Red‐Emitting Rhodamines with Hydroxylated, Sulfonated, and Phosphorylated Dye Residues and Their Use in Fluorescence Nanoscopy

Abstract

Fluorescent dyes emitting red light are frequently used in conventional and super-resolution microscopy of biological samples, although the variety of the useful dyes is limited. We describe the synthesis of rhodamine-based fluorescent dyes with absorption and emission maxima in the range of 621-637 and 644-660 nm, respectively and demonstrate their high performance in confocal and stimulated emission depletion (STED) microscopy. New dyes were prepared by means of reliable chemical transformations applied to a rhodamine scaffold with three variable positions. They feature polarity, water solubility, variable net charges, improved stabilities of N-hydroxysuccinimidyl (NHS) esters, as well as large fluorescence quantum yields in dye solutions and antibody conjugates. The photophysical and imaging properties of dyes containing three different polar groups, namely primary phosphate, sulfonic acid (in two different positions), and hydroxyl were compared. A dye with two primary phosphate groups was explored as a valuable alternative to dyes with "classical" sulfonic acid groups. Due to the increased net charge of the phosphorylated dye (q=-4 at pH 8), it demonstrated a far better electrophoretic mobility compared with analogues with two sulfonic acid groups (q=-2). As an example, one fluorescent dye was designed to be especially convenient for practical use. It is characterized by sufficiently high chemical stability of the NHS ester, its simple isolation, handling, and solubility in aqueous buffers, as well as in organic solvents. All these features, accompanied by a zero net charge in conjugates, were accomplished by the introduction of hydrophilic groups of two types: two hydroxyl groups and one sulfonic acid residue.

Novel red fluorophores with superior performance in STED microscopy

“…These are, to our knowledge, the highest values that have so far been reported for near-IR emitting dye conjugates with proteins. The net charges of the new dyes were varied; in conjugates of KK9046 and STAR635 they are positive and zero, respectively, which is favorable for cell permeability (Kolmakov, et al 2012). As expected, the polar groups − sulfonic acid, hydroxyl and primary phosphate residues − increase the solubility in water and aqueous buffers relative to Atto647N.…”

“…While the syntheses of the new dyes and all the related data will be detailed elsewhere (Kolmakov et al 2012), Figure 2 shows the properties of the new red-emitting compounds and the benchmark dyes Atto647N and KK114. The required spectral properties and fluorescence quantum yields of the new dyes in solution are similar to the benchmark dyes.…”

Lens-based fluorescence nanoscopy

“…(d) Higher-order photobleaching from the first excited electronic state S 1 , as exemplified for the organic dyes pDI and pTDI and for eGFP. Absorption spectra of S 0 (blue) and S 1 (red) and fluorescence development of new bright and photostable dyes, often specialized for STED applications, has and continuously will enhance the applicability and flexibility of experiments using this superresolution technique Kolmakov et al 2010aKolmakov et al , 2010bKolmakov et al , 2012Mitronova et al 2010;Wurm et al 2012).…”

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

“…These include long-established STED fluorophores such as Chromeo 488, Atto 565, and Atto 647 N, as well the recent rhodamine derivatives, carbopyronine derivatives, or Abberior STAR series fluorophores which are now commercially available (e.g., Abberior STAR 635P) [34,52,81,82,99,100]. Multicolor imaging strategies include the use of common excitation and depletion wavelengths with closely emitting fluorophores (e.g., that can be separated through spectral unmixing techniques, as was described above for two-color fluorescent protein imaging).…”