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 nonbound 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 better suited for imaging the superficial tissues because of the short penetration distance of green light in tissue and 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 four common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and Av-rhodamine 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 implications for the design of tumor-targeted fluorescent probes that rely on cellular internalization for cancer detection.

Figure 1

The chemical structures of the four green fluorescence dyes compared in this study.

Figure 2

Optical characteristics of avidin-conjugated BODIPY, FITC, OreG and RhodG. 5 μg Av-BODIPY, Av-FITC, Av-OreG or Av-RhodG in 390 μL PBS (pH 7.4) or the mixture of sodium dihydrogen phosphate and phosphate (pH 3.3) were placed in a nonfluorescent 96-well plate and spectral fluorescence image was obtained. The ratio of BODIPY, FITC, OreG or RhodG molecules to avidin was 0.8 in all cases a: Emission spectra. All 4 dyes had the same emission peak at a wavelength of 550 nm but the fluorescence intensity was different with Av-FITC the highest followed by Av-OreG, Av-RhodG and Av-BODIPY. b: Fluorescence intensities of Av-BODIPY (B), Av-FITC (F), Av-OreG (O) and Av-RhodG (R) were 28, 238, 170 and 159, respectively, in arbitrary units (a.u.) with Av-FITC the highest and Av-BODIPY the lowest under pH 7.4. However, under the acidic condition (pH 3.3), fluorescence intensities of Av-BODIPY, Av-FITC, Av-OreG and Av-RhodG were 35, 33, 122 and 199 (a.u.), respectively, with Av-FITC the lowest and Av-RhodG the highest.

Figure 3

Serial fluorescence microscopy images of SHIN3 ovarian cancer cells. SHIN3 cells were incubated with each of the four fluorophores for 1 hour and fluorescent microscopy was then performed immediately and at 4 and 8 hours after washing with PBS. Av-RhodG showed that the size of intracellular fluorescent dots as well as the fluorescence intensity progressively increased with time, while the other 3 dyes showed a minimal change. Original magnification: x200. Photographic exposure time: Av-BODIPY, Av-FITC and Av-OreG (immediate, 4 and 8 hours) = 2s, Av-RhodG (immediate and 4 hours) = 2s, Av-RhodG (8 hours) = 1s.

Figure 4

Histograms for flow cytometric analysis of SHIN3 cancer cells immediately (red) and at 24 hours (green) after washout of each of the 4 green dyes as well as the control SHIN3 cells without treatment with dye (black). Samples with Av-BODIPY, Av-OreG and Av-RhodG showed a significant rightward shift both immediately and at 24 hours after washing dyes compared with the untreated control samples. Av-RhodG demonstrated the highest fluorescence both immediately (MFI 652) and 24 hours after (MFI 130) washing. The percent cells in M2 increased from 0.44% to 99.9% for Av-BODIPY, 96.6% for Av-OreG and 99.3% for Av-RhodG after 96-hour incubation with each of the 4 dyes. Av-FITC did not show a significant rightward shift with percent cells in M2 from 0.44% to 9.7%.

Figure 5

In vivo spectral fluorescence images of tumor-bearing mice 3h after intraperitoneal injection with Av-BODIPY (B), Av-FITC (F), Av-OreG (O) and Av-RhodG (R). Upper: Autofluorescence image. Middle: Green dye fluorescence image. Lower: Composite image (red: autofluorescence, green: green dye fluorescence). Spectral fluorescence image of the peritoneal cavities clearly visualized the disseminated tumor foci (yellow arrows) in mice incubated with Av-BODIPY, Av-OreG or Av-RhodG whereas Av-FITC failed to visualize tumor foci (yellow arrow) due to insufficient fluorescence intensity. Closeup image of the peritoneal membranes demonstrates peritoneal implants histologically confirmed to be metastatic deposits as small as 1 mm in diameter in all mice including Av-FITC injected mouse (blue arrows). The fluorescence intensity of Av-RhodG was the highest and that of Av-FITC was the lowest of all when compared visually.

Figure 6

Semi-quantitative assessment of in vivo fluorescence intensity. a: ROI was drawn inside the intestine on the unmixed green fluorescence image (the same image as the unmixed green fluorescence image of Figure 5). B: Av-BODIPY. F: Av-FITC. O: Av-OreG. R: RhodG. b: Histogram of fluorescence intensity of an ROI drawn on each of the peritoneal membranes instilled with 4 green dyes, Av-BODIPY, Av-FITC, Av-OreG and RhodG. The dynamic range of the fluorescence intensity was split into equal-sized 256 bins (1–256). Then for each bin (horizontal axis), the number of pixels from the data set that fall into each bin (vertical axis) are counted. The shape of the plot distribution ≤40 in arbitrary unit (a.u.) is almost the same among the 4 histograms. c: Regression lines of 4 green dyes. The regression lines were calculated from the data sets (fluorescence threshold values 41–241, total number of pixels within the threshold rage 10–100,000 in common logarithm). The slopes of Av-BODIPY, Av-FITC, Av-OreG and Av-RhodG were -0.022, −0.056, −0.028 and −0.006, respectively. Av-RhodG has the highest slope value consistent with it being the brightest fluorophore whereas, Av-FITC had the lowest slope value, and Av-BODIPY and Av-OreG were intermediate.

Figure 6

Semi-quantitative assessment of in vivo fluorescence intensity. a: ROI was drawn inside the intestine on the unmixed green fluorescence image (the same image as the unmixed green fluorescence image of Figure 5). B: Av-BODIPY. F: Av-FITC. O: Av-OreG. R: RhodG. b: Histogram of fluorescence intensity of an ROI drawn on each of the peritoneal membranes instilled with 4 green dyes, Av-BODIPY, Av-FITC, Av-OreG and RhodG. The dynamic range of the fluorescence intensity was split into equal-sized 256 bins (1–256). Then for each bin (horizontal axis), the number of pixels from the data set that fall into each bin (vertical axis) are counted. The shape of the plot distribution ≤40 in arbitrary unit (a.u.) is almost the same among the 4 histograms. c: Regression lines of 4 green dyes. The regression lines were calculated from the data sets (fluorescence threshold values 41–241, total number of pixels within the threshold rage 10–100,000 in common logarithm). The slopes of Av-BODIPY, Av-FITC, Av-OreG and Av-RhodG were -0.022, −0.056, −0.028 and −0.006, respectively. Av-RhodG has the highest slope value consistent with it being the brightest fluorophore whereas, Av-FITC had the lowest slope value, and Av-BODIPY and Av-OreG were intermediate.

Figure 6

Semi-quantitative assessment of in vivo fluorescence intensity. a: ROI was drawn inside the intestine on the unmixed green fluorescence image (the same image as the unmixed green fluorescence image of Figure 5). B: Av-BODIPY. F: Av-FITC. O: Av-OreG. R: RhodG. b: Histogram of fluorescence intensity of an ROI drawn on each of the peritoneal membranes instilled with 4 green dyes, Av-BODIPY, Av-FITC, Av-OreG and RhodG. The dynamic range of the fluorescence intensity was split into equal-sized 256 bins (1–256). Then for each bin (horizontal axis), the number of pixels from the data set that fall into each bin (vertical axis) are counted. The shape of the plot distribution ≤40 in arbitrary unit (a.u.) is almost the same among the 4 histograms. c: Regression lines of 4 green dyes. The regression lines were calculated from the data sets (fluorescence threshold values 41–241, total number of pixels within the threshold rage 10–100,000 in common logarithm). The slopes of Av-BODIPY, Av-FITC, Av-OreG and Av-RhodG were -0.022, −0.056, −0.028 and −0.006, respectively. Av-RhodG has the highest slope value consistent with it being the brightest fluorophore whereas, Av-FITC had the lowest slope value, and Av-BODIPY and Av-OreG were intermediate.

Figure 7

In vivo spectral fluorescence close-up images of a peritoneally disseminated SHIN3 ovarian cancer-bearing mouse 6h after intraperitoneal injection with Av-RhodG. A white light image (a), a green fluorescence image (b), a composite image of green fluorescence (green) and auto fluorescence (red) (c), and an uncalculated image obtained with a 545–555 nm band pass filter. Strong green florescence derived from Av-RhodG allows us to clearly show SHIN3 ovarian cancer submillimeter implants in an uncalculated image taken with a 545–555 nm band pass filter without the use of the wave length-resolved technique (d) as well as in a spectrally calculated green fluorescence image (b).