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

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

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)

What we are talking about? Bioluminescence Firefly

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

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

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

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

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.

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

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.

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

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

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

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

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

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.

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

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

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

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

What we are talking about? Effect of pH pH light-emitted energy 6.8 40 7.3 55 7.8 100 % 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

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

What we are talking about? Spectral response of GFP and derivatives YFP Excitation Emission GFP 490 nm 510 nm YFP 515 nm 525 nm dsRED 555 nm 580 nm dsRED

What we are talking about? GFP Excitation and Emission

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

What we are talking about? Autofluorescence of hairy skin

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

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

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!

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