Applications for LV200 |
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 Unrivalled sensitivity and resolution in
bioluminescence microscopy
Extremely light tight enclosure
Environmental control for ultra-long live
cell studies
Compatible with a broad range of
cameras and objectives
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Ultra Long Gene Expression Analysis
 Chronobiology (the study of biological timing or circadian rhythm) was arguably the first research area to use bioluminescence microscopy. Measuring the upregulation and downregulation of a gene over days or even weeks needs a reporter system with a short half-life, stable substrates and an analysis method lacking any toxic effect to the cells. The luciferin-luciferase bioluminescent system covers these requirements perfectly. Circadian timing is a complex process, and in mammals most vital processes are
subject to circadian variations. Thus sleep-wake cycles, locomotor activity, heartbeat, blood pressure, renal plasma flow, body temperature, sensorial perception and the secretion of many hormones fluctuate during the day in an orderly fashion. The daily timing of these physiological parameters persists under constant conditions and must therefore be controlled by one or more circadian pacemakers.
Example: Expression of Dbp transcription factor
Mouse NIH3T3 fibroblasts, stably expressing the full-length circadian Dbp gene fused to luciferase, were visualised for four days in the Olympus LV200. Image A shows the low-resolution display of the whole time series. Image B shows a display of individual cells. The luminescence signal can be measured for any cells over time (see ).
Microscope settings: Objective 20x, Hamamatsu ImageEM, no pixel binning, EM gain 150, 15 min acquisition time (courtesy of M. Stratmann, U. Schibler, Department of Molecular Biology, University of Geneva, Switzerland).
Promoter Studies in Tissue Slices
 Hugh Piggins, Alun Hughes and Clare Guilding at the University of Manchester’s Faculty of Life Sciences are looking at the long-term expression of the protein Period-2 (PER2) in mice, a protein encoded by the PER2 gene, a key CLOCK gene. They have been using the Olympus LV200 to look at long-term expression patterns of the PER2 protein, a process that requires acutely cultured brain slices to be incubated and imaged for extended periods of time. Recording images once every 3 minutes for up to 7 days, they can then analyse the gross expression of PER2 over these times within the entire culture, as well as view each individual cell (A) to look for variations from and similarities to the gross expression (B, C).
Gene Expression and Promotor Analysis using Heat Shock Response
 If cells are exposed to environmental stress (heat, toxic substances and oxygen deficiency), stress proteins such as HSP70 (Heat Shock Protein) are induced. In the stable state, HSP70 binds to HSF (Heat Shock Factor), which is a transcription factor of HSP70 promoter, and HSF is in an inactive form. High temperature greater than 40°C dissociates HSP70-HSF complex, and the HSF binds to HSP70 promoter to induce HSP70 gene expression. Then, HSP70 binds to intracellular proteins to prevent structural damage by heat. This series of reactions is called the heat shock response.
In this study, we applied the luminescence imaging system for detection of the HSP70 promoter activity on HeLa cells using GL3 luciferase (Promega) as a reporter. At 24 hours after transfection cells were treated for 1 hour at 43°C, and then 200 μM D-luciferin (Promega) was added before the imaging experiment. Images were acquired using LUMINOVIEW attached to a cooled charge-coupled device (CCD) camera DP-30 (Olympus). The figure shows a superimposed brightfield and luminescence image of transfected cells after heat shock activation (above). Individual transfected cells show huge difference in Luciferase expression (below).
Intracellular Protein Localisation with Luminescence Microscopy
 In luminescence imaging signals decrease drastically with increasing magnification. Thanks to the high sensitivity of Luminoview LV200 a clear signal can be observed even with high magnification and enables for the first time a subcellular resolution in luminescence microscopy.
The nuclear localization signal (NLS), an endoplasmic reticulum (ER) localization signal (calreticulin) and a peroxisom localization signal (SKL) sequence are fused with the luciferase gene in the mammalian expression vector. HeLa cells are transfected with these vectors and luminescence images are recorded after overnight incubation. As a result, localization of the luciferase tagged signal sequence is visualized in the different cellular compartments using luminescence imaging.
Ca2+ Imaging with Luminescence using Photina® Photoprotein
The measurement of intracellular Ca2+ concentrations is one of the key assays in drug development, signalling and molecular physiology. Ca2+ dependent Photo proteins like Aequorin, Obelin, BRET probes or Photina® can be used to measure mobilized Ca2+ without using fluorescent excitation. Due to the enormous step in sensitivity the LV200 opens new possibilities for Ca2+ measurements on a single cell resolution. The high S/N ratio, lack of background and phototoxic effects are only some advantages over standard fluorescence methods.
The video shows CHO cells expressing Photina® protein (Axxam, Milan, Italy). The cells are stimulated by adding 10µM ATP. This leads to a phospholipase C activation through endogenous P2Y receptor and G-protein activation. Phospholipase C cleaves PIP2 in Diacylglycerol (DAG) and Inositol-3-phosphat (IP3). IP3 releases intracellular Ca2+ which is stored in the ER. The Ca2+ depended reaction between Photina® protein and the substrate Coelenterazine leads to a bioluminescent signal.
 Figure: The bioluminescence signal created by Ca2+ depended Photina® protein (see above) was measured using the Olympus Luminoview LV200 and a Hamamatsu ImageEM® camera. The signal of 5 individual cells is displayed after activation with 10µM ATP. The experiment was performed by Dr. Marc Spehr, Inst. of Zellphysiologie, Ruhr-Universitaet Bochum.
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