1. Luminescence

2. Light Two ways of light emission:
“Hot” Light: Thermal excitation of atoms or molecules (eg incandescent lamp)
“Cold” Light: Non-thermal excitation of molecules (eg Fluorescence, luminescence)

3. Luminescence
In Chemiluminescence the source of energy to excite the molecule is a chemical reaction, frequently oxidation.
In Bioluminescence the energy generating chemical reaction is catalysed by an enzyme

4. Examples of Bioluminescence
Arabidopsis
Ctenophore Deiopea
Photinus pyralis

5. Examples of Chemiluminescence
1,2-Dioxetanes Glow
Luminol (Enhanced Chemiluminescence) Glow
Acridinium Ester Flash

6. Cellular Luminescence /Phagocytosis in fact CL but applied to living cells usually injection of stimulus luminol or lucigenin

7. Flash and Glow Type Kinetics

8. For chemistry of Acridinium Ester and other flash kinetic reactions (e
For chemistry of Acridinium Ester and other flash kinetic reactions (e.g. Aequorin based Calcium assays) reagent injectors are required

9. Reagent Injectors
injection of µl may take ~ 100 ms (> time for flash to reach maximum):
intrinsic rise-time of flash is slowed down
until injection is completed, mixture is in a transient state of rapidly changing concentrations

10. Reagent Injectors 2
good precision only with reproducible volume, time and pressure profile
rotation and shaking do not mix fast enough
“jet” type injection with % precision
sequential luminescent reactions like DLR assay
injection of a internal standard following for e.g. low amounts of ATP or simultaneous measurement of ATP, ADP and AMP
materials used
TEFLON, TEFZEL
Polypropylen
Advantage: stainless steel can harm live cells

11. For chemistry of Enhanced Luminol and other Glow kinetic reactions reagent injectors are NOT required

12. Advantages of Glow chemistry
Instrumentation is much simpler
Samples can be re-measured

13. Dis-Advantages of Glow chemistry
Sensitivity is usually less good

14. Advantages of Chemiluminescence
Best sensitivity with large dynamic range
Non radioactive
Low cost
Low background
No protein-quenching problems (fluorescence)

15. Detection limits of different labels (immunoassays)
low light detection

16. Dynamic Range
Log RLU/OD
Log Concentration

17. How is the light produced?
Chemical Energy
Light
Excited State
Ground State

18. Light Example of a Reporter Gene Reaction
Excited states: Luciferin-Luciferase-AMP complex
Oxy-Coelenterazine-Luciferase complex

19. PhotoMultiplier Tube PMT
Light Detection
PhotoMultiplier Tube PMT
photoelectric effect
release of secondary electrons per dynode
up to 14 dynodes

20. Spectral sensitivity

22. Types of measurement modes
current measurement: output signal depends on
applied high voltage
amplifier gain
digitizing factor converting current (voltage) into RLU´s
photon counting:
released photoelectrons = photons(cathode) x QE
stability of signal (less temperature dependent)
so-called RLU-factors for identical RLU reading of different luminometers
lower dark current (1/2 of current mode)
possibility to discriminate background via threshold

23. Amplitude-Time Function
3 categories of pulses:
Amplitudes between A + B signals (RLU)
Smaller than B: noise events from dynodes
Greater than A: radioactivity in material, cosmic radiation

24. Dynamic Range Photon Counting
> 106 decades/orders of magnitude
Itrue = Im
Imax
1 -
photon counter: saturation effect due to its dead-time
approx. Im/Itrue = 0.5

25. Dynamic Range Current Counting
Photon Counting

26. Advantages of Photon Counting vs Current Measurement
No need to calibrate
No need to set HV range
Single Dynamic range (current mode typically has 3 settings)
More stable backgrounds
Improved sensitivity
Instrument is stable over years

27. Detection Limit A * 3BG S = SA - SBG
minimum amount of analyte detectable
characteristic of both luminometer and reagent quality
S =
A * 3BG
SA - SBG
Sometimes 2 is used
S = sensitivity (same unit as A)
A = amount/concentration of analyte in g, mol ...
SA= result of measurement of A in cps
BG = mean value for background in cps
BG = standard deviation of background

28. Some competitors claim they use photon counting but do not !
If we are measuring the light we are in a sense counting the photons
If they have to set the High Voltage then Current mode is used

29. Units
RLU ... Relative Light Units
a priori: one cannot expect identical
RLU readings from different
luminometers when
measuring the same sample

30. Crosstalk (X-talk) spillover of light between wells
quasi constant signal Sm
apparent signals in neighboring wells
Sd diagonal position
Sa adjacent position
CTa = Sa / Sm
CTd = Sd / Sm

31. Cross talk is minimized by:
Design of the detector optics
Using opaque microplates
White (or black)
Cross talk is <10-5
Black plates at least 10x lower signal
Compared to white plates