Optimizing light throughput in image cytometry
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| Year of publication | 2007 |
| MU Faculty or unit | |
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| Description | Image cytometry applications often suffer from the lack of light due to the use of fluorescence mode. Low light levels typically cause poor signal-to-noise ratio or long exposure times. Signal level in fluorescence set-up is determined by many factors, such as power of the light source and its spectral characteristics, transmission curves of excitation/emission filters, transmission curves of and dichroic/polychroic mirrors, excitation/emission spectra of imaged fluorochrome(s), numerical aperture of the objective and its transmission curve, quantum efficiency curve of the light detector. Thus, resulting light throughput is given by the composition of spectral characteristics (functions of wavelength) of many components. Proper computation and optimization of light throughput is a non-trivial task that requires knowledge of all these curves for all available hardware components as well as all available fluorochromes. Such computation is unfortunately not performed by the users and is typically replaced, for example, by visual comparison of fluorochrome spectral curves with spectral curves of optical filters when choosing fluorochromes or filters. Quantum efficiency curve of the detector or spectral characteristics of the white light source are usually not taken into account at all. In our laboratory, we have developed software for the computation of the overall light throughput of an optical set-up. It can assist users in choosing hardware components for their system as well as choosing suitable fluorochromes. For each component the user enters the available choices (e.g. list of available optical filters, list of available fluorochromes, list of available cameras in camera-based systems, etc.) and the computer suggests a list of suitable combinations ordered according to the resulting signal level. Contrast between signal from emitted fluorescence and signal from remaining excitation light is also taken into account. The software runs on a web server and can be used from a web browser for: choosing a suitable fluorochrome when ordering probes or staining kits, choosing suitable optical filters (excitation/emission) to be installed or purchased, choosing suitable mirrors (dichroic/polychroic) to be installed or purchased, choosing suitable light detector (especially in camera-based systems), comparing light throughput and contrast for fluorochromes imaged simultaneously, comparing light throughput and contrast for different combinations of hardware components available in the lab. This research has been supported by the Ministry of Education of the Czech Republic (Projects No. MSM0021622419 and No. LC535). |
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