Measurement of absolute values of chemiluminescence and bioluminescence and the

se 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

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)

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 spec

trum analyzer

③ ③ Luminescence collection efficiency Luminescence collection efficiency with micro wellwith micro well• Collection efficiency with white and transparent micro well to

lens system calibrated by thin transparent plate cell.

CalibrationCalibration

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

Collection

efficiencyPoint light source approximation

Calibrated by reference transparent plate cell

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

　　　　　　　　　　　　　　　　　　　　　　　　　　　　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 ））

　　　　　　　 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

θ

a : 10 (mm) r : 74 (mm) 　

lens

volume ＝ 4 x 4 x 0.79mm3

iris

Top view

Side view

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

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+

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

　　P

hot

olu

min

esce

nce

inte

nsi

ty (

ph

oton

/0.5

nm

)P

hot

olu

min

esce

nce

inte

nsi

ty (

ph

oton

/0.5

nm

) 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 53.196

white 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

Concentration（Ｍ )

Volume （ μｌ） Final (M)

Final (M)Methods in Enzymology

(vol.305)

Luminol1.0×10-6 ～

1.0×10-750

5×10-7 ～5×10-8

1.0×10-6 ～1.0×10-8

H2O2 0.1 2 2×10-31.0×10-3 ～

1.0×10-5

Horseradishperoxidase

1.0×10-6 50 5×10-7 2.0×10-7

＊

ルミノール

++ ｈｈ ννH2O

2

HRP

＊

Luminol

Anionic luminol

Process of light emission : Process of light emission : LuminolLuminol

reference ： LUMIKA CORPORATION Home page

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 1.631

clear 1.00E- 06 3.01E+13 3.4385E+07 7.31E- 05 5.03.E+11 1.563

clear 1.00E- 07 3.01E+12 4.331E+06 7.31E- 05 6.34.E+10 1.969

clear 1.00E- 07 3.01E+12 3.790E+06 7.31E- 05 5.54.E+10 1.723

clear 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

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

　　　　　　

Future plansFuture plans• Measure firefly quantum yield again using bufferMeasure firefly quantum yield again using buffer

which 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.