Flow Cytometry

Multicolor Detection

Flow Cytometry optical interference filters are available both off-the-shelf and customized to your specifications to support a wide variety of fluorophores, tandems, multi-color cell sorting and analysis applications. While three or four fluorochrome analysis had become standard, simultaneous detection of more than 18 colors is now possible. As the number of lasers and fluorescent detection channels in instruments increases, applications require filters designed to maximize multiple dye discrimination.

The ability of modern multicolor flow cytometers to simultaneously measure up to 18 distinct fluorophores and to collect forward and side scatter information from each cell allows more high quality data to be collected with fewer samples and in less time. The presence of multiple fluorescing dyes excited by an increasing number of lasers places high demands on the interference filters used to collect and differentiate the signals. These filters are and typically a series of emission filters and dichroic filters designed to propagate the scattered excitation light and fluorescence signal through the system optics and deliver to the detectors.

Filter Selection Considerations

In multichannel systems, the emission filters’ spectral bandwidths must be selected not only to optimize collection of the desired fluorescent signal, but also to avoid channel cross talk and to minimize the need for color compensation that inevitably results from overlapping dye emission spectra. For example, suppose a system is being configured to simultaneously count cells that have been tagged with a combination of FITC and PE. If either of these dyes were used alone, a good choice of emission filter would be a 530BP50 for FITC and a 575BP40 for PE. 

These wide bands would very effectively collect the emission energy of each dye transmitting the peaks and much of each dye’s red tail. There is a possibility of two problems if used simultaneously. First, there will be significant channel cross talk since the red edge of the 530BP50 FITC filter would be coincident with the blue edge of the 575BP40 PE filter. Second, because the red tail of FITC overlaps with most of the PE emission, a high percentage of color correction will be needed to remove the input that the FITC tail will make to the signal recorded by the PE channel. A narrower FITC filter (XHC522/31) that cuts off at 535nm would provide good channel separation.  This will not however reduce the need for color compensation. To achieve this a narrower PE filter is required. By moving the blue edge of the PE filter to 565nm and the red edge to 585nm, Omega Optical recommends the resulting XHC574/26 filter, which transmits the peak of the PE emission spectrum. Because it is more selective for PE, it transmits much less of the FITC red tail. The result is that the need for compensation due to FITC in the PE channel will be greatly reduced.

Emission filters

The selection of emission band placement and width is made more complicated by the presence of multiple excitation lasers. If all of the sources are on simultaneously, then in addition to cross talk and color compensation concerns, the interference filters will need to block all excitation wavelengths to OD5 or greater. If the lasers are fired sequentially, the complexity is reduced since each emission filter need only provide deep blocking for the laser that is on at the particular time a given channel is collecting energy.

Dichroic Filters

Dichroic filters must exhibit very steep cut-on edges to split off fluorescent signals that are in close spectral proximity. Specifying the reflection and transmission ranges of each dichroic in a multichannel system requires complete knowledge of all the emission bands in the system and of their physical layout. Most often, obtaining optimal performance requires flexibility in the placement of the individual channels and the order in which the various signals are split off.

System Configuration

Filter recommendations for a custom multicolor configuration require a complete understanding of the system. This includes the dyes that are to be detected, the laser sources that will be exciting the dyes, the simultaneity of laser firings, and the physical layout of the detector channels. With this information, optimum interference filters can be selected that will provide the highest channel signal, the lowest excitation background, channel cross-talk and the need for color correction.

Since the emission spectra of fluorescent dyes tend to be spectrally wide, there is considerable spectral overlap between adjacent dyes. This becomes more the case as the number of channels is increased and the spectral distance between dyes is reduced. The result of this overlap is that the signal collected at a particular channel is a combination of the emission of the intended dye and emission contributions from adjacent dyes, Color compensation is required to substract the unwanted signal contribution from adjacent dyes. Through our work with researchers in the Flow Cytometry community we have established specific band shape characteristics that minimize the need for color compensation. By creating narrower pass bands and placing them optimally on emission peaks, we have reduced the relative contribution of an adjacent dye to a channels signal, thereby producing a purer signal with less need for color compensation. Flow Cytometry filters are manufactured to fit all research and clinical instruments including models by Accuri, Beckman Coulter, BD Biosciences, Bay Bio, ChemoMetec A/S, iCyt, Life Technologies, Molecular Devices, Partec and others. Our products are manufactured with the features required to guarantee excellent performance in Cytometry applications while keeping the price low.

Available sizes: 25, 15.8, and 12.5mm

Shape: Please specify round or square

Thickness: Ring ≤ 6.7mm

AOI: Please specify 45° or 11.25°  

Note to Instrument Designers:

With laser sources all of the output is linearly polarized. The dichroics’ performance will be different depending on the orientation of the lasers polarization. Omega Optical designs for minimum difference between polarization states, though it should be expected that the effective wavelength of the transition will vary by up to 10nm. Engineers at Omega Optical will gladly assist in discussing how to address this issue.

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