1-Piece Ostomy Pouch,One Piece Ostomy Bags,One Piece Ostomy Pouch,1 Piece Ostomy Bag Henan Maidingkang Medical Technology Co.,Ltd , https://www.mdkmedicales.com In the multi-function microplate reader, we have two technical options for separating monochromatic light of a specific wavelength - a filter or a grating. Filter systems tend to have better sensitivity than gratings because of their better light transmission and wider bandwidth. The benefit of a grating system is the flexibility to provide wavelength selection without the need to purchase or install filters of a specific wavelength. Despite this, many users have found that many applications do not achieve the desired results on a raster system, and some applications may not even be possible at all.
On the microscope, we selected two filters with a wavelength of 480 nm, a bandwidth of 30 nm, a wavelength of 535 nm, and a bandwidth of 45 nm to detect the signals of mTFP1 and YFP, respectively.
On CLARIOstar, we used LVF gratings to select the same wavelength and bandwidth as the starting point. After optimization, we obtained the same FRET efficiency on the microplate reader as the confocal microscope, thus achieving microscopic analysis to high-throughput microplates. Seamless transformation of analysis.
The revolution of multi-function microplate reader technology - excellent performance of LVF grating in FRET and BRET analysis
For example, in the detection of fluorescent proteins such as GFP, mKate, mOrange and CFP-YFP, the grating type microplate reader does not achieve the desired effect, especially in the dual emission analysis such as FRET and BRET. One obvious reason why grating-type microplate readers can't achieve these applications is that we need more than 30nm bandwidth. In fact, in some BRET analysis, we need a bandwidth of 60 to 100nm. At present, traditional gratings often have narrow fixed bandwidth or The limited adjustable bandwidth (up to 30nm) does not meet the test requirements at all. Another reason is that the signal of these applications is weak, and the emitted light is far less than the commonly used dyes, such as FITC or rhodamine, and the light transmission efficiency of the grating is far lower than the filter. The light intensity loss is too large, which is bound to reduce the sensitivity. .
In order to satisfy both the flexibility of wavelength selection and the higher sensitivity, engineers at BMG LABTECH in Germany have developed a revolutionary linear gradient filter grating, referred to as the LVF grating (Figure 1).
LVF grating technology consists of a linear gradient short pass filter and a linear gradient long pass filter. When two linear gradient filters are properly arranged, we can not only separate the light of a specific wavelength, but also achieve 8 to 100 nm. Any choice of bandwidth. LVF gratings make it easy to perform analyses that previously required filters, such as FRET and BRET.
In addition to the wider bandwidth, there are two reasons for the high sensitivity of LVF gratings. One is that the LVF grating is based on the transmission principle of the filter rather than the diffraction of the conventional grating. Second, due to the difference in optical path design, the LVF grating passes through the direct Optical components replace fiber optics to conduct signals (fiber conduction causes loss of light intensity); therefore, LVF gratings provide 5 to 10 times more light than conventional diffraction gratings, greatly increasing sensitivity. At present, LVF grating has been applied to BMG's latest generation multi-function microplate reader - CLARIOstar.
A great challenge for researchers is how to convert microscope-based fluorescent protein analysis into microplate-based analysis. Now with the LVF grating we can choose the same wavelength and bandwidth as the microscope on the multi-function microplate reader, thus achieving a seamless connection between the microscope and the microplate reader.
For example, Figure 2 shows the detection of FRET between mTFP1 and YFP in HEK293 cells using confocal microscopy and CLARIOstar, respectively.
Another explanation is that the application of LVF gratings comparable to filters is BRET. Like FRET, BRET also needs to detect two transmitted optical signals at the same time. The difference is that it requires a larger bandwidth (60 to 100 nm). Therefore, due to bandwidth. Limitation, the traditional grating type microplate reader is basically unable to achieve BRET analysis. However, CLARIOstar's LVF gratings can achieve comparable results for the filters (Figure 3).
LVF grating is the only microplate reader technology on the market that can achieve arbitrary wavelength and arbitrary bandwidth selection. This breakthrough design enables CLARIOstar to realize arbitrary fluorescence and luminescence detection, so that "any wavelength, any bandwidth, and any detection" "Everything can be done easily!