M. A. Proskurnin

Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

Light-absorbing endogenous cellular proteins, in particular cytochrome c, are used as intrinsic biomarkers for studies of cell biology and environment impacts. To sense cytochrome c against real biological backgrounds, we combined photothermal (PT) thermal-lens single-channel schematic in a back-synchronized measurement mode and a multiplex thermal-lens schematic in a transient high resolution (ca. 350 nm) imaging mode. These multifunctional PT techniques using continuous-wave (cw) Ar+ laser and a nanosecond pulsed optical parametric oscillator in the visible range demonstrated the capability for label-free spectral identification and quantification of trace amounts of cytochrome c in a single mitochondrion alone or within a single live cell. PT imaging data were verified in parallel by molecular targeting and fluorescent imaging of cellular cytochrome c. The detection limit of cytochrome c in a cw mode was 5 x 10(-9) mol/L (80 attomols in the signal-generation zone); that is ca. 10³ lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 x 10(-21) mol) in the ultrasmall irradiated volumes limited by optical diffraction effects. For the first time, we demonstrate a combination of high resolution PT imaging with PT spectral identification and ultrasensitive quantitative PT characterization of cytochrome c within individual mitochondria in single live cells. A potential of far-field PT microscopy to sub-zeptomol detection thresholds, resolution beyond diffraction limit, PT Raman spectroscopy, and 3D imaging are further highlighted.

Photothermal lens detection of gold nanoparticles: theory and experiments.

An approach for mode-mismatched two-beam (pump–probe) photothermal lens detection of multipoint light-absorbing targets in solution (e.g., gold nanoparticles) is developed for continuous-wave intensity-modulated laser-excitation mode. A description of the blooming of the thermooptical element (thermal lens) upon absorption of the excitation laser radiation is based on the summation of individual thermal waves from multiple heat sources. This description makes it possible to estimate the irregularities of the temperature (and, thus, the refractive index) profile for a discrete number of nanoparticles in the irradiated area and a change in the concentration and particle size parameters. Experimental results are in good agreement with theoretical dependences of the photothermal signal on nanoparticle size and concentration and excitation laser power. Calibration plots for particles from 2 to 250 nm show long linear ranges, limits of detection of gold nanoparticles at the level of hundreds of nanoparticles with the current setup, and the photothermallens sensitivity coefficient increases as a cubic function of particle size. Further improvements are discussed, including increasing the sensitivity thresholds up to one nanoparticle in the detected volume.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof‐of‐concept, we characterized high‐speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.

Thermooptical detection in microchips: From macro‐ to micro‐scale with enhanced analytical parameters

In this paper, we compared the methods of photothermal spectroscopy used in different spatial scales, namely thermal‐lens spectrometry (TLS) and thermal‐lens microscopy (TLM) to enhance the performance parameters in analytical procedures. All of the experimental results were confirmed by theoretical calculation. It was proven that the design for both TLM and TLS, despite a different scale for the effect, is governed by the same signal‐generating and probing conditions (probe beam diameter at the sample should be equal to the diameter of the blooming thermal lens), and almost does not depend on the nature of the solvent. Theoretical and experimental instrumental error curves for thermal lensing were coincident. TLM obeys the same law of instrumental error as TLS and shows better repeatability for the same levels of thermal‐lens signals or absorbances. TLS is more advantageous for studying low concentrations in bulk, while TLM shows much lower absolute LODs due to better repeatability for low amounts. The behavior of the thermal‐lens signal with different flow rates was studied and optimum conditions, with the minimum contribution to total error, were found. These conditions are reproducible, are in agreement with the existing theory of the thermal response in thermal lensing, and do not significantly affect the design of the optimum scheme for setups. TLM showed low LODs in solvent extraction (down to 10–8 M) and electrokinetic separation (10–7 M), which were shown to be governed by discussed instrumental regularities, instead of by microchemistry.

Improving the dispersity of detonation nanodiamond: differential scanning calorimetry as a new method of controlling the aggregation state of nanodiamond powders

Detonation nanodiamond (ND) is a suitable source material to produce unique samples consisting of almost uniform diamond nanocrystals (d = 3-5 nm). Such samples exist in the form of long stable aqueous dispersions with narrow size distribution of diamond particles. The material is finding ever increasing application in biomedicine. The major problem in producing monodispersed diamond colloids lies in the necessity of deagglomeration of detonation soot and/or removing of clusters formed by already isolated core particles in dry powders. To do this one must have an effective method to monitor the aggregation state or dispersity of powders and gels prior to the preparation of aqueous dispersions. In the absence of dispersity control at various stages of preparation the reproducibility of properties of existing ND materials is poor. In this paper we introduce differential scanning calorimetry (DSC) as a new tool capable to distinguish the state of aggregation in dry and wetted ND materials and to follow changes in this state under different types of treatment. Samples with identical X-ray diffraction patterns (XRD) and high resolution transmission electron microscopy (HRTEM) images gave visibly different DSC traces. Strong correlation was found between dynamic light scattering (DLS) data for colloids and DSC parameters for gels and powders of the same material. Based on DSC data we improved dispersity of existing ND materials and isolated samples with the best possible DSC parameters. These were true monodispersed easily dispersible fractions of ND particles with diameters of ca. 3 nm.

Determination of Soluble Iron Species in Seawater Using Ferrozine

Ferrozine was used for the simultaneous determination of soluble iron(II) and iron(III) species in seawater. It was shown that iron forms complexes with dissolved organic matter in seawater, which hinders the color development of ferrozine chelates. To eliminate this factor, samples were heated and exposed to UV irradiation. The developed procedures were used for determining soluble iron species in samples of surface and silt seawater. The results agree with the data obtained by atomic absorption spectrometry.

Optimisation of the optical scheme of a dual-beam thermal lens spectrometer using expert estimation

An expert estimation of the measurement results was applied to optimise the optical-scheme configuration of a dual-beam thermal lens spectrometer with a single-channel photodetector system to achieve the longest linear calibration range. A combination of experimental justification and the expert estimation provides an increase in the linear range of analytical procedures up to two to three times and enhances the repeatability compared to a non-optimised design.

Model for continuous-wave laser-induced thermal lens spectrometry of optically transparent surface-absorbing solids

A theoretical model for cw laser-induced thermal lens spectrometry of optically transparent surface-absorbing solids is developed. In the model, the sample is represented as a set of discrete layers with certain thicknesses and light absorptivities. The bloomed thermo-optical element in the sample is described with a summation of heat-flux functions for all the layers. The model employs simple mathematical expressions and can be used for both steady-state and time-resolved thermal lens experiments. Good coincidence of the experimental and theoretically predicted signal dependences is achieved. This model is verified for volume-absorbing samples (colored optical glasses) and used successfully to calculate absorbances and concentrations for various surface-absorbing samples.

Optimization of Instrumental Parameters of a Near-Field Thermal-Lens Detector for Capillary Electrophoresis

The optical scheme of a near-field dual-beam mode-mismatched thermal-lens detector for capillary electrophoresis with a crossed-beam configuration employing a multimode HeCd laser (325 nm) as an excitation source was optimized. It is shown that a multimode laser can be successfully used as an excitation source in thermal lensing with minimal deviations in thermal responses from Gaussian excitation sources. An equation for diffraction thermal-lens theory for near-field measurements is deduced, and the experimental results agree with the deduced equation. The temperature rise in the capillary was estimated, and the exponential decrease of the signal with time for static conditions and low flow velocities was explained. The optimum configuration of the detector from the viewpoint of the maximum sensitivity and beam sizes was found. The detector provides a significant improvement in the detection limits for model compounds absorbing at 325 nm (nitrophenols) compared to the results obtained with a commercial absorbance detector operating at the same wavelength.

Pesticide analysis by MEKC on a microchip with hydrodynamic injection from organic extract

An integrated microchip for monitoring carbamate pesticides in environmental water using continuous flow chemical processes is under development, i. e., the integration of hydrolysis, azo-derivatization, liquid-liquid extraction, electrophoretic separation, and quantification. The separation of the derivatives of four carbamate pesticides (carbaryl, carbofuran, propoxur, and bendiocarb) extracted in the continuous flow of a 1-butanol phase was studied in a silica microchip using micellar EKC. A baseline separation of four pesticide derivatives was achieved on a silica chip using hydrodynamic injection with electroosmotic gating. Detection using a thermal lens microscope showed good linearity in the concentration range of 10(-6 )-10(-5 )M with an LOD of 5 x 10(-7) M, which is superior to that of conventional CE with UV absorption detection at a level of 10(-4) M.