ISSP Univ. of TokyoA, CRESTB・JST ATTO Corp.C, AISTD, Shizuoka Univ.E 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

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)

Calibration with micro well 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.

Comparison between conventional system and our system Detector Photomultiplier Cooled CCD camera Calibrated by NBS standard lamp Total radiation standard lamp Standard color temperature lamp Power meter + lasers OSA + W lamp features Glass tube technique 　 High voltage aging effect 　 High DC noise(dark current, 1/f noise) Semiconductor technique 　 Low voltage 　 Low aging effect 　 Low DC noise optics － Lens,　mirror Collection efficiency Point light source approximation Calibrated by reference transparent plate cell

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

Calibration ② CCD sensitivity & wavelength dependence Laser (nm) CCD conversion rate (photon/count) 633 6.04 532 8.61 476.5 9.23 457.9 10.64 Spectroscopy （CCD ＆ spectrometer） Image (CCD)

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

Collection efficiency with white & transparent micro well Intensity (count/s/μl) Normalized by transparent plate cell η （％） Transparent plate cell (4×4×0.79mm3) 1.00 0.256 White micro well (4×4×8.5mm3) 6.00 1.536 0.73 0.197

Process of light emission : Firefly Luciferase(enztme), ATP,　Mg+ Luciferin (substrate) 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

ホタルルシフェリン量子収率測定 発光過程 Luciferin＋ATP------------- 　　　 　　　　　Luciferyl-ＡＭＰ・Luciferase+ Ｐ-P 　　　　　Luciferyl-ＡＭＰ・Luciferase------------------ 　　　　 　　　　　　Product　+　Luciferase +　ｈν Ｍｇ2+，Luciferase O2+ 発光過程 Concentration（Ｍ) Volume（μｌ） Final (M) Seliger et al.(1959) Luciferin (SIGMA/PROMEGA/DOJINDO) 1.0×10-7 5 5.0×10-9 4.2×10-9 Luciferase (WAKO) 2.0×10-5 1.0×10-6 4.2×10-8 MgSO4 0.1 5.0×10-3 4.2×10-3 ATP 1.0×10-3 50 5.0×10-4 4.2×10-4 Glygly (pH7.6) 0.025 35 8.75×10-3 2.1×10-2

Luminescence spectrum and Quantum Yield : Firefly 　Photoluminescence intensity (photon/0.5nm) Luminescence spectrum and Quantum Yield : Firefly With white micro well Photons　 8.24×1010 Molecules　　2.77×1011 QY　　29.01％

Process of light emission : Luminol Methods in Enzymology (vol.305) +ｈν H2O2 HRP ＊ ルミノール Luminol Anionic luminol ＊ reference：LUMIKA CORPORATION Home page Concentration（Ｍ) Volume（μｌ） Final (M) Methods in Enzymology (vol.305) Luminol 1.0×10-6～ 1.0×10-7 50 5×10-7 ～ 5×10-8 1.0×10-8 H2O2 0.1 2 2×10-3 1.0×10-3 ～ 1.0×10-5 Horseradishperoxidase 1.0×10-6 5×10-7 2.0×10-7

Luminescence spectrum and Quantum Yield : Luminol Transparent micro well Photons　 5.79×1011 molecules　　 3.01×1013 QY 　　1.84％

Summary Firefly luciferin Our result Early study Comment Luminol 1.7％ 1.24％ [Lee & Seliger (1965)] while　Lee & Seliger estimate that collection efficiency is 1, we estimate it is 0.7. (1.7×0.7≒1.2) Firefly luciferin 30～50％ 88±25％ [Seliger ＆ Mcelroy （1959）] 　We observed large fluctuation in yieelds, similarly to the early result.This is due to pH instability of glycylglycin buffer. <Problems of early study> 　・ stability of buffer 　・Purity of luciferin(optical isomer) 　・Lee et al. measurements →about60%(private communication) 　・Measurements.

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.