Figure 1.
Schematic illustration showing the experimental setups and steps involved in the loading of colloidal GNP suspensions into mES cells at 3 differentiation stages, i.e., undifferentiated single cells, EBs (cell aggregates), and terminally differentiated cardiomyocytes (A).
Representative optical microscopic images of undifferentiated mES cells (B), cardiomyocyte tissue (C), and EBs (D) after GNP loading.
Figure 2.
Illustration of the experimental setup employed for SERS spectra measurements.
He-Ne laser excitation (632.8 nm) was delivered to a sample placed on an inverted microscope, and this sample was used for SERS spectral measurements from single ES cells, EBs, and cardiomyocyte tissue samples. The SERS spectra were acquired by detection of scattering signals sent through a pinhole and delivered into the polychromator and CCD camera.
Figure 3.
TEM imaging and GNP cytotoxic effects on ES cell viability and proliferation.
TEM images showing GNP localization in ES cells, EBs, and cardiomyocyte organelles (A–C). It was observed that 40-nm GNPs accumulated in the mitochondria, secondary lysosome, and other cytoplasmic organelles (A), 60-nm particles localized in the mitochondria and secondary lysosome (B), and 100-nm particles accumulated in the mitochondria and endoplasmic reticulum in the cytoplasm (C). G, M, L, N, and C indicate GNP, mitochondria, secondary lysosome, nucleus, and cytoplasm, respectively. ER indicates the endoplasmic reticulum, and small dark spheres represent ribosomes attached to the ER. TEM images captured at a voltage of 80 kV with a Jeol 1200EX electron microscope. Cytotoxic effects of GNPs (40, 60, and 100 nm) on ES cell viability for 2 h (D) and on cell proliferation for 24 h (E). The effect of the GNP concentration on ES cell proliferation for 72 h (F) at varying ES cell densities. The experiments were performed in a 96-well plate using the MTS assay. Figure 3D and 3E show the values at the end of 3 h of incubation with the MTS reagent, while Figure 3F shows the results after 4 h of incubation. The cytotoxic effects of GNPs on ES cell viability and proliferation after 24 h (G) at varying cell densities. The experiments were performed in a 96-well plate using the NRU assay.
Figure 4.
SERS spectra characteristic of the 3 differentiation stages of mES cells.
The dark-field microscopic image of undifferentiated single ES cells loaded with 100 nm GNPs (A), and the corresponding SERS spectrum from the particle encircled inside the ES cell (D). The bright-field microscopic image of EBs treated with 100 nm GNPs and gold nanoaggregates visible in the dark (B). The corresponding SERS spectrum measured from the encircled area of EB (E). The bright-field microscopic image of 12-d-grown cardiomyocyte tissues loaded with 100 nm GNPs appeared as aggregates in the circled area (C) and in the corresponding SERS spectrum (F). Images captured by a digital camera (Canon, Japan) after excitation with a He-Ne laser (632.8 nm) at a power of 130 µW using a ×100 objective lens. SERS spectra of both ES cells and EBs acquired with 30-s laser acquisitions. The SERS spectrum of the cardiomyocyte tissue samples was acquired with 1-s acquisitions.
Table 1.
Tentative assignment of SERS peaks derived from undifferentiated ES cells, EBs, and differentiated cardiomyocyte tissues [51]–[63].
Figure 5.
Spatiotemporal measurements of the SERS spectra derived from 100-nm GNP aggregates in 12-d-grown cardiomyocyte tissue in glass-bottom chamber dishes (A).
The SERS spectra (B–D) from different GNP aggregates. The spectra were acquired at an exposure of 1 s employing He-Ne (632.8 nm) laser excitations at a power of 130 µW, as shown by the arrows in Figure 5A.
Figure 6.
Quantitative SERS profiling of DNA/RNA, protein translation activities (amide I, amide II, amide III), and mitochondrial metabolic activities in single ES cells, EBs, and cardiomyocytes.
Calculations are expressed as percentage values. Thirty samples were used to measure the DNA/RNA and protein translation activities, and 20 samples were taken to determine the mitochondrial activities from each stage of ES cell differentiation.