Skip to main content

Contact us!

Home

User account menu

  • Log in

Main navigation

  • Home
  • Product Types

    • Band-pass Filters
    • UV Filters
    • Long-pass and Short-pass Filters
    • Dichroics, Beamsplitters and Mirrors
    • Fiber Tip Optical Coatings
    • Neutral Density
    • Spectral Shaping and Photopic Filters
    • Transparent Conductive Oxides
  • Capabilities&OEM

    • General Capabilities
    • Design, Engineering and Manufacturing Capabilities
    • Quality Assurance and Metrology
    • Coating Methods and Materials
    • Research & Development Group
  • Applications

    • Aerospace and Defense
    • Amateur Astronomy
    • Astronomy
    • Color Imaging
    • Flow Cytometry
    • FRET
    • Industrial & Commercial
    • Laser Filters
    • Life Science
    • Machine Vision
    • Microscopy
    • Photolithography
    • Raman Spectroscopy
    • PCR and RT-PCR
  • Resources

    • International Distributors
    • Glossary
    • Library
    • Behind the Science Blog
  • About

    • Staff Directory
    • Omega's History
    • Careers
    • Directions
    • Events
    • COVID-19 response
  • Build-a-Filter
  • Contact
  • Store

    • Filter by Type

      • All Filters
      • Bandpass
      • Beam Splitter
      • Dichroic
      • Dual Band
      • Fluorescence Sets
      • Glass
      • Longpass
      • Mirrors
      • Neutral Density
      • Notch
      • Others
      • Quad Band
      • RapidBand
      • RapidEdge
      • Shortpass
      • Triple Band
      • Non-Filter Products
    • Order Information

      • Check Your Order

Flow Cytometry

Scatterplot and medical vial

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

flowcytometry_graph1.gif

 

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

flowcytometry_graph2.gif

Excitation LaserFluorophoresRecommended Filter


DAPI, AMCA, Hoechst 33342 and 32580, Alexa Fluor® 350, Marina Blue®424DF44


Alexa Fluor® 405, Pacific Blue™449BP38


Pacific Orange545BP40

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 525525WB20

405, 457 or 488    Qdot 565565WB20

405, 457 or 488    Qdot 585585WB20

405, 457 or 488    Qdot 605605WB20

405, 457 or 488    Qdot 625625DF20

405, 457 or 488    Qdot 655655WB20

405, 457 or 488    Qdot 705710AF40

405, 457 or 488    Qdot 800 for single color   800WB80

405, 457 or 488    Qdot 800 for multiplexing with Qdot™ 705840WB80

488GFP (for separation from YFP, also for separation from Qdots 545 and higher)  509BP21

488GFP, FITC, Alexa Fluor® 488, Oregon Green® 488, Cy2®, ELF®-97, PKH2, PKH67, Fluo3/Fluo4, LIVE/DEAD Fixable Dead Cell Stain  525BP30

488GFP, FITC, Alexa Fluor® 488, Oregon Green® 488, Cy2®, ELF-97, PKH2, PKH67, YFP535DF45

488YFP (for separation from GFP)  550DF30

488 or 532PE, PI, Cy3®, CF-3, CF-4, TRITC, PKH26   574BP26

488 or 532PE, PI, Cy3®, CF-3, CF-4, TRITC, PKH26   585DF22

488 or 532Lissamine Rhodamine B, Rhodamine Red™, Alexa Fluor® 568, RPE-Texas Red®, Live/Dead Fixable Red Stain   614BP21

488 or 532Lissamine Rhodamine B, Rhodamine Red™, Alexa Fluor® 568, RPE-Texas Red®, Live/Dead Fixable Red Stain   610DF30

488 or 532Lissamine Rhodamine B, Rhodamine Red™, Alexa Fluor® 568, RPE-Texas Red®, Live/Dead Fixable Red Stain  630DF22

488 or 532PE-Cy5®  660DF35

532PE-Cy5.5®, PE-Alexa Fluor® 700  710DF40

633APC, Alexa Fluor® 633, CF-1, CF-2, PBXL-1, PBXL-3  660BP20

633Cy5.5®, Alexa Fluor® 680, PE-Alexa Fluor® 680, APC-Alexa Fluor® 680, PE-Cy5.5®  710DF20

633Cy7® (for separation from Cy5® and conjugates)  740ABLP

633PE-Cy7®, APC-Cy7®  748LP

633Cy7®, 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.
 

ApplicationRecommended 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.

View Products
Fluorescence Sets
Contact Us
Contact us for more information
Additional Reading
Optical Density + Flow Cytometry
Fluorescence Sets Flyer
Imaging of Intrinsic Fluorescence
Omega Optical, LLC

21 Omega Drive
Brattleboro, VT 05301
USA
U.S. Toll-Free: 1-866-488-1064
International: +1 (802) 251-7300
Sales and Support: sales@omegafilters.com
Press: marketing@omegafilters.com

Terms and Conditions
Site Map
Careers
Privacy Policy
Events
ITAR Registered
ITAR Registered
ISO 9001:2015 Certified
ISO 9001:2015 Certified
Made in the USA
Made in the USA
Twitter Facebook LinkedIn

Products listed on the website may be limited to the current inventory. Omega cannot guarantee their availability in the future. Contact us for details, or to request pricing of a custom part.

© 2014 - 2021 Omega Optical, LLC. All Rights Reserved.