Flavylium- and Silylrhodapolymethines In Excitation Multiplexing

Abstract

Creation of a flavylium polymethine dye set enabled selection of two fluorophores that match common lasers for exciting in the near-IR II region. Using these, researchers cast a broad net to catch any wavelength emission in the near-IR II region, and relied on selective excitation to multiplex; this is a paradigm shift away from multiplexing via discrimination of emission wavelengths. Excitation multiplexing with flavylium dyes is a new and exciting strategy, but not yet a perfect one; it requires discrete water soluble fluorophores, including one that is turned on at 808 nm.

1. Photophysical Characteristics Of The Flav7 Dye Family

In 2017, the Sletten group at UCLA disclosed new near-IR dye structures based on flavylium polymethines, hence a family of dyes based around “Flav7” (Figure 1) was born.^[1] More recently, they have elaborated on these designs and, in conjunction with Bruns (Helmholtz Zentrum München), applied them in an intriguing way.^[2] Their work features systematic substitutions on the Flav7 framework to reveal positional dependence on the wavelengths of absorption and emission. Placing a dimethylamino group at the 5, 8, 6, and 7-positions in this series, gives progressive red-shifts in λ_max for absorption and fluorescence (partly because steric effects disfavor Me₂N coplanarity with the aromatic ring when that group is at the 5 and 8-positions). Those studies showed how wavelengths of absorption could be adjusted via simple modifications, setting up further modifications and their application in excitation multiplexing. Thus a series of Flav7 derivatives with different 7-substitutions were prepared to target absorption at the wavelengths of two common lasers: 980 and 1064 nm. These Flav7 dyes shows were brighter than commercial SWIR dyes such as IR-26, and they are about as bright as ICG. (Table 1).

Figure 1.

a) Scheme showing an electromagnetic spectrum showing NIR—I and -II regions. b) Structure of Flav7 (top) and an absorption/emission maxima “map” showing the influence of introducing two dimethylamino substituents at identical places on each flavylium ring. (values shown are in dichloromethane)^[3].

Table 1.

Photophysical characteristics of the Flav7 dye family with references from which values are taken.

Compound	λmax,abs [nm]	E (λmax) [M−1 cm−1]	λmax,em [nm]	ΦF [%]	Brightness[a] [M−1 cm−1]	Ref.
Flav7[b]	1027	241,000	1053	0.61	1470	[1]
MeOFlav7[b]	984	190,000	1008	0.52	990	[3]
JuloFlav7[b]	1061	238,000	1088	0.46	1180	[3]
ICG[c]c	787	194,000	818	0.66	1200	[5]
IR-26[d]	1080	171,000	1114	0.05	86	[6]

^[a]Brightness = ε × Φ_F.

^[b]In dichloromethane.

^[c]In ethanol.

^[d]In 1,2-dichloromethane.

2. Excitation Multiplexing

Fluorescence multiplexing typically involves detection at different wavelengths for a single laser excitation source, therefore relying on Stokes’ shift variance for differentiation. Sletten’s palate of flavylium dyes enable different near-IR II lasers (980 and 1064 nm) to selectively excite two different dyes and resolve their outputs by detecting in a broad wavelength span in the shortwave IR range. Specifically, MeOFlav7 and JuloFlav7 (Figure 2) were chosen to be multiplexed with indocyanine green (ICG) in a three-color system involving excitation at 780, 980, and 1064 nm and blanket detection in the 1150–1700 nm range. A characteristic of excitation imaging is it can be performed at rapid frame rates fast enough to observe moving animals, in other words, images of live, conscious mice. This is highly significant because normal imaging involving anesthesia impacts mouse physiology; what is observed for the animals in induced sleep may not be the same as when they are conscious and active.^[3] This paper shows fun images of mice serially injected with the three dyes such that false-stained stripes of red, green and blue cover the animal perpendicularly from head to tail (see Figure 3g in that paper).

Figure 2.

a) Essentials of excitation-based multiplexing. b) Novel dyes developed to be excited at around 980 and 1064 nm. (values shown are in dichloromethane).^[3]

Figure 3.

Comparison of silylrhodapentamethine and rhodapentamethine dyes (values shown are in dichloromethane).^[4]

A dark shadow surrounds flavylium dyes, however, that is they are extremely hydrophobic. Consequently, the mouse studies described above featured the fluorophores formulated in micelles doped with polyethylene glycol for water solubility. This is a major limitation because the pharmacokinetics of localization in vivo and in cellulo is dominated by the micelle and it will be hard to overcome that bias using targeting groups.

3. Silylrhodapolymethine Dyes

Use of ICG is a weakness of the work described above since only the red-tail of its absorption spectrum is excited by the 785 nm laser; consequently, other dye systems are being investigated. Flayvlium fragments are one of several heterocyclic components that can be conjugated to polymethine termini to form near-IR dyes. Recognizing this, Sletten has expanded her dye palette to include fluorophores which we call silylrhodapolymethines. Figure 3 compares how absorbance and emission characteristics of a silylrhodamine fluor compares with the corresponding rhodamine systems, both on a pentamethine framework; unsurprisingly based on the characteristics of the parent fragments, the silyl-derivatives are red-shifted relative to the oxygen analogs.

The silylrhodapentamethine showed absorbance peaks characteristic of both the silylrhodamine and the polymethine fragments at 663 and 938 nm. The system is solvatochromatic, the lower wavelength absorbance became more prevalent in polar solvents.^[4] Dual absorbance peaks and solvatochromaticity are an effect of incomplete resonance between one silylrhodamine and the rest of the dye. Our interpretation is that this reflects solvent dependent fractions of the conformer populations are twisted. When the conditions are changed to promote overall planarity, the system is more delocalized, less polar, and the red absorption dominates. However, polar solvents favor twisted conformations in which the whole system has a dipole moment, and the silylrhodamine fragments act as distinct donors. Unlike absorption, emission from the dyes is wavelength independent because, we think, the silylrhodamine can act as a donor in the twisted state wherein energy transfers rapidly to the polymethine acceptor.

Discussion of detailed photophysical characteristics should not distract readers from the main point of this paper. Sletten’s work with silylrhodamine tri- and penta-methines is part of a search for dyes that absorb strongly when excited at 808 nm, corresponding to a common near-IR laser. Finding a fluor that does this is important because ICG is not a good accomplice for MeOFlav7 and JuloFlav7 in excitation multiplexing. ICG has a λ_max,abs that is not well matched with 808 nm, and its fluorescence in the near-IR II region is only the weak tail of its fluorescence emission.

We predict this research is a beginning that precedes many more developments in this area. Those developments will involve water soluble modifications of the flavylium systems, and design of another dye to absorb maximally around 808 nm, then emit with a large Stokes’ shift.