Two-Photon Fluorescence Vascular Bioimaging with New Bioconjugate Probes Selective toward the Vascular Endothelial Growth Factor Receptor 2

Abstract

We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in “whole” mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 μm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.

Two‐Photon Fluorescent Probes for Bioimaging

“…For deep tissue 2PFM imaging, whole tumor ex vivo imaging experiments showed excellent results, allowing the visualization of vasculature over 800 μm deep in the tumor tissue (Figure 11). 12 The selectivity of probe 54‐ anti ‐VEGFR‐2 towards the tumor is evidenced by the excellent contrast observed in the micrographs in Figure 11. There was very little nonspecific fluorescence within the tumors attributable to autofluorescence.…”

“…VEGFR‐2 is the major mediator of VEGF‐driven responses in endothelial cells and it has been considered to be crucial in angiogenic processes. Shown in Scheme are two 2PA fluorophores conjugated with the monoclonal DC‐101 antibody ( 53‐ anti ‐VEGFR‐2 and 54‐ anti ‐VEGFR‐2 ), which targets VEGFR‐2 12. For the convenience of the photophysical studies, corresponding model compounds 53‐Bu and 54‐Bu were synthesized.…”

“…C) 2PFM image (excitation: 810 nm; emission: spectral scanner window 480–620 nm). Reprinted with permission from ref 12…”

“…The availability of hardware led to a wide range of bioimaging studies carried out by numerous researchers. Examples of applications of 2PFM includes: visualization of kidney dynamics,3 neurobiology and brain studies,4 cardiovascular imaging,5 the chemical and structural imaging of the eye,6 architecture and chemical phenomena in the skin,7 imaging and analysis of dynamic processes in living cells and tissues,8 clinical two‐photon microendoscopy,9 snoninvasive diagnostic procedures for the detection of malignancy in organs, cancer imaging,10 lysosomal11 and vascular imaging,12 and diagnostic tools for tissue engineering and drug delivery,13 to name a few.…”

SNAP‐Tag‐Based Subcellular Protein Labeling and Fluorescent Imaging with Naphthalimides

“…Various fluorescentdyes and probes have been applied for biological detection, targeted labeling, and tissue imaging by using two-photon technology. [5] The highly specific fluorescent labeling of certainc ells, organelles, and bio-macromolecules with two-photon fluorophores in complex biological systemsi sac hallenge, but it is of great significance for biological research. Genetically encoded technologiesp rovide methods for the specific fluorescentl abeling of as ubcellular protein of interest by fusion with af luorescent protein or polypeptide/protein tag, for example, Tetracysteinet ag, [6] SNAP-tag, [7] Halotag, [8] and TMP-tag.…”