technologies
Please find below a description of the technologies behind FLIM and its main application FRET, as well as the technologies we use in the development of our intensified CCD camera's and other products.
technology categories
FLIM, Fluorescence Lifetime Imaging Microscopy
FLIM is the abbreviation for "Fluorescence Lifetime Imaging Microscopy" and is the technique that maps the spatial distribution of lifetimes within microscopic images of fixed as well as of living cells. The fluorescence lifetime is the signature of a fluorescent material; it is the exponential decay in emission after the excitation of a fluorescent material. In other words, the lifetime is the average time that fluorescent molecules spend in the excited state.
In the picture two lifetime images (combined with intensity) show that FLIM can be used to detect FRET. Two centrosomal proteins (microtubule organising centers) are labelled with Alexa488 and Alexa568. In the left picture both fluorophores are present and do show FRET (so the conclusion can be drawn that both proteins interact with each other). Namely the lifetime of Alexa488 at the centrosomes is lower (yellow; 2,3ns) than outside the centrosomes (red; 2,7ns). In the right picture only Alexa488 is present and no FRET is shown (red; 2,7ns). Courtesy of Prof. Diaspro, Genua, Italy.
FRET, Fluorescence Resonance Energy Transfer
FRET is the abbreviation for "Förster or Fluorescence Resonance Energy Transfer" and is a useful tool to quantify molecular dynamics like interactions of two fluorophores by microscopy. The proteins under investigation are labelled with donor fluorophores or acceptor fluorophores. Interaction between the two fluorophores is accompanied by direct energy transfer from donor to acceptor (FRET). When FRET occurs, it means that the two proteins of interest are in such close proximity that they can interact with each other.
Image intensifier
An image intensifier is a device that intensifies low light-level images to light levels that can be seen with the human eye or can be detected by a video camera. An image intensifier is a vacuum tube, having an input window on which inside surface a light sensitive layer called the photocathode has been deposited. Photons are absorbed in the photocathode and give rise to emission of electrons into the vacuum. These electrons are accelerated by an electric field to increase their energy and focus them. After multiplication by an MCP (multi channel plate), that is applied in second and third generation image intensifiers, these electrons will finally be accelerated towards the anode screen. The anode screen contains a layer of phosphorescent material that is covered by a thin aluminium film. When striking the anode the energy of the electrons is converted into photons again. Because of the multiplication and increased energy of the electrons the output brightness is higher as compared to the original input light intensity.
CCD camera sensitivity
At low light levels standard CCD/CMOS cameras are not sensitive enough to capture useful images. There are ways to increase the sensitivity of such cameras. The first method is to allow the CCD to integrate for much longer times. In order to prevent high background noise, CCD cooling is applied when using long exposure times. A second method is to use an image intensifier to boost the input signal.
(Intensified) High speed camera
High-speed cameras require a high brightness of the object because of the relative short exposures of the camera. The higher the frame rates the shorter the exposure time per frame. In many applications an increase of the illumination can compensate the shorter exposure time per frame. However, in some applications the object itself is emitting light at an intensity that is too low for high-speed imaging (combustion research, imaging of dynamic phenomena in fluorescent biological cells, low intensity PIV). In other applications the heat generated by a powerful light source can have an unacceptable influence on a measurement (Micro Fluidics).