Measurement of absolute values of chemiluminescence and bioluminescence and these quantum yields
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Measurement of absolute values of chemiluminescence and bioluminescence and the
se quantum yields
ISSP Univ. of TokyoA, CRESTB ・ JSTATTO 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
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ContentsContents• 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)
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① ① OpticsOptics• Reflectance and Transmittance measured by rock-in detection
② ② Sensitivity of detectorSensitivity of detector• Sensitivity of CCD calibrated by 4 kinds of lasers • Wavelength dependence calibrated by W lamp and optical spect
rum analyzer
③ ③ Luminescence collection efficiency Luminescence collection efficiency with micro wellwith micro well• Collection efficiency with white and transparent micro well to l
ens system calibrated by thin transparent plate cell.
CalibrationCalibration
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Comparison between Comparison between conventional system and our systemconventional system and our system
Conventional Our systemDetector 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
Collectionefficiency
Point light source approximation Calibrated by reference transparent plate cell
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121mm87mm
↑White or Transparentmicro well ( 4×4×8.5mm3 )full 100μ l
lens(f=50)
← Substrate solution
↑Long cut filter
Trigger solution →
cooledCCD PCSpectro-
meter
IrisMirror
Grating : spectroscopyMirror : imaging of spectrally integrated light
Experimental setupExperimental setup
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Calibration ② Calibration ② CCD sensitivity & wavelength CCD sensitivity & wavelength dependencedependence
Laser (nm) CCD conversion rate (photon/count)
633 6.04
532 8.61
476.5 9.23
457.9 10.64
ImageImage(CCD)(CCD)
SpectroscopySpectroscopy(( CCD CCD & & spectrometerspectrometer ))
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NAext = sinθ=0.1347
NAint = sinθ/nint =0.1011
solid angle η={1-(1-NAint 2)1/2} / 2
=0.256(%)
Cover glass 0.1mm
Plastic plate 0.79mm
Mask
substrate solution
4mm
referencereference :: collection efficiency can be accurately estimated collection efficiency can be accurately estimated for a transparent plate cellfor a transparent plate cell
Reference substrate solutionReference substrate solutionピッカジーン LT7.5 ( TOYO B-Net )Half-life more than 7.5hours
Calibration ③ Collection Calibration ③ Collection efficiencyefficiency
i
θ
nint=1.33
next =1.0
Substrate solution→
a
r
θa : 10 (mm) r : 74 (mm)
lens volume = 4 x 4 x 0.79mm3
iris
Top view
Side view
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Intensity(count/s/μl)
Normalized by transparent plate cell
η (%)Transparent plate cell
(4×4×0.79mm3)1.00 0.2560.256
Whitemicro well
(4×4×8.5mm3) 6.001.5361.536
Transparentmicro well
(4×4×8.5mm3)0.73 0.1970.197
Collection efficiency Collection efficiency with white & transparent micro wellwith white & transparent micro well
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reference : KIRIYA CHEMI Home page
Luciferase(enztme),
ATP, Mg+
Luciferin (substrate)
pH < 7
pH > 7.6
Process of light emission : Process of light emission : FireflyFirefly
oxyluciferin
Singlet exited state Singlet exited state
Singlet exited state
Red lightRed light
Yellow green Yellow green lightlight
Ground state
Ground state
O2+
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ホタルルシフェリン量子収率測定ホタルルシフェリン量子収率測定
Luciferin + ATP------------- Luciferyl- AMP・ Luciferase+ P -P
Luciferyl- AMP・ Luciferase------------------
Product + Luciferase + hh νν
Mg 2+ , Luciferase
O2+
発光過程発光過程
Concentration(M )
Volume( μ l) Final (M) 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 5 1.0×10-6 4.2×10-8
MgSO4 0.1 5 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
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Pho
tolu
min
esce
nce
inte
nsity
(pho
ton/
0.5n
m)
Phot
olum
ines
cenc
e in
tens
ity (p
hoto
n/0.
5nm
) Luminescence spectrum Luminescence spectrum and Quantum Yield : and Quantum Yield : FireflyFirefly
With white micro well Photons 8.24×1010
Molecules 2.77×1011
QY 29.01 %spectrometer マイクロウェル 分子数 CCD total count 変換換算値 Photon数 (%)量子効率
全光 white 2.77E+11 1.290E+08 8.7545.E- 04 6.440E+10 53.196white 2.77E+11 1.330E+08 8.7545.E- 04 6.420E+10 54.845white 2.77E+11 1.220E+08 8.7545.E- 04 5.890E+10 50.309white 2.77E+11 1.110E+08 8.7545.E- 04 5.340E+10 45.773white 2.77E+11 1.020E+08 8.7545.E- 04 4.910E+10 42.062white 2.77E+11 1.010E+08 8.7545.E- 04 4.850E+10 41.650white 2.77E+11 7.727E+07 8.9079.E- 04 8.674E+10 31.315white 2.77E+11 9.347E+07 8.9079.E- 04 1.049E+11 37.880white 2.77E+11 8.634E+07 8.9079.E- 04 9.692E+10 34.990white 2.77E+11 8.045E+07 8.9079.E- 04 9.031E+10 32.603white 2.77E+11 7.617E+07 8.9079.E- 04 8.551E+10 30.871clear 2.77E+11 1.442E+07 1.0904.E- 04 1.322E+11 47.742clear 2.77E+11 1.151E+07 1.0904.E- 04 1.056E+11 38.121clear 2.77E+11 1.291E+07 1.0904.E- 04 1.184E+11 42.749
分光 white 2.77E+11 2.991E+06 3.7223.E- 05 8.035E+10 29.008white 2.77E+11 3.644E+06 3.7223.E- 05 9.790E+10 35.341white 2.77E+11 3.714E+06 3.7223.E- 05 9.978E+10 36.020clear 2.77E+11 5.629E+05 5.1432.E- 06 1.094E+11 39.5109clear 2.77E+11 4.886E+05 5.1432.E- 06 9.500E+10 34.2956
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Concentration(M )
Volume ( μl) Final (M)
Final (M)Methods in Enzymology
(vol.305)
Luminol 1.0×10-6 ~1.0×10-7
50 5×10-7 ~5×10-8
1.0×10-6 ~1.0×10-8
H2O2 0.1 2 2×10-3 1.0×10-3 ~1.0×10-5
Horseradishperoxidase
1.0×10-6 50 5×10-7 2.0×10-7
*ルミノール
++ hh ννH2O2
HRP
*Lumin
olAnionic luminol
Process of light emission : Process of light emission : LuminolLuminol
reference : LUMIKA CORPORATION Home page
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Luminescence spectrum Luminescence spectrum and Quantum Yield : and Quantum Yield : LuminolLuminol
Transparent micro well Photons 5.79×1011
molecules 3.01×1013
QY 1.84 %
spectrometer マイクロウェル Luminol (M)濃度 分子数 CCD total count 変換換算値 Photon数 (%)量子効率 (%)量子効率 平均全光 clear 1.00E- 07 3.01E+12 3.5897E+06 7.31E- 05 5.25.E+10 1.632
clear 1.00E- 07 3.01E+12 3.5891E+06 7.31E- 05 5.25.E+10 1.631clear 1.00E- 06 3.01E+13 3.4385E+07 7.31E- 05 5.03.E+11 1.563clear 1.00E- 07 3.01E+12 4.331E+06 7.31E- 05 6.34.E+10 1.969clear 1.00E- 07 3.01E+12 3.790E+06 7.31E- 05 5.54.E+10 1.723clear 1.00E- 07 3.01E+12 4.416E+06 7.31E- 05 6.46.E+10 2.007clear 1.00E- 07 3.01E+12 4.478E+06 7.31E- 05 6.55.E+10 2.035
分光 clear 1.00E- 06 3.01E+13 1.898E+06 3.43E- 06 5.79.E+11 1.839clear 1.00E- 06 3.01E+13 1.680E+06 3.43E- 06 5.12.E+11 1.328clear 1.00E- 06 3.01E+13 1.423E+06 3.43E- 06 4.34.E+11 1.379 1.71± 0.25
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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)
Fireflyluciferin
30 ~ 50 %
88±25 %[Seliger & Mcelroy ( 1
959 ) ]
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.
SummarySummary
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Future plansFuture plans• Measure firefly quantum yield again using bufferMeasure firefly quantum yield again using bufferwhich is available around pH8. 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.
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