Self-assembling nanoclusters in living systems: application for integrated photothermal nanodiagnostics and nanotherapy

The photothermal effect of an ultratrace amount of nonfluorescent molecules in liquid was determined by optimizing the optical arrangement for a thermal lens microscope. The optimized experimental setup could be determined from the evaluation of probing volume and the concentration of the sample solutions even when the expectation of the molecule number in the probing region was less than a single molecule. The minimum expectation, which is explained as being the time average, was 0.4 molecule of Pb(II) octaethylporphyrin (OEP) in benzene. The concentrations in the 9.7 × 10-11−7.8 × 10-10 M region used in this work corresponded to the expected number of 0.4−3.4 molecules, and the calibration curve in this region showed good linearity. Taking into account the enhancement factor of solvent, the molar absorption coefficient of solute, and the optimization of the optical arrangement, the present result, which was the determination limit of 0.34, was consistent with that previously reported. The relation betwe...

Determination of Subyoctomole Amounts of Nonfluorescent Molecules Using a Thermal Lens Microscope: Subsingle-Molecule Determination

We developed a microsystem for cell experiments consisting of a scanning thermal lens microscope detection system and a cell culture microchip. The microchip system was good for liquid control in microspace, and this results in secure cell stimulation and coincident in vivo observation of the cell responses. The system could detect nonfluorescent biological substances with extremely high sensitivity without any labeling materials and had a high spatial resolution of ∼1 μm. This system was applied to monitoring of cytochrome c distribution in a neuroblastoma−glioma hybrid cell cultured in the microflask (1 mm × 10 mm × 0.1 mm; 1 μL) fabricated in a glass microchip. Cytochrome c release from mitochondria to cytosol during the apoptosis process was successfully monitored with this system. The cytochrome c detected with this system was estimated to be ∼10 zmol. We concluded that the system was suitable for measuring the distribution of chemical substances in a single cell because the microchip is good for liq...

Single-Cell Analysis by a Scanning Thermal Lens Microscope with a Microchip: Direct Monitoring of Cytochrome c Distribution during Apoptosis Process

High-resolution nonlinear laser spectroscopy based on absorption saturation, Lamb-dip and spectral hole-burning phenomena have contributed much to basic and applied photonics. Here, a laser spectroscopy based on nonlinear photothermal and photoacoustic phenomena is presented. It shows ultrasharp resonances and dips up to a few nanometres wide in broad plasmonic spectra of nanoparticles. It also demonstrates narrowing of absorption spectra of dyes and chromophores, as well as an increase in the sensitivity and resolution of the spectral hole-burning technique. This approach can permit the study of laser-nanoparticle interactions at a level of resolution beyond the spectral limits, identification of weakly absorbing spectral holes, spectral optimization of photothermal nanotherapy, measurements of tiny red and blue plasmon resonance shifts, multispectral imaging and multicolour cytometry.

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Ultrasharp nonlinear photothermal and photoacoustic resonances and holes beyond the spectral limit

Super-resolution fluorescence microscopy enables imaging of fluorescent structures beyond the diffraction limit. However, this technique cannot be applied to weakly fluorescent cellular components or labels. As an alternative, photothermal microscopy based on nonradiative transformation of absorbed energy into heat has demonstrated imaging of nonfluorescent structures including single molecules and ~1-nm gold nanoparticles. However, previously photothermal imaging has been performed with a diffraction-limited resolution only. Herein, super-resolution, far-field photothermal microscopy based on nonlinear signal dependence on the laser energy is introduced. Among various nonlinear phenomena, including absorption saturation, multiphoton absorption, and signal temperature dependence, signal amplification by laser-induced nanobubbles around overheated nano-objects is explored. A Gaussian laser beam profile is used to demonstrate the image spatial sharpening for calibrated 260-nm metal strips, resolving of a plasmonic nanoassembly, visualization of 10-nm gold nanoparticles in graphene, and hemoglobin nanoclusters in live erythrocytes with resolution down to 50 nm. These nonlinear phenomena can be used for 3D imaging with improved lateral and axial resolution in most photothermal methods, including photoacoustic microscopy.

Super‐Resolution Nonlinear Photothermal Microscopy

Application of coaxial beam photothermal microscopy to the analysis of a single biological cell in water

We have developed a novel thermal lens microscopy coupled with an optical microscope, and presently applied it to the ultratrace molecule detection in a single biological cell (mouse hybridoma) in-vitro. The determination results obtained for individual cells were calibrated by the average values determined by absorption spectrophotometry. The determination limit of the thermal lens microscope, defined as twice the standard deviation of the calibration curve, was 37.8 amol/cell, which was more than one order smaller than that of a fluorescence microscope under our experimental conditions. This superiority should come from escaping light scattering by a cell membrane and by cytoplasm, which is inevitable in fluorescence spectrometry. The absolute determination limit of the thermal lens microscopy was calculated down to sub-attomole level. In addition, the method is more widely applicable, even to non-fluorescent samples without chemical preparation, and therefore, the thermal lens microscope has proved to be very useful in directly quantifying ultratrace chemical species in a single biological cell in-vitro.

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Masashi Shibata

Papers

Application of coaxial beam photothermal microscopy to the analysis of a single biological cell in water

Sub-Attomole Molecule Detection in a Single Biological Cell in-vitro by Thermal Lens Microscopy