Photoblueing of organic dyes can cause artifacts in super-resolution microscopy

Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy

“…In prior work, we established that the in-house synthesized samples of 2 do not contain detectable 1 , and we have repeated the same analysis to show that 3 does not contain 2 or 1 . 14 …”

“… 13 Furthermore, the yield of this reaction had been estimated to be approximately 2% or less under all conditions examined previously. 13 , 14 However, despite the widespread use of the cyanine scaffold, the chemistry underpinning this “photoblueing” artifact had remained opaque. We recently found that the irradiation of the simplest pentamethine cyanine, 2 , leads to formation of the corresponding trimethine cyanine, 3 ( Figure 1 b).…”

“…We recently found that the irradiation of the simplest pentamethine cyanine, 2 , leads to formation of the corresponding trimethine cyanine, 3 ( Figure 1 b). 14 This reaction, involving the formal excision of the ethene diradical from a polymethine chain, has no precedent in the photochemical literature.…”

Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy

“…We found that Cy3 NHS ester was more favorable than ATTO 647N NHS ester as a fiducial marker, as the excitation and emission spectra of ATTO 647N embedded in a hydrogel were blue-shifted when they were exposed to excitation lasers for an extended period of time (Supplementary Figs. 36-37, see Supplementary Note 5 for details) 47 . Cy3 NHS ester also enabled three-dimensional registration between two z-stack images of an expanded brain slice acquired from different staining and imaging rounds (Supplementary Fig.…”

Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging

“…S7b and c † ). Recently, a study 36 published during the revision of our manuscript reported that Cy5 becomes dioxetane species (em max = 434 nm), photooxidative cleavage products (em max = 210 nm, 345 nm, 370 nm), and Cy3 (em max = 540 nm) after the blue-conversion, although the mechanism behind its blue-conversion has not been fully described. However, unlike their study, we observed that Cy5 produced the blue-converted species with their emission peaks majorly at 607 nm, 567 nm, and 523 nm ( Fig.…”