How to Choose a Label
To obtain the best possible results utilizing a fluorescent label several factors have to be considered.
First is the source of excitation: To reduce interference due to autofluorescence of the sample an excitation wavelength above 550 nm or even 600 nm is preferable. Besides reduced background, the red spectral region is also advantageous when working with living cells, because damage is reduced.
Secondly the label should show strong absorption at the excitation wavelength, as well as high fluorescence quantum yield. The product of extinction coefficient and fluorescence quantum yield is often called the „brightness“ of a dye.
The fluorescence efficiency of dyes is highest in the blue and green region of the spectrum. Here the quantum yield reaches in some cases almost the theoretical limit of 100 %. Towards longer wavelengths the efficiency of the emission drops drastically, in particular so in aqueous solution. However, ATTO-TEC has been able to develop labels that show high quantum yield even around 650 nm. For instance: ATTO 647N fluoresces in aqueous solution twice as strong as the older cyanine dye Cy5TM.
Relative fluorescence intensities of ATTO 647N and Cy5TM. Aqueous solution in PBS with an
absorbance of 0.04 at the respective absorption maximum in a 1 cm cell. Excitation of fluorescence
at the intersection of both absorbance spectra (same absorption) at 22 °C.
Finally the emission spectrum of the label should match the transmission of the applied filter set. The filter set, in turn, must be chosen such that it rejects the excitation light scattered by the sample, yet transmits the fluorescence as effectively as possible.
For example, when using a diode laser of wavelength 635 nm as excitation source and a filter set with high transmittance between 650 nm and 750 nm, ATTO 647N would be a very good choice. As can be seen from the list of ATTO-labels, ATTO647N has a high extinction coefficient at 635 nm, a wavelength close to the maximum of the absorption curve, as well as an excellent quantum yield of fluorescence (ηfl = 65 %).
The table below provides an overview of some frequently used excitation sources and recommended ATTO-labels.
| Light Source | Emission Line | Suitable Dye |
|---|---|---|
| Mercury arc lamp | 365 nm 405 nm 436 nm 546 nm 577 nm |
ATTO 390 ATTO 425, ATTO 430LS ATTO 425, ATTO 430LS, ATTO 465 ATTO 550, ATTO 565 ATTO Rho12, ATTO Rho101, ATTO 590 ATTO Rho13, ATTO 594, ATTO 610, ATTO Rho14 |
| Argon-Ion laser | 488 nm 514 nm |
ATTO 488, ATTO 490LS ATTO 514, ATTO 520 ATTO 514, ATTO 490LS, ATTO 520 ATTO 532, ATTO 542 |
| Nd:YAG laser, frequency doubled |
532 nm | ATTO 532, ATTO Rho6G, ATTO 542 ATTO 550, ATTO 565, ATTO Rho11, ATTO Rho12 |
| He-Ne laser | 633 nm | ATTO 643, ATTO 633, ATTO 647, ATTO 647N, ATTO 655 |
| Krypton-Ion laser | 647 nm 676 nm |
ATTO 643 ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680 ATTO 680, ATTO 700, ATTO 725, ATTO 740 |
| Diode laser | 635 nm | ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655 |
Properties of Fluorescent Labels
It is to be noted, however, that besides the already discussed optical considerations other factors may be important selecting a label, e.g. pH-dependence of the optical and chemical properties of the dye, solubility, photo- and chemical stability, size of chromophore or length of the linker, and several others concerning/matching the demands for the application in hand. These properties can be highly relevant with respect to the suitability of dyes as labels.
Most important, the dye must remain intact during irradiation. Many common labels, e.g. Fluorescein derivative FITC, show very low photo-stability. As a result sensitivity and quality of imaging are limited if high-intensity laser excitation is used and processes are to be observed over long periods of time. This is a serious draw-back with microscopy and other techniques based on the confocal principle, e.g. in single cell detection applications. In contrast to some widely used older dyes, the new patented ATTO-labels are designed to be much more stable under prolonged irradiation.
Photo-stability of ATTO 655 compared to common Cy5TM in water.
Radiation of a 250 W tungsten-halogen lamp focussed into a 1 cm cell.
Absorbance vs. time of illumination.
Many common fluorescent labels deteriorate even without any irradiation (i.e. in the dark), in particular when exposed to small concentrations of ozone present in the laboratory atmosphere. Under identical conditions of ozone exposure the dyes ATTO 647N and ATTO 655 last up to 100 times longer than dyes like the cyanine dyes Cy5TM and Alexa647TM. This is very important in microarray applications, where the dye molecules are located at the surface and thus are directly exposed to the atmosphere.
Nowadays, compact and powerful laser diodes are covering the whole visible and near infrared part of the spectrum. They found their way into many applications/devices as very efficient excitation sources and more and more substitute classical light sources.
If there is no label available with an absorption maximum exactly matching the wavelength of the excitation source, a label with slightly longer wavelength should be chosen. The absorbance will be smaller, but the larger difference between excitation wavelength and fluorescence spectrum, which is always independent of excitation wavelength, has the advantage of better discrimination against scattered excitation light.