![]() ![]() These color-separation masks are optimized for maximal inclusion of localizations and minimal crosstalk between the channels. By applying binary masks to exclude or include populations of the intensity distribution, the colors are assigned to each paired localization before multicolor rendering ( Figure 1d). Depending on their emission spectra, localizations from each dye display a spatially distinct population within the 2D intensity histogram of long and short channel intensity values ( Figure 1c). By using a custom open-source software tool (SD-Mixer2 (16)), the single-molecule localizations are “paired”, while nonpaired localizations, from which a large part is random noise, are excluded ( Figure 1b). (12−15) The SD-mode uses spectrally overlapping fluorophores excited by a single laser line and a simple dichroic-based emission splitter to image short and long wavelength components of the emission on two sides of the same camera ( Figure 1a). We recently developed a robust multicolor dSTORM approach that achieved high localization precision with fast, simultaneous, dual-color acquisition based on spectral demixing (SD). ![]() (8,9) However, these improved multicolor DNA-PAINT approaches were still limited by the fluid exchange that was required for sequential acquisition. By optimizing the DNA sequences and buffer conditions, the acquisition time was dramatically shortened. The combination of long exposure times that are needed to average out the noise from diffusing imagers and the sequential color channel acquisition leads to long total acquisition periods. Each fluid exchange, including the washing steps, is prone to cause extended sample drift through thermal or pressure gradients and therefore requires precise drift-correction and multichannel registration. ![]() Exchange-PAINT, however, relies on the complete exchange of the imagers containing buffer in between the acquisition phases of each channel. (4) This approach reaches very high localization precisions and is insensitive to chromatic errors by using the same dye in each acquisition round. In the recently developed “Exchange-PAINT” method, multiplexing is realized by sequential fluid exchange of the imaging buffer that contains distinct imagers for each channel. ![]() Multicolor DNA-PAINT can be achieved by using multiple docking/imager strand pairs with orthogonal sequences. We demonstrate high localization precision (3–6 nm) and multicolor registration of dual- and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures. By using newly designed probes and a novel multichannel registration procedure, we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). However, multicolor DNA-PAINT has primarily been realized by “Exchange-PAINT”, which requires sequential exchange of the imaging solution and thus leads to extended acquisition times. The oligonucleotide-based SMLM approach “DNA-PAINT” robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. ![]()
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