Measurement of absolute values of chemiluminescence and bioluminescence
Measurement of absolute values of chemiluminescence and bioluminescence and these quantum yields ISSP Univ. of Tokyo. A, CRESTB・JST ATTO Corp. C, AISTD, Shizuoka Univ. E Yoriko Ando. A, B, N. Yamada. C, T. Irie. C, T. Enomoto. C H. Kubota. B, C, K. Niwa. E, K. Ohmiya. D, E, H. Akiyama. A, 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 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 Calibrated by Conventional Our system Photomultiplier Cooled CCD camera NBS standard lamp Total radiation standard lamp Standard color temperature lamp Glass tube technique features Power meter + lasers OSA + W lamp Semiconductor technique Low voltage High voltage aging effect Low aging effect High DC noise(dark current, 1/f noise) Low DC noise optics － Lens, mirror Collection efficiency Point light source approximation Calibrated by reference transparent plate cell
Experimental setup Iris lens(f=50) Mirror 87 mm Spectrometer 121 mm cooled ＣＣＤ ＰＣ ↑ Trigger solution → Long cut filter ← Substrate solution ↑ White or Transparent micro well（4× 4× 8. 5 mm 3） full 100μｌ 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 Image （CCD ＆ spectrometer） (CCD)
Calibration ③ Collection efficiency reference：collection efficiency can be accurately estimated for a transparent plate cell Side view Cover glass 0. 1 mm Mask Plastic plate 0. 79 mm substrate solution Reference substrate solution ピッカジーンLT 7. 5 （TOYO B-Net） Half-life more than 7. 5 hours 4 mm Top view volume＝ 4 x 0. 79 mm 3 lens a iris θ i r a : 10 (mm) r : 74 (mm) Substrate solution→ θ NAext = sinθ=0. 1347 next =1. 0 NAint = sinθ/nint =0. 1011 nint=1. 33 solid angle η={1 -(1 -NAint 2)1/2} / 2 =0. 256(%)
Collection efficiency with white & transparent micro well Intensity (count/s/μl) η （％） Normalized by transparent plate cell Transparent plate cell (4× 4× 0. 79 mm 3) White micro well (4× 4× 8. 5 mm 3) Transparent micro well (4× 4× 8. 5 mm 3) 1. 00 0. 256 6. 00 1. 536 0. 73 0. 197
Process of light emission : Firefly Luciferase(enztme), ATP, Mg+ Luciferin (substrate) O 2+ oxyluciferin p. H < 7 Singletexitedstate Red light Ground state p. H > 7. 6 Singlet exited state Yellow green light Ground state reference：KIRIYA CHEMI Home page
ホタルルシフェリン量子収率測定 Ｍｇ2+，Luciferase 発光過程 Luciferin＋ATP------- Luciferyl-ＡＭＰ・Luciferase+ Ｐ-P O 2+ Luciferyl-ＡＭＰ・Luciferase--------- Product + Luciferase + ｈν Luciferin (SIGMA/PROMEGA/DOJIN DO) Luciferase Concentration（Ｍ ) Volume（μｌ） Final (M) Seliger et al. (1959) 1. 0× 10 -7 5 5. 0× 10 -9 4. 2× 10 -9 2. 0× 10 -5 5 1. 0× 10 -6 4. 2× 10 -8 0. 1 5 5. 0× 10 -3 4. 2× 10 -3 1. 0× 10 -3 50 5. 0× 10 -4 4. 2× 10 -4 0. 025 35 8. 75× 10 -3 2. 1× 10 -2 (WAKO) Mg. SO 4 ATP Glygly (p. H 7. 6)
Photoluminescence intensity (photon/0. 5 nm) 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 ルミノール Luminol Anionic luminol ＊ H 2 O 2 + ｈν HRP reference：LUMIKA CORPORATION Home page Concentration（Ｍ) Luminol 1. 0× 10 -6～ 1. 0× 10 -7 Volume（μｌ） Final (M) Methods in Enzymology (vol. 305) 50 5× 10 -7 ～ 5× 10 -8 1. 0× 10 -6～ 1. 0× 10 -8 1. 0× 10 -3 ～ 1. 0× 10 -5 2. 0× 10 -7 H 2 O 2 0. 1 2 2× 10 -3 Horseradishperoxidase 1. 0× 10 -6 50 5× 10 -7
Luminescence spectrum and Quantum Yield : Luminol Transparent micro well Photons 5. 79× 1011 molecules 3. 01× 1013 QY 1. 84％
Summary Our result Luminol 1. 7％ Early study Comment 1. 24％ while Lee & Seliger estimate that collection efficiency is 1, we estimate it is 0. 7. (1. 7× 0. 7≒ 1. 2) [Lee & Seliger (1965)] We observed large fluctuation in Firefly 30～ 50％ luciferin yieelds, similarly to the early result. This is due to p. H instability of glycylglycin buffer. 88± 25％ <Problems of early study> [Seliger ＆ Mcelroy （1959） ・ stability of buffer ] ・Purity of luciferin(optical isomer) ・Lee et al. measurements →about 60%(private communication) ・Measurements.
Future plans • Measure firefly quantum yield again using buffer which is available around p. H 8. • 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.
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