|
In vivo imaging in general allows a non-invasive insight into living organisms and helps to understand metabolic processes and disease related changes. Especially bioluminescence imaging (BLI) and biofluorescence imaging (BFI) enable monitoring of gene expression or disease progression in living organisms due to outstanding sensitivity.
In 1989 Berthold Technologies introduced its first low light imaging instrument – the LB 980 Luminograph. The first in-vivo gene expression experiments in plants and animals performed on this instrument date back to the year 1993.
BLI utilizes light emitted by luciferase enzymes. Today bioluminescence markers can be tailored to any gene, enabling detailed research of gene function. BFI utilizes proteins, which fluoresce under illumination, either applied as exogenous reagents or endogenously expressed. Both BLI and BFI have contributed to the understanding of disease mechanisms and the development of new treatments.
Cameras Todays generation of low light imagers from Berthold Technologies, the LB 983 NightOWL II, is equipped with cooled slow scan CCD cameras:
- Front-illuminated NC 320
is best suited for fluorescence applications with 3 million pixels for high resolution. Micro-lenticular array on top of CCD chip enhance light collection to maximum quantum efficiency of 85 %
- Back-illuminated NC 100
with high full well capacity for large dynamic range is best suited for luminescence applications. Midband coating enhances the quantum efficiency in the spectral range between 440 to 770 nm (80% QE) ideally to luciferase and GFP emissions. Efficient Peltier cooling keeps chip temperatures low, reducing the dark current and thereby enhancing the signal to noise ratio.
Applications*
Whole animals and plants can be imaged as well as blots, gels, microplates, cell culture dishes and arrays regardless luminescent or fluorescent markers are used. Optical calibration ensures the comparability of all images captured with the NightOWL.
Detection of weak light signals with CCD cameras can be exordinarily achieved with high quantum efficiencis and extremely low noise levels to enable long exposure times. The camera and cabinet design are the key to superior imaging performance, complemented by scientific evaluation software for quantification.
Cabinet
The NightOWL cabinet is extremly light-tight preventing any interferences from ambient light. NightOWL is the first imager with an motor-driven camera inside the cabinet. Positioning is reproducible with +/- 100 µm allowing very precise height correction. Optimum resolution and focus of the sample is achieved by automatic positioning of the camera according to the actual sample size. The NightOWL cabinet has enuogh space to install special light sources or to place transilluminators, heaters, coolers etc. These devices may even be switched on and off through the software and the built-in sockets. The flange option provides light-tight access to the inner part for tubings and cables.
Fluorescence excitation unit
The unique optical system from the LB 940 Mithras Multimode Reader has been integrated in the NightOWL II model. The light beam is kept constant for each fluorescent measurement, which is ideal with the ringlight epi illumination. If the ringlight is always set at the same height, the excitation energy on the sample will always be the same. The lamp energy can be set by a lamp factor in the software. This allows calibration of the imaging system for each fluorophor. Comparison of the amounts of different fluophors in one sample becomes very easy.
Fluorescence illumination
can be mounted in the cabinet for fluorescence applications. Different illumination systems such as gooseneck fibre optics or ringlights are available with the corresponding filters ranging from 300 nm up to 1100 nm. Tungsten halogen lamp (340-700 nm) is used for illumination.
Filter
Berthold Technologies offers a complete range of filters between 340nm up to 1100nm. Filters have to be used in fluorescence and as well BRET or FRET applications. If a transilluminator is used only an emission filter is needed. Berthold Technologies offers filter pairs for NightOWL for following standard dyes amongst others:
| Filter Recommendations |
|
Excitation |
Order No. |
Emission |
Order No. |
| GFP |
475/20 nm |
53183 |
520/10 nm |
39805 |
| Cy5 |
630/20 nm |
50097 |
680/30 nm |
49180 |
| Cy5.5 |
630/20 nm |
50097 |
700/20 nm |
50479 |
| Cy7 |
700/20 nm |
50475 |
780/20 nm |
50476 |
| ICG |
740/30 nm |
50480 |
820/30 nm |
50481 |
| dsRed |
530/20 nm |
38536 |
600/20 nm |
50477 |
| Qdot700® |
630/20 nm |
50097 |
700/20 nm |
50479 |
| Qdot800® |
630/20 nm |
50097 |
820/30 nm |
50481 |
| mCherry |
550/10 nm |
39796 |
620/10 nm |
40540 |
*Some techniques for generating and/or detecting light in biological subjects are patented and may require licences from third parties. Users are advised to independently determine for themselves wheter their activities infringe any valid patent.
|