Which dyes work?

It is a commonly held misconception that for a fluorophor to work with the Dark Reader it must have an excitation maximum between 420 - 500 nm and an emission maximum above ~ 520 nm.

While these are useful guidelines, it should be emphasized that the DR can also be effectively used to detect fluorophors that have maxima well outside outside these ranges.

The more general criteria for visualizing a fluorophor with a Dark Reader transilluminator or hand lamp are:

  • a PORTION of the excitation spectrum (ex) is between about 420 - 500 nm and
  • a PORTION of the emission spectrum (em) is above about 520 nm.

Excitation spectra of SYBR Green and RFP
For example, consider the ex/em maxima of SYBR Green (494/521 nm) and RFP (558/583 nm) as shown to the left.

From the ex/em information alone, one would predict SYBR Green to work well (correct!), but RFP to be feeble (incorrect!).

In fact, RFP works very well - see the transgenic fish movie! The reason is evident from a more detailed consideration of the RFP excitation spectrum (left) which reveals substantial excitation between 400 - 500 nm.
Excitation spectra of SYBR Green and RFP
The broader ex/em criteria outlined above encompass many commonly used fluorophors, besides the popular SYBR and SYPRO stains such as:

Pro-Q® Diamond phosphoprotein stain,
Pro-Q Emerald 488 glycoprotein stain,
fluorescein and rhodamine derivatives,
Cy2 and Cy3,
GFP variants such as EGFP, EYFP and RFP/dsRed,
alkaline phosphatase substrates such as AttoPhos® and ECF®,
dimeric cyanine stains such as YOYO® and TOTO®
many of the Alexa® dye series
Deep Purple protein stain

There are numerous other dyes that can be used effectively with Dark Reader units and this list is by no means exhaustive.
Clare Chemical Research

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