1. 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

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 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.

4. 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

5. 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

6. 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)

7. 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→

8. 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

9. 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

10. 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

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

12. 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

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

14. 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.

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.