Oxygen consumption of single bovine embryos probed by scanning electrochemical microscopy

Oxygen consumption of individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). A probe microelectrode was used to scan near a single embryo surface in a culture medium to monitor the oxygen reduction current at 37 degrees C, under a water-saturated atmosphere of 5% CO2 and 95% air. The oxygen concentration profiles near the embryos were in good agreement with the theoretical spherical diffusion. When an embryo reached the stage of a morula with a 74-microm radius on day 6 after in vitro fertilization, the oxygen concentration difference (deltaC) between the bulk solution and the morula surface was 6.90 +/- 1.35 microM. The oxygen consumption rate (F) of the single morula was estimated to be (1.40 +/- 0.27) x 10(-14) mol s(-1). After the SECM measurement, the embryo was continuously cultured for another 2 days and grew to the stage of a blastocyst with a 100-microm radius. For the blastocyst, the deltaC values for the inner cell mass side and the trophoblast side were 16.40 +/- 1.83 and 9.14 +/- 1.68 microM, respectively. The oxygen consumption rate of the blastocyst was found to be in the range of (2.50 +/- 0.46) x 10(-14) mol s(-1) < F < (4.49 +/- 0.50) x 10(-14) mol s(-1). We have carried out SECM measurements for 19 embryos, and the results were compared in detail with these from an optical microscopic observation. The deltaC values for the morulae on day 6 after in vitro fertilization were strongly related to the morphological embryo quality. The morulae showing a larger deltaC value developed into blastocysts of a larger size, and the deltaC value after the subsequent 2 days of cultivation was found to be increased.     

Measurement of gene expression from single adherent cells and spheroids collected using fast electrical lysis

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 microm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 micros between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 microL of the lysed solution was collected using a micro-injector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the single-cell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids-the aggregated cellular masses formed during the three-dimensional culture -- as a model system to isolate small cellular clusters from tissues and organs.     

Photoelectrochemical and photocatalytic properties of biphasic organic p- and n-type semiconductor nanoparticles fabricated by a reprecipitation process

The visible-light-responsive photoelectrochemical and photocatalytic properties of nanoparticles of C(60), partially hydrolyzed aluminum phthalocyanine chloride (denoted as AlPc), and a composite of the two are reported. The three types of nanoparticles were obtained through a reprecipitation method from N-methyl-2-pyrrolidone solutions of C(60), aluminum phthalocyanine chloride (AlPcCl), and their mixture, respectively. The nanoparticle composite's ultraviolet-visible absorption, diffuse-reflectance and Fourier transform IR spectra, X-ray diffraction pattern, and scanning electron microscopy image are all similar to the sum of those of the C(60) and AlPc particles, respectively. The nano-ordered composite exhibits p/n junctionlike photoelectrochemical characteristics, which were investigated in comparison with those of vapor-deposited C(60) (n-type), AlPcCl (p-type), C(60)/AlPcCl (n/p), and AlPcCl/C(60) (p/n) electrodes. The nanoparticle composite further shows photocatalytic activity for the decomposition of trimethylamine to carbon dioxide in a suspension system.