A Comparison of the Emission Efficiency of Four Common Green Fluorescence Dyes after Internalization into Cancer Cells

Abstract

In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting non-bound fluorophores to be washed away or absorbed. The choice of fluorophores for this task must be carefully considered. For depth of penetration, near infrared probes are ordinarily preferred but suffer from relatively low quantum efficiency. Although green fluorescent protein has been widely used to image tumors on internal organs in mice, green fluorescent probes are suitable for imaging the superficial tissue because of the short penetration distance of green light in tissue but the highly efficient production of signal. While the fluorescence properties of green fluorophores are well known in vitro less attention has been paid to their fluorescence once they are internalized within cells. In this study, the emission efficiency after cellular internalization of 4 common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and AvRhodamine green) were compared after each conjugate was incubated with SHIN3 ovarian cancer cells. Using the lectin binding receptor system, the avidin-fluorophore conjugates were endocytosed and their fluorescence was evaluated with fluorescence microscopy and flow cytometry. While fluorescein demonstrated the highest signal outside the cell, among the four fluorophores, internalized Av-Rhodamine green emitted the most light from SHIN3 ovarian cancer cells both in vitro and in vivo. The internalized Av-Rhodamine green complex appeared to localize to the endoplasmic vesicles. Thus, among the four common green fluorescent dyes, Rhodamine green is the brightest green fluorescence probe after cellular internalization. This information could have implication for the design of tumor targeted fluorescent probes that rely on cellular internalization for cancer detection.

Platinum Compounds for High‐Resolution In Vivo Cancer Imaging

“…To this end, intravital imaging enables measurements of drug distribution at subcellular resolution while simultaneously tracking mitotic arrest [12] , apoptosis [14] , and DNA damage (reported herein) at the single-cell level. Although previous fluorescent-Pt compounds have been described elsewhere [15-17] , they use fluorescein derivatives, which are generally not ideal for intravital imaging due to photobleaching and poor fluorescence in intracellular environments [18] . In contrast, BODIPY-FL (BFL) has been shown to exhibit excellent properties for in vivo imaging [19] , including environmentally robust fluorescence; structural stability in vivo; high photostability; high brightness; and favorable cell permeability [20-22] .…”

A Target Cell–Specific Activatable Fluorescence Probe for In vivo Molecular Imaging of Cancer Based on a Self-Quenched Avidin-Rhodamine Conjugate

“…2D). These results indicate that both Av-0.5ROX and Av-3ROX are internalized into the cells (8,(12)(13)(14); however, the self-quenched Av-3ROX was activated once it was internalized and increased markedly in fluorescence likely as a consequence of proteolysis in the lysosome (Fig. 1).…”

Fluorescent Dye Conjugates for Optical Imaging of Cancer

“…207,208 A subsequent study compared the emission efficiency after cellular internalization of four common green fluorophores conjugated to avidin. 209 Among the four dyes tested, rhodamine green was found to be the brightest after cellular internalization. Therefore, galactosyl serum albumin-conjugated rhodamine green was investigated for fluorescence imaging of human ovarian adenocarcinoma in mice, which also allowed the visualization of sub-millimeter-sized ovarian tumor implants.…”