and divides this resulting estimated brightness
by that of the similarly calculated brightness of the intended fluor. So, dividing the Opal 520 brightness
obtained with the 540 filter set by the Opal 540 brightness obtained with the same set will provide the
estimate of the relative overlap of Opal 520 signal into the Opal 540 imaging channel, keeping in mind
the above, bulleted caveats.
Values assume a uniform white light source across a given excitation passband. In reality, intensity varies
somewhat across the spectrum of all light sources, especially arc lamps. Often, the variation is negligible over
a 20-40nm region typical of these passbands. LED light sources with integrated excitation filters could
potentially result in very different values.
The Modified #1 Opal 520 Filter set is intended for use with a Zeiss Colibri LED light source. Because of the
discontinuous nature of the illumination provided by the two relevant LEDs and their dedicated excitation filters,
an excitation filter with a wider passband is used in the modified set. This is an estimate, and does not take into
consideration the gap between the two dedicated excitation filters, which is not expected to skew the data in
one direction.

3. 1. Opal 520 Filter set with Opal 520 & estimated Opal 540 overlap
Spectra of Opal 520 filter set with Opal 520
Spectra of Opal 520 filter set with Opal 520 (blue) & 540 (green)

4. Integrals of Opal 520 filter set with Opal 520 (black) & 540 (red) spectra
a. Estimated fluorescence intensity calculations with Opal 520 filter set
1. Opal 520 set with Opal 520
ET485-25x_X_Opal520_EX = 1846.799
ET520-20m_X_Opal520_EM = 1657.313
1846.799 * 1657.313 = 3,060,724
2. Opal 520 set with Opal 540
ET485-25x_X_Opal540_EX = 641.678
ET520-20m_X_Opal540_EM = 739.438
641.678 * 739.438 = 474,481
b. Estimated overlap of Opal 540 into Opal 520 filter set expressed as fraction of Opal
520 signal
1. Opal 540 signal intensity/Opal 520 signal intensity
474,481/3,060,724 = 15.5%

5. 2. Opal 520 Modified #1 Filter set with Opal 520 & estimated Opal 540
overlap
Spectra of Opal 520 filter set with Opal 520
Spectra of Opal 520 filter set with Opal 520 (blue) & 540 (green)

6. Integrals of Opal 520 filter set with Opal 520 (black) & 540 (red) spectra
c. Estimated fluorescence intensity calculations with Modified #1 Opal 520 filter set
3. Modified #1 Opal 520 set with Opal 520
ET480-40x_X_Opal520_EX = 2526.081
ET520-20m_X_Opal520_EM = 1657.313
2526.081 * 1657.313 = 4,186,507
4. Modified #1 Opal 520 set with Opal 540
ET480-40x_X_Opal540_EX = 875.408
ET520-20m_X_Opal540_EM = 739.447
875.408 * 739.447 = 647,318
ci. Estimated overlap of Opal 540 into Modified #1 Opal 520 filter set expressed as
fraction of Opal 520 signal
2. Opal 540 signal intensity/Opal 520 signal intensity
647,318/4,186,507 = 15.5%

7. 3. Opal 540 Filter set with Opal 540 & estimated Opal 520 & 570 overlap
Spectra of Opal 540 filter set with Opal 540
Spectra of Opal 540 filter set with Opal 540 (green) & 520 (blue) & 570 (red)

8. Integrals of Opal 540 filter set with Opal 540 (black) & 520 (blue) spectra
Integrals of Opal 540 filter set with Opal 540 (black) & 570 (red) spectra

9. a. Estimated fluorescence intensity calculations with Opal 540 filter set
1. Opal 540 set with Opal 540
ZET520-20x_X_Opal540_EX = 1676.235
ET550-20x_X_Opal540_EM = 1237.60
1676.235 * 1237.605 = 2,074,517
2. Opal 540 set with Opal 520
ZET520-20x_X_Opal520_EX = 267.952
ET550-20x_X_Opal520_EM = 934.952
267.952 * 934.952 = 250,522
3. Opal 540 set with Opal 570
ZET520-20x_X_Opal570_EX = 994.071
ET550-20x_X_Opal570_EM = 375.881
994.071 * 375.881 = 373,652
b. Estimated bleedthrough of Opal 520 into Opal 540 filter set expressed as fraction of
Opal 540 signal
1. Opal 520 signal intensity/Opal 540 signal intensity
250,522/2,074,517 = 12.1%
c. Estimated bleedthrough of Opal 570 into Opal 540 filter set expressed as fraction of
Opal 540 signal
1. Opal 690 signal intensity/Opal 650 signal intensity
373,652/2,074,517 = 18.0%

10. 4. Opal 570 Filter set with Opal 570 & estimated Opal 540 & 620 overlap
Spectra of Opal 570 filter set with Opal 570
Spectra of Opal 570 filter set with Opal 540 (green) & 570 (blue) & 620 (red)

11. Integrals of Opal 570 filter set with Opal 570 (black) & 540 (green) spectra
Integrals of Opal 570 filter set with Opal 570 (black) & 620 (purple) spectra

12. a. Estimated fluorescence intensity calculations with Opal 570 filter set
1. Opal 570 set with Opal 570
ET546-22x_X_Opal570_EX = 1788.68
ET585-30m_X_Opal570_EM= 1937.907
1788.68 * 1937.907 = 3,466,295
2. Opal 570 set with Opal 540
ET546-22x_X_Opal540_EX = 272.647
ET585-30m_X_Opal540_EM = 598.914
272.647 * 598.914 = 163,292
3. Opal 570 set with Opal 620
ET546-22x_X_Opal620_EX = 672.421
ET585-30m_X_Opal620_EM = 581.637
672.421 * 581.637 = 391,105
b. Estimated bleedthrough of Opal 540 into Opal 570 filter set expressed as fraction of
Opal 570 signal
1. Opal 540 signal intensity/Opal 570 signal intensity
163,292/3,466,295 = 4.71%
c. Estimated bleedthrough of Opal 620 into Opal 570 filter set expressed as fraction of
Opal 570 signal
1. Opal 620 signal intensity/Opal 570 signal intensity
391,105/3,466,295 = 11.3%

13. 5. Opal 620 Filter set with Opal 620 & estimated Opal 570 & 650 overlap
Spectra of Opal 620 filter set with Opal 620
Spectra of Opal 620 filter set with Opal 620 (blue) & 570 (red) & 650 (green)

14. Integrals of Opal 620 filter set with Opal 620 (black) & 650 (red) spectra
Integrals of Opal 620 filter set with Opal 620 (black) & 570 (green) spectra

15. a. Estimated fluorescence intensity calculations with Opal 620 filter set
1. Opal 620 set with Opal 620
ET580-20x_X_Opal620_EX = 1601.33
ET615-30m_X_Opal620_EM = 2666.805
1601.33 * 2666.805 = 4,270,435
2. Opal 620 set with Opal 650
ET580-20x_X_Opal650_EX = 540.75
ET615-30m_X_Opal650_EM = 250.784
540.75 * 250.784 = 135,611
3. Opal 620 set with Opal 570
ET580-20x _X_Opal570_EX = 86.18
ET615-30m_X_Opal570_EM = 759.733
86.18 * 759.733 = 65,474
b. Estimated overlap of Opal 650 into Opal 620 filter set expressed as fraction of Opal
620 signal
1. Opal 650 signal intensity/Opal 620 signal intensity
135,611/4,270,435 = 3.18%
c. Estimated overlap of Opal 570 into Opal 620 filter set expressed as fraction of Opal
620 signal
1. Opal 650 signal intensity/Opal 620 signal intensity
65,474/4,270,435 = 1.53%

16. 6. Opal 650 Filter set with Opal 650 & estimated Opal 620 & 690 overlap
Spectra of Opal 650 filter set with Opal 650
Spectra of Opal 650 filter set with Opal 650 (green) & 620 (blue) & 690 (red)

17. Integrals of Opal 650 filter set with Opal 650 (black) & 620 (blue) spectra
Integrals of Opal 650 filter set with Opal 650 (black) & 690 (red) spectra

18. a. Estimated fluorescence intensity calculations with Opal 650 filter set
1. Opal 650 set with Opal 650
CT620-20x_X_Opal650_EX = 1701.678
ET660-30m_X_Opal650_EM = 2416.943
1701.678 * 2416.943 = 4,112,859
2. Opal 650 set with Opal 620
CT620-20x_X_Opal620_EX = 176.534
ET660-30m_X_Opal620_EM = 1029.187
176.534 * 1029.187 = 181,686
3. Opal 650 set with Opal 690
CT620-20x_X_Opal690_EX = 534.942
ET660-30m_X_Opal690_EM = 168.792
534.942 * 168.792 = 90,294
b. Estimated bleedthrough of Opal 620 into Opal 650 filter set expressed as fraction of
Opal 650 signal
1. Opal 620 signal intensity/Opal 650 signal intensity
181,686/4,112,859 = 4.42%
c. Estimated bleedthrough of Opal 690 into Opal 650 filter set expressed as fraction of
Opal 650 signal
1. Opal 690 signal intensity/Opal 650 signal intensity
90,294/4,112,859 = 2.20%

19. 7. Opal 690 Filter set with Opal 690 & estimated Opal 650 overlap
Spectra of Opal 690 filter set with Opal 690
Spectra of Opal 690 filter set with Opal 690 (red) & 650 (green)

20. Integrals of Opal 690 filter set with Opal 690 (black) & 650 (green) spectra
a. Estimated fluorescence intensity calculations with Opal 690 filter set
1. Opal 690 set with Opal 690
ET665-30bp_X_Opal690_EX = 2233.772
CT725-60m_X_Opal690_EM= 2849.773
2233.772 * 2849.773 = 6,365,743
2. Opal 690 set with Opal 650
ET665-30bp_X_Opal650_EX = 243.787
CT725-60m_X_Opal650_EM= 736.052
243.787 * 736.052 = 179,439
b. Estimated overlap of Opal 650 into Opal 690 filter set expressed as fraction of Opal
690 signal
1. Opal 650 signal intensity/Opal 620 signal intensity
179,439/6,365,743 = 2.82%