The LSM 5 family: an integrated imaging and spectroscopic platform for the study of cellular dynamic processes

The elucidation of diffusion processes and molecular interactions and their relation to compartments and structures will be essential to understand cellular functions in detail. Often it is not the average signal that is of interest but the behaviour of single molecules which behave as individuals. Fluorescence based assays have revolutionized the way we can observe molecules at work in their natural cellular settings and they have now also become available for single molecule studies. These technologies comprise Fluorescence Fluctuation Analysis (FFA) including Fluorescence Correlation Spectroscopy (FCS), Fluorescence Redistribution After Photobleaching (FRAP), Foerster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging (FLIM). Especially for dual colour experiments and when dealing with delicate samples the employment of multiphoton microscopy using the aforementioned technologies can be of great benefit. Ideal instruments to study single molecules would therefore need to accommodate equipment that allow for fast time resolution, adequate detectors and lasers as well as integrated work flows. In this contribution we discuss the newest developments in commercial instrumentation and software at Carl Zeiss towards highly sensitive imaging in combination with spectroscopic analysis.

[1]  Karsten König,et al.  Modern laser scanning microscopy in biology, biotechnology and medicine. , 2003, Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft.

[2]  George H Patterson,et al.  Photobleaching and photoactivation: following protein dynamics in living cells. , 2003, Nature cell biology.

[3]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[4]  M E Dickinson,et al.  Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. , 2001, BioTechniques.

[5]  Atsushi Miyawaki,et al.  Lighting up cells: labelling proteins with fluorophores. , 2003, Nature cell biology.

[6]  K. König,et al.  Fluorescence lifetime imaging by time‐correlated single‐photon counting , 2004, Microscopy research and technique.

[7]  K. Fujita.  [Two-photon laser scanning fluorescence microscopy]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[8]  A. Miyawaki,et al.  Mechanisms of protein fluorophore formation and engineering. , 2003, Current opinion in chemical biology.

[9]  Ammasi Periasamy,et al.  Protein localization in living cells and tissues using FRET and FLIM. , 2003, Differentiation; research in biological diversity.

[10]  Petra Schwille,et al.  Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy. , 2003, Methods.

[11]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[12]  J. Lippincott-Schwartz,et al.  Studying protein dynamics in living cells , 2001, Nature Reviews Molecular Cell Biology.

[13]  Petra Schwille,et al.  Fluorescence correlation spectroscopy and its potential for intracellular applications , 2007, Cell Biochemistry and Biophysics.

[14]  J. Lichtman,et al.  Optical sectioning microscopy , 2005, Nature Methods.

[15]  Horst Wallrabe,et al.  Imaging protein molecules using FRET and FLIM microscopy. , 2005, Current opinion in biotechnology.