Measurement of absolute values of chemiluminescence and bioluminescence and these quantum yields

1. Measurement of absolute values of chemiluminescence and bioluminescence and these quantum yields ISSP Univ. of TokyoA, CRESTB・JST ATTO Corp.C, AISTD, Shizuoka Univ.E Yoriko AndoA,B, N.YamadaC, T.IrieC, T.EnomotoC H.KubotaB,C, K.NiwaE, K.OhmiyaD,E, H.AkiyamaA,B

2. Contents • Development of system measuring quantum yield of bioluminescence and chemiluminescence • Quantum yield of Luminol early study : 1.24% by Lee & Seliger (1965) • Quantum yield of Firefly bioluminescence early study : 88% by Seliger & Mcelroy (1959)

3. Calibration ① Optics • Reflectance and Transmittance measured by rock-in detection ② Sensitivity of detector • Sensitivity of CCD calibrated by 4 kinds of lasers • Wavelength dependence calibrated by W lamp and optical spectrum analyzer ③ Luminescence collection efficiency with micro well • Collection efficiency with white and transparent micro well to lens system calibrated by thin transparent plate cell.

4. Comparison between conventional system and our system

5. Iris lens(f=50) Mirror cooled ＣＣＤ ＰＣ Spectro- meter ↑ Long cut filter Trigger solution → ← Substrate solution Grating : spectroscopy Mirror : imaging of spectrally integrated light 　　 ↑ White or Transparent micro well（4×4×8.5mm3） full 100μｌ Experimental setup 121mm 87mm

6. Calibration ② CCD sensitivity & wavelength dependence Spectroscopy （CCD ＆ spectrometer） Image (CCD)

7. θ next =1.0 i nint=1.33 lens a r Reference substrate solution ピッカジーンLT7.5 （TOYO B-Net） Half-lifemore than 7.5hours a : 10 (mm) r : 74 (mm) θ Calibration ③ Collection efficiency reference：collection efficiency can be accurately estimated for a transparent plate cell Side view Mask Plastic plate 0.79mm substrate solution Cover glass 0.1mm 4mm Top view volume＝4 x 4 x 0.79mm3 iris NAext = sinθ=0.1347 NAint = sinθ/nint =0.1011 solid angleη={1-(1-NAint2)1/2} / 2 =0.256(%) Substrate solution→

8. Collection efficiency with white & transparent micro well

9. Luciferase(enztme), ATP,Mg+ Luciferin (substrate) Process of light emission : Firefly O2+ oxyluciferin pH < 7 Ground state Singlet exited state Singlet exited state Red light pH > 7.6 Yellow green light Ground state Singlet exited state reference：KIRIYA CHEMI Home page

10. 発光過程 Ｍｇ2+，Luciferase Luciferin＋ATP------------- Luciferyl-ＡＭＰ・Luciferase+ Ｐ-P Luciferyl-ＡＭＰ・Luciferase------------------ Product+Luciferase +ｈν O2+ ホタルルシフェリン量子収率測定

11. Photoluminescence intensity (photon/0.5nm) Luminescence spectrum and Quantum Yield : Firefly With white micro well Photons8.24×1010 Molecules2.77×1011 QY29.01％

12. ＊ H2O2 +ｈν HRP Process of light emission : Luminol ルミノール Luminol Anionic luminol ＊ reference：LUMIKA CORPORATION Home page

13. Luminescence spectrum and Quantum Yield : Luminol Transparent micro well Photons5.79×1011 molecules3.01×1013 QY 1.84％

14. Summary

15. Future plans • Measure firefly quantum yield again using buffer which is available around pH8. • Development and spread high sensitivity system measures absolute values of luminescence(?). • Development of system measuring quantum yield of photoluminescence(?). ηchemiluminescence＝ ηreaction× ηsinglet exited state× ηluminescence • Quantitative micro-spectrophotometry image measurment of living cells.