Spectral demixing

Simultaneous Multicolor Nanoscopy
Despite the different Innovative solutions that have emerged recently1, SMLM lacks straightforward multi-color capability due to significant technical challenges: the different photoswitching characteristics of fluorophores which require adapted buffer specific for the different spectral ranges; significant chromatic aberration at the nanoscale which hampers the reconstruction at such localization precision and accuracy; the uncontrollable drifts between colors in conventional multicolor sequential acquisitions.

Different methods have been proposed by the SMLM community, based on spectral demixing algorithm2-7.  In this scenario, Abbelight focused on the development of an easy and robust ratiometric approach to spectrally separate the contribution of red emitting dyes.


The emission light is splitted with a long pass dichroic into two separate pathways before reaching the two cameras; the number of photon is evaluated for each detection on both cameras; a photon ratio R is then calculated for each detection; the detection is then assigned to a particular color on the basis of the measured R and the a priori knowledge of the characteristic ratio for each fluorophore.

A three-step protocol is therefore required:

1.Calibration on single color samples. To measure the characteristic intensity ratio distribution R for each fluorophore, necessary to identify and hence correctly assign a particular detection to a proper l channel. Single color measurements are also crucial to evaluate crosstalk, the percentage of detections that are assigned to the wrong color.

2.Detection on multicolor samples. As usual in SMLM strategy, is based on the localization of PSFs and a fitting procedure to measure the x,y,z positions with nm accuracy.

3. λ assignment before final reconstruction. This step becomes straightforward by choosing the ratio ranges with respect to crosstalk and rejected molecules. The goal is to minimise crosstalk to best separate different structures, while rejecting the least of detections for a good quality reconstruction.

(A) Optical scheme ; (B); example of PSF recordings on Cam1 and Cam2 (C top), zoom in (C middle) and 2D gaussian PSF representation to evaluate centre position and photon counting (C bottom); photon ratio distribution and example of ranges chosen for l assignement (D).

Which fluorophores can I use?

Any red emitting fluorophores can be used. We characterised and recommend AF647, CF660, CF680 dyes as they present the best and most similar photoswitching characteristics.

Does it work in 3D?

Multicolor three-dimensional nanoscopy is possible with the spectral demixing strategy here presented, allowing important insight into the relative organization of cellular structures. Abbelight strategy to obtain the axial information is to deform the PSF by introducing two cylindrical lenses in the optical path (fig.2). Via astigmatism the z information is encoded in the PSF deformation: by measuring the deformation the z position of the emitter from the focal plane can thus be retrieved.

Which system can I use?

This SMLM modality is available in the dedicated product SAFe RedSTORM as well as abbelight highest range product SAFe 360.

3D spectral demixing examples: two cylindrical lenses (CL) are inserted in the two detection pathways before reaching the cameras (as shown in the optical scheme) in order to encode the axial information within the PSF deformation; An example of 3D spectral demixing reconstruction is presented in the image on the right: cos7 cells are labelled for tubulin-AF647 (greyscale) and Clathrine-CF680 (red); below the.cross-section of the line as indicated in the image are also shown. Scale bar 80nm.


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