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

Helmerich, Dominic A.; Beliu, Gerti; Matikonda, Siddharth S.; Schnermann, Martin J.; Sauer, Markus

doi:10.1038/s41592-021-01061-2

Cited by 58 publications

(54 citation statements)

References 24 publications

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“…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 …”

Section: Methodsmentioning

confidence: 99%

“… 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).…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.

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“…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 …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…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.…”

Section: High-precision Registration Of Images Using Fluorophore Nhs-ester Staining As Fiducial Markersmentioning

confidence: 99%

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

Sim

et al. 2021

Preprint

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Nanoscale imaging of all anatomical structures over whole vertebrates is needed for a systematic understanding of human diseases, but this has not yet been achieved. Here, we demonstrate whole-ExM, which enables nanoscale imaging of all anatomical structures of whole zebrafish larvae by labeling the proteins of the larvae with fluorophores and expanding them four-fold. We first optimize the fluorophore selection and labeling procedure to visualize a broader range of anatomical structures. We then develop an expansion protocol for zebrafish larvae having calcified body parts. Through this process, we visualize the nanoscale details of diverse larvae organs, which have corresponding organ counterparts in humans, over the intact larvae. We show that whole-ExM retains the fluorescence signals of fluorescent proteins, and its resolution is high enough to visualize various structures that can be imaged only with electron microscopy. Whole-ExM would enable the nanoscale study of the molecular mechanisms of human diseases.

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“…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.…”

mentioning

confidence: 95%

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

et al. 2021

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

Cited by 58 publications

References 24 publications

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

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

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

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

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