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Introduction into Bioluminescence Imaging Over 25 years experience in
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Bioluminescence Firefly
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From Bioluminescence to reporter gene assays
In 1985 an ATP dependent firefly luciferase was cloned. Genom of firefly luciferase was determined This luc operon and meanwhile lux operon from bacterial luciferase was transferred into in the genom of plasmids to transfect cells and bacteria
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From Bioluminescence to reporter gene assays
You start the Lu-Lu-reaction How you can optimize your in-vivo imaging? Substrate concentration Quantum efficiency Spectral response Absorption and Scattering Effect of pH After establishing a stable cell line
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1. Substrate Concentration
Firefly luciferase follows Michaelis-Menten kinetics, and as a result maximum light output is not achieved until the substrates and co-factor are present in large excess Luciferin Luciferin Luciferase AMP Oxyluciferin Luciferase AMP O2 CO2 Light Luciferase PPi ATP
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1. Substrate Concentration
ATP is always present. Injection of transfected tumor cells or bacteria Injection of Luciferin Injection of anesthetics Acquisition time ~ 5 min.
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1. Substrate Concentration
Luciferin degradation in mice
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1. Substrate Concentration
The yellow curve was the more often obtained. But sometimes, the signal remained stable during more than 30 minutes (red curve) or it took more than 30 minutes to rich the maximum luciferase activity (blue curve). This experiment point out the necessity to perform time courses using the sequence acquisition mode to reduce signal variability of in vivo measure-ments. The bioavailability of luciferin within the animal could be different from an experiment to another.
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Comparison of different bacteria
2. Quantum efficiency Comparison of different bacteria 109 cells each of different bacteria strains intramuscular inoculated top left: Salmonella top right: Vibrio fischeri bottom left: Shigella bottom right: E. coli constitutive expression of the Vibrio fischeri luxABCD operon each Courtesy Szalay, Loma Linda, CA
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Comparison of different luciferases
2. Quantum efficiency Comparison of different luciferases QE of LU-LU reactions: Photinus pyralis 90% Photorhabdus luminescens 70% Vibrio harveyi 15% Multiplied with the quantum efficency of the camera (QE = 80 – 90%) Maximal total QE = 80% Firefly LU bac LU
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2. Quantum efficiency + 3. Spectral response
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4. Absorption and Scattering
In-vitro / in-vivo Comparison with Firefly LU-LU In-vitro PBS, pH 7,8 firefly LU In-vivo
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4. Absorption and Scattering
Hemoglobin Water
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4. Absorption and Scattering
Melanin Living epidermis Blood vessels and body fat
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4. Absorption and Scattering
Effect of depth in-vivo In 1 cm depth 100-fold is absorbed at 650 nm Light at 550 nm is strongly absorbed by the tissue. Scattering is much more at higher wavelenghts 650 nm 590 nm 550 nm 650 nm 590 nm 550 nm
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5. Effect of pH pH light-emitted energy 6.8 0.40 7.3 0.55 7.8 1.00
pH influences QE of firefly LU-LU reaction very significant Blood of bigger mammalians has a very good controlled pH value of 7.4 Rodents could have also pH 7.2
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From Biofluorescence to reporter gene assays
The green fluorescent protein (GFP) from Aequorea victoria was discovered in 1962. Knowledge of the structure, mechanism and applications of GFP developed very rapidly after cloning of the GFP gene in 1992. GFP gene was transferred into in the genom of plasmids to transfect cells and bacteria
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1. Spectral response GFP YFP Excitation Emission GFP 490 nm 510 nm
YFP 515 nm nm dsRED 555 nm nm dsRED
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1. Spectral response GFP Excitation and Emission
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2. Absorption and Scattering
Effect of depth in-vivo Effect like bioluminescence, even worse. Excitation and emission light is strongly absorbed by the tissue. 650 nm 590 nm 550 nm 650 nm 590 nm 550 nm
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3. Autofluorescence
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3. Autofluorescence Chlorophyll a and b
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Luminescence vs. Fluorescence (1)
Basics of Optical Imaging Luminescence vs. Fluorescence (1) Excellent spatial info Can be measured in vivo & postmortem No temporal results Depth of analysis ~1-2 mm (highly absorbed λ) Non-invasive, real time Temporal up/down regulation observed Internal organs can be imaged Little spatial info when internal organs are imaged (highly scattered λ) Advantages Disadvantages
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Luminescence vs. Fluorescence (2)
Basics of Optical Imaging Luminescence vs. Fluorescence (2) Camera Specs for Luminescence Fluorescence QE Full well capacity Readout noise Resolution Dark current (for long exposure) Pixel size big small There will be no camera optimal for both technologies!
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Summary Best QE of the camera in the desired spectral range
Best QE of the LU-LU reaction Best QE in the plasmid Absorption, scattering, pH, substrate concentration etc. have to be carefully watched Avoid black mice
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Thanks for Your attention! Over 25 years experience in Imaging
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