mRNA imaging in cells using TAM

To this end, we applied the TAM to image and quantify single mRNA molecules in turbid biological environments such as cells and tissues, using AuNPs as probes. Figure 4.6 shows the basic scheme of the detection. AuNPs used here are synthesized in our group and have a sphere shape and the mean size of 28 nm (see SEM image in the inset of Figure 4.6). The AuNP is first chemically treated to obtain mono-dispersed colloidal solutions, following the protocol described in Chapter 4.3.1. Then the chemically-modified AuNPs are conjugated with single strand oligonucleotides which are complementary to the targeting transcript, human epidermal growth factor receptor 2 (Her2) mRNA. The overexpression of Her2 gene is regarded to associate with the tumorigenesis of 15-20% of invasive breast cancers and can be targeted by antibody based therapies (e.g. Herceptin), whereas Her2-negative tumors require other therapeutic alternatives.

The expression levels of Her2 mRNA have been used as predictive markers for diagnosis or in therapeutics for malignant tumors.³³ The oligonucleotide-AuNP conjugates are incubated with fixed cells and mRNAs are quantified when being hybridized with the conjugate and detected in TAM images of AuNPs without background. To validate the capability of quantification, we perform the detection in two cell lines, MCF-7 and SK-BR-3, in which Her2 mRNA is rarely expressed and overexpressed, respectively.

Figure 4.7 Quantitative detection of Her2 mRNA in cancer cell lines. (a) Pump-probe scanning microscopy with 0 ps delay (left column) to show the location of cells in SRS channel, and with 2 ps delay (middle column) to show the AuNPs in TAM channel, in MCF-7 cells. Doted profiles imply the boundary of cells. The right column is the merged image of SRS and TAM channels.

(b) mRNA detection in SK-BR-3 cells, the assignment of channels is the same as described in (a).

Figure 4.7 shows the expression levels and localization patterns of Her2 mRNA in MCF-7 (a) and SK-BR-3 cells (b). Pump beam at 800 nm and probe beam at 1045 nm are selected to match the Raman peak 2950 cm^-1, a vibration frequency of C-H3 bond, which is the major component in lipids and proteins. The TAM utilizes the same pump beam but different probe beam working at 1066 nm, as suggested by Figure 4.5d. The left panel of Figure 4.7 is the pump-probe image without delay (0 ps), which shows both cells and AuNPs. From the SRS image one can observe the shape of nucleus and cytoplasmon from the chemical vibration of proteins and lipids. The middle panel is the TAM image with a 2 ps delay, which only shows the localization of AuNPs without any information of cells; dashed lines are marked boundary of cells obtained from the SRS image. The right panel is the merged image of prior panels. The binding specificity of the oligonucleotide-AuNP conjugates is validated by the control experiments which imaging the

MCF-7 and SK-BR-3 cells with chemically-treated AuNPs, as shown in Figure 4.8 and Figure 4.9. The TAM image successfully removes the interference of cellular background and only provides the signal of mRNAs with a ultra-high SNR. From Figure 4.7, it is observed that the MCF-7 cells have a low expression level of Her2 mRNAs, while SK-BR-3 cells have over-expressed Her2 mRNAs. In addition, it is also observed that the AuNP not only localized in the cytoplasma of cells, which was observed in our previous working using 80 nm BTO nanocrystals and represent the location of mature mRNAs,⁸ but also exists in the nucleus, suggesting that the oligonucleotide-AuNP conjugates penetrate into the nucleus and hybridize to the nascent mRNA.

Figure 4.8 Pump-probe images of MCF-7 cells incubated with bare AuNPs (upper) and

AuNP_Her2 conjugates (bottom) showing the binding specificity of the conjugates. Left row is the SRS image with 0 ps delay; middle row is the TAM image with 2 ps delay, white dashed lines indicate the boundary of cells; right row is the merged image of both channels. Very few AuNPs can be observed within the cells for the nonspecific binding of bare AuNPs.

Figure 4.9 Pump-probe images of SK-BR-3 cells incubated with bare AuNPs (upper) and AuNP_Her2 conjugates (bottom) showing the binding specificity of the conjugates.

To ensure that the detection of oligonucleotide-AuNP conjugates is specifically in the cells, other than “laying on” the cellular surfaces, we performed the three-dimensional (3D) stack-imaging of MCF-7 cells incubated with oligonucleotide-AuNP conjugates, the results are illustrated in Figure 4.10. The 3D SRS and TAM images confirm that the AuNPs are located in cells with a random distribution inside the cell.

Providing that the TAM can offer background-free detection of Her2 mRNAs with a single molecule sensitivity, we quantify the expression levels of Her2 mRNA in MCF-7 cells and SK-BR-3 cells. The statistics of the quantification results and the heterogeneity of the mRNA copy per cell are plotted in Figure 4.10. The Her2 mRNA number per cell for the MCF-7 cell line has a mono-component distribution with the peak around 10 per cell, while the SK-BR-3 cells have a two-component distribution for the heterogeneity of the copy number of the Her2 mRNA.

Statistics shows that the average number of Her2 mRNA in MCF-7 and SK-BR-3 cell lines is

estimated as 11.29±4.47 per cell and 203.19±80.48 per cell, respectively. Our quantification results with the TAM and AuNP probes agree well with past values obtained either from fluorescence-based technique[108]or SHG-based technique.[117] Furthermore, the TAM results are validated by the FISH approach, with which the Her2 mRNA is quantified in the same cell lines (Figure 4.11). From the FISH images, it is clear that SK-BR-3 cells have a much higher expression of Her2 mRNA than MCF-7 cells do, and the corresponding quantification results are in the same order as our TAM technique and AuNP probe.

Figure 4.10 Analysis of the Her2 mRNA in single cells using the pump-probe scanning

microscopy. (a). Three-dimensional (3D) profiles of pump-probe images with 0 ps (left) and 2 ps (right) delay. (b). Quantification of Her2 mRNA in MCF-7 and SK-BR-3 breast cancer cell lines.

Left figure shows the statistical comparison; middle and right figures illustrate the heteogeneity of the number of Her2 mRNA copies in each MCF-7 and SK-BR-3 cell, respectively. Solid lines are gaussian fittings.

Figure 4.11 Confocal images of Her2 mRNA in MCF-7 cells (upper) and SK-BR-3 cells (bottom) using the standard FISH approach. Left row (blue) is the DAPI channel showing the cell nucleus, middle row (red) is the FISH channel showing the localizations of Her2 mRNA. The right row is the merged images.