Functional Fluorescence Microscopy Imaging (fFMI)
Our research focuses on quantitative characterization of the kinetics of molecular interactions in live cells [1-3] and the development of new imaging modalities [4-6]. Our mission is to understand how reaction-diffusion processes are integrated in live cells/tissues to form dynamical biochemical networks and how the functional dynamics of these regulatory networks is impaired in common diseases of the Central Nervous System (CNS). To this aim, we use advanced fluorescence microscopy and fluorescence correlation spectroscopy based techniques with high spatio-temporal resolution and single-molecule sensitivity. These methods are particularly suited for non-destructive quantitative characterization of molecular trafficking and interactions in live cells, which is a prerequisite for understanding the function of biological molecules.
The laboratory for Functional Fluorescence Microscopy Imaging (fFMI) is situated in the Center for Molecular Medicine (CMM), L5:02, room 020a, where the following instruments are available:
- ConfoCor 3 system, Carl Zeiss, Jena, Germany, individually modified to allow fluorescence imaging with single-molecule sensitivity in live cells. This LSM 510 system consists of an inverted microscope for transmitted light and epifluorescence (Axiovert 200 M) with 6 objectives (5X Plan-Apochromat/0.16, 10X Plan-Neo/0.3, 20X Plan-Apo/0.75 with DIC capability, C-Apochromat 40X/1.2 NA W Corr, C-Apochromat 63x/1.2 W Corr, Plan-Apochromat 100x/1.40 Oil DIC), 3 lasers with 6 laser lines (Ar/ArKr laser with four lines 458, 477, 488 and 514 nm, HeNe 543 nm and HeNe 633 nm), HBO and Halogen lamps, 3 photomultiplier tubes (PMTs), scanning module LSM 510META modified to enable detection in the imaging mode by using silicone avalanche photodiodes (SPCM-AQR-1X; PerkinElmer) and FCS module with 3 detection channels.
- ConfoCor 2 system, Carl Zeiss, Jena, Germany for quantitative in vitro studies of molecular interactions. The ConfoCor2 system consists of an upright microscope with the C-Apochromat 40X/1.2 NA W Corr UV-VIS-IR objective, 3 lasers (Ar/ArKr laser with four lines 458, 477, 488 and 514 nm, HeNe 543 nm and HeNe 633 nm), a Perkin Elmer APD detection system and computer working station.
- Microscope stage cell incubator consisting of heating Insert P, a solid heating element with uniform heat distribution and a high thermal conductivity, the CTI-Controller 3700 digital, with an IR absorption based CO2-sensor for continuous monitoring of CO2 concentration in the circulating air stream, and the Tempcontrol 37-2 digital, temperature regulator with two independent channels designed to maintain cells at the desired temperature and under desired atmosphere on a microscope stage.
- State-of-the-art home built microscope specially designed for quantitative ultra-fast confocal fluorescence microscopy imaging of dynamical systems without scanning via massively parallel Fluorescence Correlation Spectroscopy (FCS).
Quantitative study of synthetic Hox transcription factor-DNA interactions in live cells.
Vukojevic V, Papadopoulos DK, Terenius L, Gehring WJ, Rigler R
Proc Natl Acad Sci U S A 2010 Mar;107(9):4093-8
GPR37 protein trafficking to the plasma membrane regulated by prosaposin and GM1 gangliosides promotes cell viability.
Lundius EG, Vukojevic V, Hertz E, Stroth N, Cederlund A, Hiraiwa M, et al
J Biol Chem 2014 Feb;289(8):4660-73
Lipoprotein complex of equine lysozyme with oleic acid (ELOA) interactions with the plasma membrane of live cells.
Vukojević V, Bowen AM, Wilhelm K, Ming Y, Ce Z, Schleucher J, et al
Langmuir 2010 Sep;26(18):14782-7
Quantitative single-molecule imaging by confocal laser scanning microscopy.
Vukojevic V, Heidkamp M, Ming Y, Johansson B, Terenius L, Rigler R
Proc Natl Acad Sci U S A 2008 Nov;105(47):18176-81
Simultaneous imaging of amyloid-β and lipids in brain tissue using antibody-coupled liposomes and time-of-flight secondary ion mass spectrometry.
Carlred L, Gunnarsson A, Solé-Domènech S, Johansson B, Vukojević V, Terenius L, et al
J Am Chem Soc 2014 Jul;136(28):9973-81
A single-photon avalanche camera for fluorescence lifetime imaging microscopy and correlation spectroscopy Vitali M, Bronzi D, Krmpot AJ, Nikolić S, Schmitt F-J, Junghans C, Tisa Set al. IEEE J. Sel. Top. Quantum Electron. 2014 20:1-10