PHOTODETECTOR CALIBRATION METHOD FOR REPORTING BIOLUMINESCENCE MEASUREMENTS IN STANDARDIZED UNITS DA BARTHOLOMEUSZ, JD ANDRADE Laboratory for the Modeling, Measurement, and Management of the Metabolome (M4 Lab) Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112-9202 Email: D.Bartholomeusz@m.cc.utah.edu ) ( r I LED Position (r) 1.0 0.8 0.6 0.4 0.2 0.0 INTRODUCTION Abstract Luminescence measurements reported in relative light units (RLUs) are dependant on specific detector used, its operating conditions, and solid collection angle. do not allow accurate comparison between experiments conducted in separate labs with different assays and detectors, without a limit of detection analysis (i.e. calibration curve). do not allow for the calculation of quantum efficiency (Q.E.). Luminescence needs to be reported in units of radiance (i.e. W/cm2/sr). Radiance units are independent of detector and collection angle (See Fig. 1). Radiance can be converted to photons/s/cm2/sr to determine Q.E. Still need a calibrated low-light light source standard for detector calibration. Luminol, radioactive liquids, and other calibration methods are available.1 Most methods often do not have similar emission spectra as common bioluminescent sources of interest. Luminescence data from calibrated detectors is still not reported in radiance (W/cm2/sr). We calibrate and use a light emitting diode (LED) as a low-level light source standard. An LED is calibrated with a silicon photodiode detector whose spectral calibration is traceable to a National Institute of Standards and Technology (NIST) standard. The LED standard is calibrated in W/cm2 as a function of the solid collection angle and is then used to calibrate the output of a photomultiplier tube (PMT) and a charge-coupled device (CCD). The calibrated detectors are used to measure and report bioluminescence in nW/cm2/sr. The method accounts for spectral properties of light sources and detector responsivities. METHOD LED Source Standard Design – Governing Equations Source intensity, I(t, r, ) (in irradiance - W/cm2) must be known as a function of time t, position r = (x, y), and wavelength  at the detection plane (Fig. 2) in order to determine detector output, ODetector(t). An LED at a steady state temperature with a constant current source and a diffuser plate has the following intensity function: Where, I(t) is a constant value for all  and r due to the constant temperature and current. Since the Lambertian diffuser plate averages any variance in I(, r) due to uneven temperatures on the surface of the LED, I() can be integrated over the emission spectrum (Eqn. 1) independent of r. I(t,r, ) is simplified by normalizing I(r) and I(), according to their respective peak intensities in position and wavelength, resulting in and (Eqn. 1). Optical power of source at the detection/image plane, P() (in radiant flux units of watts or photons/s), is the integration of the intensity, Iimage, normal to the detector, over the detector area, A (Eqn. 2).2 ODetector is the detector output from an incident source power, Pimage, on detection plane and is a function of the detector’s normalized spectral responsivity, SDetector() (Eqn. 3) LED Source Standard Design – Experimental Set-Up (Fig. 3) Black walled cuvette with clear, flat bottom Built in protective window at known distance from the detector surface LED Diffuser Plate 250 m aperture Imaging/Detection Surface Lens + - d1 d2 <5 Constant DC supply with precision load resistor This same set up was used for each detector with the same d1 and d2 referenced to the imaging/detection surface of the Si, PMT, and CCD detectors. FFL Bioluminescent solution Detector housing Detector surface Collection angle, , determined by ray tracing Figure 5. Normalized intensity profile as a function of position for the focused LED image on the CCD. Figure 6. Cuvette with bioluminescent solution directly above detector surface. Figure 3. Setup for measuring the detector output of the Silicon photodetector, OSi,, and calibrating the LED light standard for subsequent calibration of the PMT based luminometer and CCD detector. The LED operated at constant current and steady state temperature, and was diffused through a Lambertian diffuser plate. This LED was chosen because it’s peak spectral properties were closest to that of the Firefly Luciferase (FFL) reaction used later. The FFL peak intensity occurs at about   560 nm while the peak intensity of the LED occurs at  = 555 nm (Fig. 5). The aperture diameter divided by d1 was much less than 0.02, effectively acting as a point source.3  < 5°, so 99% of light was within ±5° radiance response curve for which the photodetector was calibrated.2 d2/d1 was chosen such that all of the magnified image fit within the detector area for all detectors. d1was greater than 5 times the lens diameter, therefore the solid collection angle,  = Aperture area  d12. Finding the Radiance of ATP/FFL Bioluminescent Source for All Detectors 1 mM ATP, 1 mM Luciferin, 1.8 M Luciferase, 250 mM Mg2+, 12.1 M BSA. Measure spectrum of ATP/FFL to find IFL(). Normalize at 555 nm and scale to detector output (Fig. 4) for all detectors. Create a uniform light source in a flat bottom cuvette (Fig. 6) to find IFFL(r). Verify uniformity of with CCD measurement using set up in Fig. 6, and normalize to unity. for a uniform source equals the detector area. Measure Odetector(t) for for all three detectors (at time t = t1). IFFL(t) changes with time for the bioluminescence reaction after mixing at time t0. Since CCD only integrates source light over time, use a short exposure, t, at a time t1, where the light emission, IFFL(t), is approximately constant (1%). This can be verified by PMT luminometer and calibrated Si detector measurements. Solve for IFFL(t1) (W/cm2) for each detector using the following equations: Calculate the solid collection angle, Detector, for each detector by ray tracing. Solve for the radiance RFFL(t1) = IFFL(t1)/ Detector. RESULTS Table 1. Conversion Constants, Collection Angle, Irradiance, and Radiance at t1 = 3.5 min for ATP/FFL CONCLUSIONS Radiance measurements obtained for an ATP/FFL bioluminescence (at t1) for each detector show that luminescence can be reported in comparable units, independent of detectors and collection angle. Standard equipment usually available to luminescence researchers was used to calibrate sources. kCCD and kPMT are dependant on detector operating conditions. Once kCCD and kPMT are determined to calibrate the detectors for all operating conditions using the set up in Fig. 3, luminescence measurements can be reported in radiance units by using ray tracing to determine the collection angle and measuring the spectral emission of the luminescent source, Iluminescence(). ACKNOWLEDGEMENTS Dr. D.A. Christensen and Dr. S.M. Blair of the University of Utah for the use of their detection equipment and guidance on the experimental setup REFERENCES O’Kane DJ, Lee J. Absolute calibration of luminometers with low-level light standards, Methods in Enzymology, Bioluminescence and Chemiluminescence, Part C, Academic Press, 2000: 87-96. Saleh BEA, Teich MC. Fundamentals of Photonics. New York: John Wiley & Sons, 1991: 44. Ryer A. Light Measurement Handbook, International Light, Inc. 1997: 26-42. Créton R, Jaffe, L. Chemiluminescence microscopy as a tool in biomedical research. Biotechniques 2001; 31:1098-1105. Calibration of the LED Source (Finding ILED(t) in W/cm2, from Eqn. 3) Determining for calibrated silicon (Si) photodetector: Normalize responsivities of each detector, SSi(), at peak  of LED (555 nm) (Fig. 4). Measure spectrum of LED, ILED() and normalize at peak . Multiply by , (Fig. 4), and integrate over  . Determining : Focus LED on CCD and expose for 0.2 seconds. Subtract background CCD count values and normalize to peak intensity (Fig. 5). Integrate normalized CCD count values, , over the CCD area. Verify that ILED(t) is constant over time using the PMT and calibrated Si photodetector. Use the calibrated photodetector to measure Osi(t). Solve for ILED(t) in W/cm2 from Eqn. 3. The LED source is now calibrated. Calculate LED radiance R(t) by normalizing ILED(t) by solid collection angle, . Calibration of the PMT at Specific Sensitivity Setting Same set up as Fig. 3. Measure OPMT(t), and use modified Eqn. 3. Scale the normalized ILED() to the spectral responsivity of the PMT, SPMT(). Solve for kPMT, the only unknown, to calibrate the PMT (convert RLUs to W/cm2). Calibration of the CCD at Specific Operating Temperature Same set up as Fig. 3. Measure OCCD(t) and use another modified form of Eqn. 3. Solve for kCCD, the only unknown, to calibrate the CCD (convert CCD counts to W/cm2/s). A 1 2 E1 = 4 W E2 = 1 W By inverse square law r1 r2 2r1 = r2 Figure 1. Determination if the radiance (R) of a source with a detector of area, A at distances r1 and r2. Due to the inverse square law,3 the radiant flux (E) is different at r1 and r2. Because of the distances, the collection angles, 1 and 2, also vary by the inverse square law. Despite the differences in E1 and E2, the radiance value of the source (R) is constant. ILED and k Values Values Error Irradiance (nW/cm2) ILED (nW/cm2) 1.27 0.07 IFFL Si 5.3 0.3 kPMT(RLU/nW) 3.0 0.9 IFFL PMT 5 1 kCCD(CCD counts/nW/s) 46107 9107 IFFL CCD 0.3692 0.0002 Collection Angle (sr) Radiance (nW/cm2/sr LED (Fig. 2) 0.03317 0.00002 RLED Si 1020 60 ATP/FFL Si (Fig. 6) 1.893 0.002 RFFL Si 2.8 0.2 ATP/FFL PMT (Fig. 6) 1.928 RFFL PMT 2.4 0.6 ATP/FFL CCD (Fig. 6) 0.1309 0.001 RFFL CCD 0.1 Equation 4. Equation 1. Normalized Source Spectra and Detector Spectral Responsivity Spectral Properties Normalized at 555 nm Normalized LED Spectra and PMT Spectral Responsivity Equation 5. y Object Plane Image/ Detection Plane Lens I(r) Source Equation 1. x Equation 2. Figure 4. (Left) LED spectral emission, PMT photodetector spectral responsivity, and LED spectral emission scaled to the spectral responsivity of the photodetector. (Right) Spectral properties of LED, ATP/FFL bioluminescence and detector spectral responsivities. The figure on the left shows how the normalized spectral emission of the LED, , was scaled to the spectral responsivity of the PMT, . This procedure was used to scale the normalized spectral emission of the LED and ATP/FFL bioluminescence to the spectral responsivities of each of the detectors to solve Eqn. 3 Equation 3. Figure 2. Diagram showing how intensity, I, power, P, and detector output, O, at the image plane are functions of time, t, position, r, and wavelength, . Photo courtesy of Ivan Polunin