Submicron Fluorescent Particles Research Articles

Single-spot spiral phase plate for tracking 3D particle motion.

We have imaged and tracked submicron fluorescent particles in three dimensions by inserting a spiral phase plate (SPP) of our design into a conventional wide-field optical microscope. Our novel SPP consists of concentric annular zones that each impose a unique quantized orbital angular momentum (OAM) on the ring of fluorescent light it intercepts. Each ring of the SPP consists of a vortex surface that produces one additional unit of OAM than the smaller ring just inside it, with the seven successive rings each contributing n = 1 to 7 quantum units of OAM respectively. The light rings interfere to produce a single-spot rotating point spread function (SS-RPSF). This image ‘spot’ rotates with the axial depth of a particle, permitting 3D mapping of the particle position. The SPP is easily incorporated into existing microscopes. We used the DIC slider here for the SPP. 3D positions of fluorescent beads were retrieved using template matching. Bead positions could be inferred over a 6 µm depth range. Best results were obtained over a depth range of 2.3 μm, with a mean absolute error of 20 nm in this range. As proof-of-concept for live-cell imaging we tracked DNA loci and determined their diffusion coefficients in the 3D environment of the nucleus of U2OS cells. We are pursuing chromatin tracking experiments to better understand how DNA damage affects chromatin motions and, reciprocally, how chromatin dynamics impacts the DNA damage response. Beyond this specific application, our SPP-based imaging approach should enable researchers to track dynamic fluorescent objects in samples with high temporal resolution because x, y, and z are determined at exactly the same time.

Characterization of sub-pollen particles in size-resolved atmospheric aerosol using chemical tracers.

Pollen grains may contain allergens that exacerbate allergic respiratory diseases like asthma and rhinitis. In the presence of water, pollen grains (10–100 μm) can rupture to produce sub-pollen particles (SPP) with diameters <2.5 μm, which in comparison to intact pollen grains, have longer atmospheric lifetimes and greater penetration to the lower lung. The current study examines SPP, fungal spores, and bacteria in size-resolved atmospheric particulate matter (PM) using chemical and biological tracers. During springtime tree pollen season in Iowa City, Iowa, fine particle (PM2.5) concentrations of fructose (a pollen chemical tracer) increased on rainy sampling periods, especially during severe thunderstorms, and peaked when a tornado struck nearby. Submicron fluorescent particles, measured by single-particle fluorescence spectroscopy, were also enhanced during rain events, particularly thunderstorms in agreement with the chemical tracer measurements. PM2.5 sucrose (a pollen chemical tracer) concentrations were higher in early spring when nighttime temperatures were closer to freezing, while fructose concentrations were higher in late spring with warmer temperatures, consistent with chemical tracers being sensitive to seasonal temperature influences. The first co-located measurements of fructose and Bet v 1 (birch pollen allergen), indicated that SPP ranged in diameter from <0.25 to 2.5 μm during rainy sampling periods and that allergens and carbohydrates exhibited distinct size distributions. Meanwhile, mannitol (a fungal spore tracer) peaked on warm, dry days following rain and was primarily in supermicron particles (>1.0 μm), which is consistent with intact fungal spore diameters (1–30 μm). Bacterial endotoxins in PM also increased during extreme weather events, primarily in supermicron particles. While the concentrations of fructose, mannitol, and endotoxin all increased in PM2.5 μm during thunderstorms, the greatest relative increase in concentration was observed for fructose. Together, these observations suggest that SPP containing starch granules and allergens (Bet v 1) were released during rainy sampling periods. This study advances the use of chemical tracers to track SPP and other bioaerosols in the atmosphere, by providing new insight to their size distribution and response to extreme weather conditions.

Liquid flow pattern around Taylor bubbles in an etched rectangular microchannel

Liquid flow around Taylor bubbles and the motion of bubble interface in a rectangular microchannel etched on a microfluidic chip were investigated using a three-dimensional particle tracking method. The Taylor bubbles were generated by releasing the dissolved air in working the liquid (water) through heating the microfluidic chip to 35–55°C and had low velocities (15–1500μm/s). Three-dimensional velocity distributions of liquid recirculation flows surrounding the Taylor bubble head and tail were obtained by tracking submicron fluorescent particles seeded in the working liquid and the motion of the bubble interface was analyzed by monitoring the motions of the particles attached on the bubble interface. The high velocity film flow through the microchannel corners acted as a liquid jet in front of bubble head and drainage into the corners behind the bubble tail to drive the liquid recirculation flows. The bubble interface near the microchannel corners was also moved by the strong liquid shear induced from the high velocity liquid flow in the microchannel corners. This high velocity liquid flow through the corners could be considered to be driven by the pressure drop over the Taylor bubble. The pressure drop resulted from the decrease of bubble surface mobility due to tracer surfactant in the gas–liquid interface.

Liquid velocity distribution in slug flow in a microchannel

The liquid velocity distributions in two-phase slug flows in a nearly rectangular microchannel etched on a microfluidic chip were investigated using a three-dimensional tracking method for submicron fluorescent particles seeded in the working liquid (water). The Taylor bubbles generated from dissolved air in the water through heating the micro-fluidic chip to 35–55°C had low velocities, so they had the very small Capillary and Reynolds numbers. The change in the Taylor bubble shape with the flow conditions such as the bubble velocity and temperature was negligible in the present study. The shapes of the fully developed Taylor bubbles were determined by analyzing the interference fringes in in-line holographic images of the microchannel section containing the bubble to estimate the bubble cross-sectional area. The three-dimensional liquid velocity distribution for the two-phase slug flow was obtained using a least-squares fit of the measured tracer particle velocities to an analytic velocity distribution for Poiseuille flow in a rectangular channel to determine the mean liquid velocity and the liquid flow rate in the slug. The liquid velocity was normalized using the measured instantaneous bubble velocity to remove the influence of the slug and the bubble velocity fluctuations on the liquid velocity distribution. The results show that the mean liquid velocity through the microchannel corners is 2.3 times the bubble velocity, which is in agreement with previous observations of the maximum liquid velocity in the corners of 3.5–3.8 times the bubble velocity.

Pattern matching for three-dimensional tracking of sub-micron fluorescent particles

A microscope objective of NA = 0.6 designed for air medium has been used to generate diffraction patterns of sub-micron fluorescent particles with various defocus distances through a thick cover glass with a mismatched refractive index. The diffraction pattern centers were located by a cross-correlation analysis between the central Airy disk of the diffraction pattern and a template with the standard Airy intensity distribution. The locating accuracy of the diffraction pattern centers was 0.02–0.04 µm (0.1–0.2 pixels). Cross-correlation analyses were also performed to match the measured particle diffraction pattern to a calculated intensity distribution with the Gibson and Lanni scalar diffraction model to determine the particle defocus distance. The matching results showed that, unlike the product cross-correlation function, the least-squares error function, which has been extensively used to recognize objects in images, could be used to match the intensity distributions with a simple weight function. The defocus distances of particles under a 1.1 mm thick cover glass with the defocus distances larger than about 13 µm could be determined at an accuracy better than 0.1 µm by intensity distribution matching.

1501 ナノ・マイクロ熱流動センシングの最前線(OS15-1 ナノ・マイクロ流体のダイナミクス,オーガナイズドセッション)

Nano- and microfluidics have been a key issue for recent miniaturized flow systems. In a small confined space like microchannels, the electrostatic potential at the wall, i.e., the zeta-potential, significantly affects the flow structure and also induces electroosmotic flows under external electric fields. This paper presents fluorescence-based optical measuring techniques for the zeta-potential and fluid velocity to investigate electroosmotic flows with nonuniform zeta-potential. The evanescent wave and volume illumination are used as excitation lights for the near-wall and bulk regions, respectively. The zeta-potential is obtained imaging the near-wall fluorescent ions excited by the evanescent wave. On the other hand, the near-wall and bulk flow velocities are measured tracing the motion of submicron fluorescent particles. The spatial and temporal resolutions of velocity measurements are improved focusing on the reproducibility of laminar flows. These techniques will contribute to further development of flow systems using electrokinetics.

Direct measurement of slip velocities using three-dimensional total internal reflection velocimetry

The existence and magnitude of slip velocities between deionized water and a smooth glass surface is studied experimentally. Sub-micron fluorescent particles are suspended in water and imaged using total internal reflection velocimetry (TIRV). For water flowing over a hydrophilic surface, the measurements are in agreement with previous experiments and indicate that slip, if present, is minimal at low shear rates, but increases slightly as the shear rate increases. Surface hydrophobicity is observed to induce a small slip velocity, with the slip length reaching a maximum of 96 nm at a shear rate of 1800. Issues associated with the experimental technique and the interpretation of results are also discussed.

Three-dimensional tracking of fluorescent particles applied to micro-fluidic measurements

The diffraction patterns of submicron fluorescent particles were obtained using an air immersion objective (M = 40, NA = 0.6) under non-design optical conditions through a thick cover glass with a mismatched refractive index. The particle diffraction patterns changed with the defocus distance out of the front focal plane in the optical axis direction. A scalar diffraction model was applied to analyze the diffraction patterns for various optical conditions and to determine the defocus distance of the particle according to the measured intensity distribution of the diffraction pattern image. The results showed that the defocus distances have a submicron accuracy (0.2–0.3 µm), which can be used to determine the three-dimensional flow velocity distribution in a micro-channel and to track the three-dimensional Brownian motion of the particles for the specified defocus conditions.

Tracking sub-micron fluorescent particles in three dimensions with a microscope objective under non-design optical conditions

A microscope objective designed for air medium has been used under non-design optical conditions to focus an exciting laser beam on sub-micron fluorescent particles with the emitted fluorescence received through a glass layer with a mismatched refractive index. The diffraction pattern with several clear interference fringes generated from the fluorescence emitted from a fluorescent particle changed with the particle's position along the optical axis. A scalar diffraction model developed by Gibson and Lanni (1991 J. Opt. Soc. Am. A 8 1601–13) was used to predict the diffraction patterns for various aberration conditions and to analyse the effects of the coherence properties of the fluorescence on the details of the diffraction pattern. The particle position along the optical axis, i.e. its defocus distance, could be determined based on the characteristic sizes of the particle's diffraction pattern to track the particle in three dimensions.

Evaluation of Electroosmotic Velocity and Zeta-Potential in Microchannel Using Submicron Fluorescent Particles

Measurements of electrophoretic and electroosmotic velocities were performed by using micron-resolution particle image velocimetry (micro-PIV) and submicron fluorescent particles. Electrophoretic velocity of particles was obtained by micro-PIV measurement in a closed cell, while electroosmotic velocity of buffer solution in a microchannel was calculated by subtracting electrophoretic verocity from observed velocity measured by micro-PIV. Both zeta-potential of particles and microchannel wall were obtained by using electrophoretic and electroosmotic velocities, respectively, which are increased with increasing pH of buffer solution, due to deprotonation. Transient process of zeta-potential at the wall surface in a T-shaped microchannel was detected by spatially-averaged electroosmotic velocities.

Spatially averaged time-resolved particle-tracking velocimetry in microspace considering Brownian motion of submicron fluorescent particles

A time-series measurement method is proposed to detect velocity fields in a microchannel taking into account Brownian motion of submicron tracer particles. The present study proposes spatially averaged time-resolved particle-tracking velocimetry (SAT–PTV), which can detect temporal variations of fluid flow and eliminate errors associated with Brownian motion without losing temporal resolution. Velocity vectors of tracer particles obtained by PTV are spatially averaged in each interrogation window of particle-image velocimetry, yielding full velocity field information with temporal resolution. Synthetic particle images, which include Brownian motion of submicron fluorescent particles in flow fields with linear velocity gradients, are generated to validate the ability of SAT–PTV to track particles. SAT–PTV correctly captures the velocity gradient profiles. The spatial resolution based on the size of the first interrogation window and the measurement depth of the microscope system is 6.7 μm×6.7 μm×1.9 μm, within which several vectors are averaged. SAT–PTV is shown to measure the velocity field of a pulsating flow generated by an electrokinetic pump.

Flow Measurements in Microspace Using Sub-Micron Fluorescent Particles. Proposal of Spatial Averaged Time-Resolved PTV Considering Brownian Motion of Tracer Particles.

A measurement technique for a flow field in a microchannel was developed using sub-micron fluorescent particles. The present study focused on a novel measurement technique so called Spatial Averaged Time-resolved Particle Tracking Velocimetry (SAT-PTV) which is able to detect the temporal variation of a fluid flow eliminating the effect of Brownian motion of the sub-micron tracer particles. Velocity vectors of individual tracer particles were averaged within a local small area, instead of temporal aerage, to realize higher time resolution. SAT-PTV method was evaluated by synthetic particle images in both a uniform flow and a flow with linear velocity gradient and the method confirmed the reduction of the measurement errors associated with Brownian motion. The validation of the present technique was performed in a microchannel flow with the time resolution of 37 ms.

B202 サブミクロン蛍光粒子を用いた界面動電現象による流れ場計測(オーガナイズドセッション10 : マイクロビジュアリゼーション)

An electrokinetic flow field in a microchannel was investigated by a PIV system using a microscope and sub-micron fluorescent particles. The present study focused on quantitative measurements of electroosmotic flow eliminating velocities of tracer particles induced by electrophoresis, which was calculated by measurement velocity in a cell, comprised of electroosmotic flow and electrophoresis. Quantitative velocity profiles of electroosmotic flow were obtained subtracting velocities of tracer particles induced by electrophoresis from measured velocities. It was observed that velocity profiles of electroosmotic flow had a reverse parabolic shape in the center region of the microchannel and were uniform near the wall.