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Flow Cytometry

Multicolor Detection

The ability of modern multicolor flow cytometers to simultaneously measure up to 20 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 typically a series of emission filters and dichroic mirrors designed to propagate the scattered excitation light and fluorescence signal through the system optics and deliver to the detectors.

Emission Filters

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. See graph 1

Graph 1

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 signifi cant 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 (525BP30) that cuts off at 535 nm would provide good channel separation. See graph 2

Graph 2

 

 

Excitation Laser Fluorophores Recommended Filter
DAPI, AMCA, Hoechst 33342 and 32580, Alexa Fluor® 350, Marina Blue® 424DF44
Alexa Fluor® 405, Pacific Blue™ 449BP38
Pacific Orange 545BP40
Quantum Dot Emission Filters The 405 laser is optimal for excitation of Quantum Dots, but the 488 line laser can also be used.
405, 457 or 488 Qdot 525 525WB20
405, 457 or 488 Qdot 565 565WB20
405, 457 or 488 Qdot 585 585WB20
405, 457 or 488 Qdot 605 605WB20
405, 457 or 488 Qdot 625 625DF20
405, 457 or 488 Qdot 655 655WB20
405, 457 or 488 Qdot 705 710AF40
405, 457 or 488 Qdot 800 for single color 800WB80
405, 457 or 488 Qdot 800 for multiplexing with Qdot™ 705 840WB80
488 GFP (for separation from YFP, also for separation from Qdots 545 and higher) 509BP21
488 GFP, FITC, Alexa Fluor® 488, Oregon Green® 488, Cy2®, ELF®-97, PKH2, PKH67, Fluo3/Fluo4, LIVE/DEAD Fixable Dead Cell Stain 525BP30
488 GFP, FITC, Alexa Fluor® 488, Oregon Green® 488, Cy2®, ELF-97, PKH2, PKH67, YFP 535DF45
488 YFP (for separation from GFP) 550DF30
488 or 532 PE, PI, Cy3®, CF-3, CF-4, TRITC, PKH26 574BP26
488 or 532 PE, PI, Cy3®, CF-3, CF-4, TRITC, PKH26 585DF22
488 or 532 Lissamine Rhodamine B, Rhodamine Red™, Alexa Fluor® 568, RPE-Texas Red®, Live/Dead Fixable Red Stain 614BP21
488 or 532 Lissamine Rhodamine B, Rhodamine Red™, Alexa Fluor® 568, RPE-Texas Red®, Live/Dead Fixable Red Stain 610DF30
488 or 532 Lissamine Rhodamine B, Rhodamine Red™, Alexa Fluor® 568, RPE-Texas Red®, Live/Dead Fixable Red Stain 630DF22
488 or 532 PE-Cy5® 660DF35
532 PE-Cy5.5®, PE-Alexa Fluor® 700 710DF40
633 APC, Alexa Fluor® 633, CF-1, CF-2, PBXL-1, PBXL-3 660BP20
633 Cy5.5®, Alexa Fluor® 680, PE-Alexa Fluor® 680, APC-Alexa Fluor® 680, PE-Cy5.5® 710DF20
633 Cy7® (for separation from Cy5® and conjugates) 740ABLP
633 PE-Cy7®, APC-Cy7® 748LP
633 Cy7®, APC-Alexa Fluor® 750 787DF43

 

Flow cytometry filters are manufactured to fit all instruments including models by Accuri, Beckman Coulter, BD Biosciences, Bay Bio, ChemoMetec A/S, iCyt, Life Technologies, Molecular Devices, Partec and others. Our flow cytometry filters are manufactured with the features required to guarantee excellent performance in cytometry applications while keeping the price low.

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 of 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.

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 detection 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 subtract 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 channel's signal, thereby producing a purer signal with less need for color compensation.

 

Application Recommended Filter
Extended reflection longpass; Reflects 451 nm, 457 nm, 477 nm, 488 nm and UV laser lines, Transmits > 525 nm. 505DRLPXR
Shortpass; Separation of FITC from PE. 560DRSP
Separation of Mithramycin from Ethidium Bromide. 575DCLP
Separation of APC from dyes with shorter wavelength. 640DRLP
Separation of PE-Cy5® and PE-Cy5.5. 680DRLP
Separation of APC from APC-Cy5.5® or APC-Cy7®. 690DRLP
Separation of PE and Cy5® from PE-Cy5.5® or PE-Cy7®. 710DMLP
Separation of Cy5.5® from Cy7® and their conjugates. 760DRLP

 

Polarization

Polarization is an important parameter in signal detection. In an optical instrument that utilizes a highly polarized light source such as a laser to generate signal in the form of both scatter and fluorescence, there will be polarization bias at the detector. Many factors such as the instrument's light source, optical layout, detector, mirrors and interference filters affect the degree of polarization bias.

Dichroic mirrors are sensitive to polarization effects since they operate at off-normal angles of incidence. Omega Optical's dichroics are designed to optimize steep transition edges for the best separation of closely spaced fluorophores, while minimizing the sensitivity to the polarization state of the incident energy.

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.