1. Introduction
Into non-invasive in-vivo
Luminescence and
Fluorescence
Imaging

2. What we are talking about? Non-invasing in-vivo imaging
Animals are alive, only anaestetized during imaging.
Most interesting applications are
• Proliferation - increased cell division and growth
• Tumor specific targets - cell surface and internal
• Targeting of therapeutic agents and cells
• Ability to invade and metastasize
• Genetic alterations - activation of oncogenes and
loss of tumor suppressor genes
• Angiogenesis - development of new blood supply

3. What we are talking about? Optical Reporter Genes
A plasmid have to be modified with
1. Firefly luciferase or
Renilla luciferase for bioluminescence
2. Fluorescent Photoproteins like GFP, YFP, RFP or m-Cherry etc. for biofluorescence
Then the plasmid is inserted in a cell.
p53 Regulatory region (enhancer)
TkGFP reporter gene
Introduced into U87 cells (human glioblastoma with normal p53 gene/signaling)

4. What we are talking about? Bioluminescence
Firefly

5. What we are talking about? Luciferases and Luciferins (1)

6. What we are talking about? Luciferases and Luciferins (2)

7. What we are talking about? Spectral peaks of Lu-Lu-reactions

8. What we are talking about? Detection of Lu-Lu-reactions
From the beginning two lines of instruments were developed at BERTHOLD:
for just the intensity with PMTs,
for 2D-localisation with cameras.
Tube or microplate
Animal

9. What we are talking about? The principle of reporter gene assys
If the plasmid in the cell becomes activated, the sequence after the promoter becomes transcripted.
After transcription and translation luciferase is coexpressed in the cells.
Addition of luciferin causes light emission.

10. What we are talking about? Activation by ligands
Luciferin
Luciferase
Ligands NR NR NR NR RE
PROMOTER
LUCIFERASE

11. What we are talking about? Non-invasing in-vivo imaging of mice
Injection of transfected tumor cells or bacteria
Injection of Luciferin
Injection of anesthetics or
gas anaesthesia (Isofluorane)
Acquisition time ~ 5 min.

12. What we are talking about? 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

13. What we are talking about? Substrate concentration in living animals
Best concentration is about 125 mg/kg bodyweight
Luciferin degradation in mice

14. Comparison of different bacteria
What we are talking about?
Quantum efficiency of the assay
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

15. What we are talking about? Quantum efficiency of CCD cameras
__ LB 980 (1989) __ LB 981 (1996) __ LB 983 (2006)

16. Comparison of different luciferases
What we are talking about?
Total 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

17. What we are talking about? Quantum efficiency and cooling
Effect of cooling
Cooling decreases the quantum effi-ciency of the CCD array.
Since dark current is already very low at -70°C, cooling temperature of the new NC 100 is set to this temperature.

18. What we are talking about? Lu-Lu-reaction in animals
In-vitro / in-vivo
Comparison with
Firefly LU-LU
In-vitro
PBS, pH 7,8
firefly LU
In-vivo

19. What we are talking about? Absorption of haemoglobin and water
Hemoglobin
Water

20. What we are talking about? Absorption of tissues and skin
Melanin
Living epidermis
Blood vessels and body fat

21. What we are talking about) Theoretical absorption in tissues
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

22. What we are talking about?
Effect of pH
pH light-emitted energy
6.8 40
7.3 55
% QE
8.3 44
8.8 37
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

23. What we are talking about? Biofluorescence
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

24. What we are talking about? Spectral response of GFP and derivatives
YFP
Excitation Emission
GFP 490 nm nm
YFP 515 nm nm
dsRED 555 nm nm
dsRED

25. What we are talking about? GFP Excitation and Emission

26. What we are talking about? Theoretical absorption in tissues
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

27. What we are talking about? Autofluorescence of hairy skin

28. What we are talking about? Autofluorescence of food
Chlorophyll a and b

29. 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

30. 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!

31. 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