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University of Connecticut Health Center Richard D. Berlin
Center for Cell Analysis & Modeling

CCAM Fluorescent Probe Information

Companies & Background Information

Visit the following sites for detailed information regarding fluorescence and fluorescent dyes.

Invitrogen - Manufacturer of numerous fluorescent probes. Site contains a comprehensive handbook, Molecular Probes, on background and technical information regarding their products.

Molecular Probes Handbook
Fluorescence Tutorials from Molecular Probes

Zeiss - see Technical Information -> Objecitves and Filter Sets Interactive fluorophore database, fluorophore data and chart.

bio status Limited - solutions for imaging, cytometry and screening

Enzo - tools for cellular analysis.

CCAM Events

Events Calendar

UV Dye Alternatives
(based on information from Invitrogen)

To our microscope users who use UV excited dyes such as DAPI or Hoechst as nuclear counterstains:
UV laser capabilities exsist only on the 780 microscope. This microscope is primarily reserved for live cell experiments that require the greater scanning control available on the 780. Experiments that rely exclusively on UV excited fluorophores will also have priority for the 780. However, there are now many choices for DNA binding dyes to use as nuclear counterstains that are available in virtually any excitation wavelength and emission color. Thus, we are requesting that those investigators performing confocal microscopy of fixed cells to switch to a visible wavelength DNA binding fluorophore and use the other microscopes whenever possible.

If you already have specimens prepared, you will of course be allowed to use the UV laser to image these specimens, however we will request that you try out some of the alternative DNA labels for future experiments.

Since the list of choices is a bit overwhelming, a few suggestions are given below. We strongly recommend visiting the Invitrogen web site (http://probes.invitrogen.com/) for additional information regarding DNA dyes.

Dye Class
Cyanine
 
Cell membrane-impermeant:
  • Dead-Cell stains -SYTOX (green, excitation 504/emission 523) is highly selective for the nucleus
  • High Affinity Stains - POPO, BOBO (excitation 570/emission 604, shows greater fluorescence enhancement upon binding to DNA, should provide brighter nuclear staining than propidium iodide.), YOYO, TOTO (excitation 642/emission 660), JOJO, LOLO (YOY-1 excitation 491/emission 509, is brighter but has weak cytoplasmic staining)
  • Nuclear Counterstains - PO-PRO, YO-PRO, TO-PRO (excitation 642/emission 661), JO-PRO
Cell membrane - permeant:
  • SYTO
Classic Nucleic Acid Stains
 
Intercalating dyes:
  • Ethidium Bromide
  • Propidium Iodide (excitation 535/emission 617,: used as nuclear counterstain for over 20 years; has weak cytoplasmic staining )
Minor-groove binders
  • DAPI
  • Hoechst dyes
Other nucleic acid stains
  • Acridine Orange
  • Hydroxystilbamidine

 

UV Dye Alternatives
(based on information from biostatus Limited )
CyTRAK Orange
  • excites at 488 nm but emits at 620 nm, you can co-excite with GFP and FITC and acquire with green (GFP, FITC) and far-red (PE, APC, Cy7)probes.
  • Stains the nucleus, but also defines the cytoplasmic area in both LIVE and fixed preparations
  • Membrane permeant
DRAQ5
  • Rapid staining of dsDNA/nuclei of LIVE or fixed cells
  • Excitation 647 nm line optimal, 488, 514, 568 and 633 nm lines, sub-optimal, emission >665 nm .
  • No fluorescence enhancement upon DNA binding
  • No photobleaching effect
DRAQ7
  • new far-red fluorescent DNA dye that ONLY stains the nuclei in DEAD and permeabilized cells
  • Combination with live cell dyes for dead/live discrimination
  • Excitation 633 & 647, emission >665 nm