Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications.

A major advantage of flow cytometry is its flexible and open instrument configuration, which is highly suitable for systems integration. This flexibility permits the coupling of auxiliary instrumentation that may offer the measurement of parameters other than those typically measured by this multiparameter measurement technique. On the basis of this advantage, we explore the principle and application of hyperspectral imaging (HSI), which has the potential to be a useful add-on feature to flow cytometry applications. Application of HSI to flow cytometry involves the acquisition of spatial information and rendering it in spectral form. In this work, we describe the development and application of an HSI system which provides both spectral and spatial information. Spectral information was generated by obtaining an entire spectrum of a single sample site within a wavelength region of interest, while spatial information was generated by recording a two-dimensional (2D) image of an area of the sample of interest at one specific wavelength. HSI is a promising additional feature to flow cytometry since it can provide both spatial (image format) and spectral information in addition to the multiparameter information already available from flow cytometry measurements.

A compact CMOS biochip immunosensor towards the detection of a single bacteria.

Recent use of biological warfare (BW) agents has led to a growing interest in the rapid and sensitive detection of pathogens. Therefore, the development of field-usable detection devices for sensitive and selective detection of BW agents is an important issue. In this work, we report a portable biochip system based on complementary metal oxide semiconductor (CMOS) technology that has great potential as a device for single-bacteria detection. The possibility of single-bacteria detection is reported using an immunoassay coupled to laser-induced fluorescence (LIF) detection. Bacillus globigii spores, which are a surrogate species for B. anthracis spores, were used as the test sample. Enzymatic amplification following immunocomplex formation allowed remarkably sensitive detection of B. globigii spores, and could preclude a complicated optical and instrumental system usually required for high-sensitive detection. Atomic force microscopy (AFM) was employed to investigate whether B. globigii spores detected in the portable biochip system exist in single-cell or multicellular form. It was found that B. globigii spores mostly exist in multicellular form with a small minority of single-cell form. The results showed that the portable biochip system has great potential as a device for single-particle or possibly even single-organism detection.

Optical nanobiosensor for monitoring an apoptotic signaling process in a single living cell following photodynamic therapy.

Optical nanobiosensors have enabled bioanalytical measurements to be undertaken within volumes as small as that of single biological cells. In this work, we use nanobiosensors to monitor a molecular signaling process, i.e., caspase-7 activation, following photodynamic therapy (PDT) induced apoptosis in breast cancer cells (MCF-7). PDT induces the mitochondrial pathway of apoptosis which triggers cytochrome c release, activation of caspases-9, -8 and -7 and cleavage of poly (ADP-ribose) polymerase (Parp) protein. Caspase-7 is an important apoptosis-related cysteine protease involved in the activation cascade of caspases and in the proteolytic cleavage of Parp protein. Caspase-7 was detected and identified intracellularly using optical nanobiosensors. Our results show the detection of caspase-7 in single living MCF-cells which in essence typifies the apoptotic event induced by a PDT drug. This work, in principle, demonstrates the minimally invasive capability of optical nanobiosensors to measure important signaling molecules and events in pathways at the single cell level.

Detection of cytochrome C in a single cell using an optical nanobiosensor.

In this work, the intracellular measurement of cytochrome c using an optical nanobiosensor is demonstrated. The nanobiosensor is a unique fiberoptics-based tool which allows the minimally invasive analysis of intracellular components. Cytochrome c is a very important protein to the process which produces cellular energy. In addition, cytochrome c is well-known as the protein involved in apoptosis, or programmed cell death. delta-Aminolevulinic acid (5-ALA) was used to induce apoptosis in MCF-7 human breast carcinoma cells. 5-ALA, a photodynamic therapy (PDT) drug in cells was activated by a HeNe laser beam. After the PDT photoactivation, the release of cytochrome c from the mitochondria to the cytoplasm in a MCF-7 cell was monitored by the optical nanobiosensor inserted inside the single cell and followed by an enzyme-linked immunosorbent assay (ELISA) outside the cell. The combination of the nanobiosensor with the ELISA immunoassay improved the detection sensitivity of the nanobiosensor due to enzymatic amplification. Our results lead to the investigation of an apoptotic pathway at the single cell level.

Optical sensor for the detection of caspase-9 activity in a single cell.

We demonstrate for the first time, the application and utility of a unique optical sensor having a nanoprobe for monitoring the onset of the mitochondrial pathway of apoptosis in a single living cell by detecting enzymatic activities of caspase-9. Minimally invasive analysis of single live MCF-7 cells for caspase-9 activity is demonstrated using the optical sensor which employs a modification of an immunochemical assay format for the immobilization of nonfluorescent enzyme substrate, Leucine-GlutamicAcid-Histidine-AsparticAcid-7-amino-4-methylcoumarin (LEHD-AMC). LEHD-AMC covalently attached on the nanoprobe tip of an optical sensor is cleaved during apoptosis by caspase-9 generating free AMC. An evanescent field is used to excite cleaved AMC and the resulting fluorescence signal is detected. By quantitatively monitoring the changes in fluorescence signals, caspase-9 activity within a single living MCF-7 cell was detected. By comparing of the fluorescence signals from apoptotic cells induced by photodynamic treatment and nonapoptotic cells, we successfully detected caspase-9 activity, which indicates the onset of apoptosis in the cells.