N-aryl pyrido cyanine derivatives are nuclear and organelle DNA markers for two-photon and super-resolution imaging

Kakishi Uno,Yoshikatsu Sato,Nagisa Sugimoto

doi:10.1038/s41467-021-23019-w

Kakishi Uno, Yoshikatsu Sato + Show 1 more

Open Access

https://doi.org/10.1038/s41467-021-23019-w

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Abstract

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences.

Highlights

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields
Staining of nuclear and organellar DNA in living cells. We demonstrate their compatibility with various optical microscopes such as two-photon excitation microscopy (2PEM), fluorescent lifetime imaging microscopy (FLIM), and separation of photon by lifetime tuning (SPLIT) method in stimulated emission depletion (STED) nanoscopy (SPLIT-STED)[22]
To assess the binding mode of PC1 to double stranded DNA, we measured circular dichroism (CD) spectrum of PC1 complexed with dsDNA

Summary

Introduction

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. Silicon-rhodamine (SiR)-Hoechst, which fused a far-red emissive SiR to Hoechst 33342, stained the nucleus with no significant phototoxicity and is compatible with stimulated emission depletion (STED) nanoscopy[12,13,14] In exchange for these preferable properties, the linking of the additional fluorescent dye decreases the DNA-binding strength of Hoechst[12,14]. In addition to not requiring UV light excitation, they display great cell permeability in various cell types and exceptional high DNA specificity for clear DNA staining of nuclear and organellar DNA in living cells We demonstrate their compatibility with various optical microscopes such as two-photon excitation microscopy (2PEM), fluorescent lifetime imaging microscopy (FLIM), and separation of photon by lifetime tuning (SPLIT) method in STED nanoscopy (SPLIT-STED)[22]

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