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.
- 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:
|Classic Nucleic Acid Stains
- Ethidium Bromide
- Propidium Iodide (excitation 535/emission 617,: used as nuclear counterstain for over 20 years; has weak cytoplasmic staining )
Other nucleic acid stains
- Acridine Orange
UV Dye Alternatives
(based on information from biostatus Limited
- 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
- 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
- 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