Detection

Conjugates

Fluorescent antibody conjugates – versatile, bright and stable tools for imaging

Atto dyes are a comprehensive series of fluorescent dyes, which:

  • cover the entire spectrum of visible light
  • match the most common output wavelengths of excitation light sources, especially mercury and xenon lamps, but also common lasers
  • provide brightest fluorescence with narrow fluorescence emission spectra
  • show extraordinary photostability

For details click here. These properties enable the parallel imaging of different targets in cells, tissues or other biological samples. Figure 1 shows characteristic absorbance- and emission spectra of the atto dye family.


Figure 1: Absorption and emission spectra of Atto 520, Atto 565, Atto 590, Atto 610, Atto 655 and Atto 680

Antibody and other fluorescent conjugates

We optimized the labelling of antibodies based on the innovative series of atto labels to provide high quality conjugates with ideal brightness and low background.

Secondary antibodies, the general work horses for immunochemistry, are offered as conjugates with several of our atto labels as well as Mega labels, which are characterized by a large gap between excitation and emission maxima. For most of the atto labels, streptavidin and biotin conjugates are also available.

Atto labels are available as reactive succinimidyl esters and maleimides, enabling straight-forward coupling by common procedures.


Figure 2: Human endothelial cells: Vimentin stained with mouse anti-vimentin and Atto 550 anti-mouse IgG (green), cadherine

Multiple staining with single excitation light

Parallel staining of different structures or target molecules in biological tissues or other samples can be complicated when fluorescence signals overlap. Thus labels with clearly distinct emission spectra are prefered. For this purpose the wide spectrum of labels offers a variety of suitable combinations.

Where laser light is used for excitation, as in the case of confocal microscopy, the suitable excitation of labels with distinct emission spectra may require two or more different lasers. This is because for most common labels (e.g. FITC, TRITC, Cy3TM, Cy5TM, Alexa labels) excitation and emission maxima are relatively close. As the laser wavelengths are far away from the maximum excitation of these dyes, fluorescence imaging works at a low efficiency.. Each additional laser source increases costs significantly, thus multiple fluorescent staining with just one excitations source would be a convenient and cost efficient methodology and provides increased flexibility.

Another potential problem with multiple fluorescent staining is the overlap of emission wavelength of dye (A) with excitation of dye (B), resulting in a lower signal intensity of dye A.

In contrast to above mentioned labels, Mega labels are characterized by a large gap between excitation and emission wavelength. All of them can be excited with argon lasers or other widely used light sources, but their fluorescence varies. Mega labels, if combined with atto dyes, enable the visualization of different structures with just one excitation source. Figure 2 shows an application of an Atto 488- and a Mega 485 labelled antibody in confocal microscopy. But such combinations can also be used on conventional fluorescence microscopes, using mercury lamp, even using standard filter sets optimized for conventional fluorophores.


Table 1: Mega labels and their spectroscopic properties

Name λmax abs [nm] λmax em [nm] εmax [l/mol cm]
Fluorescent Red Mega 480 480 640 40000
Fluorescent Red Mega 485 485 559 20000
Fluorescent Red Mega 500 500 612 90000
Fluorescent Red Mega 520 520 664 50000

Rat Stomach
Figure: Rat stomach: Actin stained with mouse anti–smooth muscle α-actin antibody and Atto 488 anti-mouse IgG (green), cytokeratin stained with polyclonal rabbit anti-cytokeratin and Mega485 anti-rabbit IgG (yellow), both labels are excited by just one laser (Argon laser).

Rat Stomach
Figure: Rat stomach: Actin stained with mouse anti–smooth muscle α-actin antibody and Atto 488 anti-mouse IgG (green), cytokeratin stained with polyclonal rabbit anti-cytokeratin and Mega520 anti-rabbit IgG (red), counterstained with DAPI (blue). Courtesy of Jacob Zbaeren, Inselspital Bern, Switzerland

Stability

For well-known and commonly used labels like fluoresceins, photostability is limited. In various applications, esp. in immunofluorescence, bleaching fluorescence intensity is a mayor concern, limiting quality and sensitivity of imaging. Photostability becomes even more important with the increasing use of laser excitation, confocal and two-photon illumination, and the increasing sensitivity of methods down to the single molecule level. Also the tracking of processes over time in living cells requires stable dyes.

Atto labels, in contrast to some of the most widely used dyes, have more rigid structures, which makes them more photostable. Atto conjugates are exceptionally stable, in several cases even outperforming the dyes, that were sofar considered the best choice in place.


Figure: Photostability of a) Atto 550 and b) Alexa 555 and c) Cy3TM at laser illumination (HeNe laser, 10 min)


Figure: Photostability of Atto labels and other dyes. Relative intensity after 5 min laser illumination with commonly used lasers. FITC, Alexa Fluor 488, Atto 488, Alexa Fluor 594 and Atto 590 excited with 488 nm, the others with 543 nm laser line. Beam extension has been reduced. With these parameters FITC is bleached immediately and to such an extent, that accurate quantification was practically impossible. Thus this bar represents a gross estimation, that less than 1% of fluorescence signal are seen after 5 min. The atto labels (blue bars) show very good photostability compared to other labels.

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