1) Application of the LuminoView LV200
2) Instrument specification
3) LuminoView LV200 booklets and user manuals
4) Theory of Bioluminescence Microscopy
1) Applications of the LuminoView LV200
- Visual inspection and quantitative analysis of any bioluminescence assay at the cellular level
- Long term life cell imaging of photosensitive samples*
(high Signal/Noise ratio without bleaching/phototoxicity)
- Imaging of samples with the high autofluorescence*
(embryos, plant cells, bacteria, parasites)
- Gene expression and promoter studies, especially time lapse monitoring of luminescence based on the reporter gene assays (fast maturation, short half live of Luciferase, quantitative measurements)
- Ca2+ imaging
(no toxic dyes, direct measurement, high Signal/Noise ratio)
- ATP and ATP coupled measurements with high special resolution in single cells and extracellular matrix (metabolic imaging and GPCR assays research)
The LV200 could be described as an imaging luminometer. It includes an inverted microscope. Based on a new optical concept and special components the LV200 allows not only to characterize the specimen by the luminescence intensity but enables high resolution imaging of cells and tissues with standard CCD and EM-CCD cameras and quantification of the luminescence structures at cellular and even sub-cellular resolution.
Optics specifically designed for luminescence:
The light path from the object to the camera is straight and as short as it can be to ensure that as much light as possible reaches the CCD chip. There is also no need for any additional mirrors, filters or lenses which absorb light and thus reducing the signal. Furthermore, the tube lens has been designed with an extremely high numerical aperture (NA), which affords a vast increase in sensitivity when compared to conventional microscope optics (tube lens optimized for luminescence imaging, 0.2x magnification). As a result, the LV200 produces signal outputs many times higher than traditional systems and can, therefore, be used with conventional CCD or EM-CCD cameras.
LV200 uses standard, high-NA objectives. This means that the whole range of magnifications and immersions is available (see Available Olympus objectives). The system contains motorized z-focus drive Adaptor for motorized z-focus.
Integrated environmental control:
The built-in system for temperature control, humidity and gas flow helps to keep the cultured cells or tissue slices in a healthy condition throughout the observation period and the unique light-tight enclosure shields the sample and optics from any external light. The LV200 is designed so that samples can be placed inside a highly accurate environmental chamber, which has independent temperature control for the stage, incubation chamber, top cover and objective. Furthermore, a water reservoir is used to maintain the correct humidity level, and CO2 flow control enables pH stability. Such environmental control enables samples to be continuously monitored over days or even weeks, without the need to move the sample between the microscope and an incubator. The system can handle small samples in 35 mm dishes.
Integrated excitation and emission filter wheels:
Motorized filter wheels enable dual-color luminescence (i.e. discrimination between probes with separate emission spectra) as well as transmitted light fluorescence imaging. With standard bright-field illumination, target areas of the sample can be found easily before switching to luminescence detection. It is, therefore, also possible to produce luminescent and fluorescent overlays on bright-field images, which enables localization and co-localization capabilities. The system contains motorized exciting filter wheel with 6 positions - 4 for standard 25 mm optical filters and motorized emission filter wheel with 6 positions - 4 for standard 25 mm optical filters, 1 for motorized shutter and 1 for bright-field.
|Filter sets||Excitation filter (nm)||Emission filter (nm)|
KL1600 LED light source
An Andor I Kon-M912 UM-CCD camera is used for image acquisition. This allows high-sensitivity and low noise imaging. This high resolution 1024 x 1024 CCD camera boasts up to 95% QEmax, high dynamic range, 13 µm pixels and exceptionally low readout noise. The iKon-M benefits from negligible dark current with industry-leading thermoelectric cooling down to -100°C enabling use of significantly longer exposure times
(Andor I Kon-M934 specification).
xcellence with graphical interface for experiment planning and execution called “Experimental Manager” (Excellence manual)
It is important that there is no ingress of external light, or any reflective surfaces within the box. Therefore the entire system is incredibly "light tight" to shields the sample and optics from external light. Thanks to this technology, most applications do not require a darkroom.
Note: Recently, Promega has introduced NanoLuc® Luciferase, a luciferase that is reported to be up to 150x brighter than the most used firefly luciferase allowing for shorter exposure times or imaging of faster cellular processes like calcium signaling.
Publikace s použitím LV200:
- Roberts et. al. (2015) Light evokes rapid circadian network oscillator desynchrony followed by gradual phase retuning of synchrony. Current Biology 25: 1-10.
- Hirobe et. al. (2014) Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide. BMC Cancer 14: 615-626.
- Lambrechts et al. (2014) A causal relation between bioluminescence and oxygen to quantify the cell niche. PLoS ONE 9: e97572.
- Han et al. (2013) Theranostic protein targeting ErbB2 for bioluminescence imaging and therapy for cancer. PLoS ONE 8: e75288.
- Saini et. al. (2012) Simulated body temperature rhythms reveal the phase-shifting behavior and plasticity of mammalian circadian oscillators. Genes Dev 26: 56-580.
- Myung et.al. (2012) Period Coding of Bmal1 Oscillators in the Suprachiasmatic Nucleus. J Neurosci 32: 8900-8918.
- Fluegge et. al. (2012) Mitochondrial Ca2+ mobilization is a key element in olfactory signaling. Nat Neurosci 15: 754-62.
- Bolinger et. al. (2011) Circadian Clocks in Mouse and Human CD4+ T Cells. PLoS ONE 6:12.
- Hirsch et al. (2011) A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5'->3' exoribonuclease (XRN) activities in Arabidopsis thaliana roots. PLoS ONE 6:e16724.
- Guilding et al. (2010) Circadian oscillators in the epithalamus. Neuroscience 169:1630-9.
- Yagitaa et al. (2010) Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro. PNAS 107: 3846-3851.
- Guilding et al. (2009) A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus. Mol Brain 2: 28-47.
- Sehadova et al. (2009) Temperature entrainment of Drosophila’s circadian clock involves the gene nocte and signaling from peripheral sensory tissues to the brain. Neuron 64:251-66.
- Kammerloher (2008) Advertising Feature; Bioluminescence microscopy for cellular level circadian analysis in the suprachiasmatic nucleus. Application note. Nature Methods 5, V-VI.