We use a novel acoustic-based flow cytometer to detect individual nanobubbles flowing in a microfluidic channel using high-frequency ultrasound and photoacoustic waves. Each individual nanobubble (or cluster of nanobubbles) flowing through the foci of high-frequency ultrasound (center frequency 375 MHz) and nanosecond laser (532 nm) pulses interacts with both pulses to generate ultrasound backscatter and photoacoustic waves. We use in-house generated nanobubbles, made of lipid shells and octafluoropropane gas core, to detect ultrasound backscatter signals using an acoustic flow cytometer. Nanobubble solutions sorted in size through differential centrifugation are diluted to 1:10,000 v/v in phosphate buffered saline solution to maximize the probability that the detected signals are from individual nanobubbles. Nanobubble populations were sized using resonant mass measurement. Results show that the amplitude of the detected ultrasound backscatter signal is dependent on the nanobubble size. The average amplitude of the ultrasound backscatter signals from at least 950 nanobubbles with an average diameter of 150 nm, 225 nm, and 350 nm was 5.1±2.5 mV, 5.3±2.3 mV, and 6.4±1.8 mV, respectively. Similarly, we detected interleaved ultrasound backscatter and photoacoustic signals from nanobubbles tagged with Sudan Black B dye. The average amplitude of the ultrasound backscatter and photoacoustic signals from these black nanobubbles with an average diameter of 238 nm is 10±11 mV and 54±75 mV, respectively. The presence of the dye on the shell suppressed unique features seen in the ultrasound backscatter from the nanobubbles without dye. At present, there is no robust commercial technique able to analyze the ultrasonic response of individual nanobubbles. The acoustic flow cytometer can potentially be used to analyze physical parameters, such as size and ultrasonic response, of individual nanobubbles.
A cell’s nucleus-to-cytoplasm (N:C) ratio is a histological metric used to stage malignant disease. Current N:C assessment methods, such as optical microscopy, are time-consuming, subjective, and low-throughput. Here, we compare the N:C ratios of prostate cancer (PC-3) cells measured by a novel microfluidic PhotoAcoustic Flow Cytometer (PAFC) to those obtained using an Imaging Flow Cytometer (IFC). PC-3 cells were stained with DRAQ-5 nuclear dye and divided into populations measured using the PAFC and IFC. The PAFC consisted of a microfluidic device integrated with a singleelement ultrasound transducer (375 MHz central frequency) and a sub-nanosecond pulsed laser (532 nm). Individual cells were 3D flow-focused through the overlapping focal region of the ultrasound and laser pulses. PAFC estimation of the cell and nucleus diameters were determined through power spectra fitting of backscattered US waves and emitted PA waves to established theoretical models. An ImageStreamX® IFC was used to acquire brightfield and fluorescent images of individual cells, which were masked, gated, and used to assess the cell (brightfield) and nucleus (fluorescence) diameter to validate the PAFC measurements. The average cell and nucleus diameters determined using the PAFC (n = 388) were 18.8 ± 3.3 μm and 14.3 ± 2.9 μm, respectively. The corresponding values from the IFC (n = 4651) were 18.3 ± 2.2 μm and 12.2 ± 1.9 μm. The N:C ratio (calculated as the ratio of the nucleus diameter to cell diameter) was 0.77 ± 0.10 using the PAFC and 0.67 ± 0.07 using the IFC. Our novel PAFC device has the potential to be used for circulation tumor cell detection using the N:C ratios of cells.
We have developed a flow cytometer based on simultaneous detection of ultrasound and photoacoustic waves from individual particles/cells flowing in a microfluidic channel. Our polydimethylsiloxane (PDMS) based hydrodynamic 3-dimensional (3D) flow-focusing microfluidic device contains a cross-junction channel, a micro-needle (ID 100 μm and OD 200 μm) insert, and a 3D printed frame to hold and align a high frequency (center frequency 375 MHz) ultrasound transducer. The focused flow passes through a narrow focal zone with lateral and axial focal lengths of 6-8 μm and 15-20 μm, respectively. Both the lateral and axial alignments are achieved by screwing the transducer to the frame onto the PDMS device. Individual particles pass through an interrogation zone in the microfluidic channel with a collinearly aligned ultrasound transducer and a focused 532 nm wavelength laser beam. The particles are simultaneously insonified by high-frequency ultrasound and irradiated by a laser beam. The ultrasound backscatter and laser generated photoacoustic waves are detected for each passing particle. The backscattered ultrasound and photoacoustic signal are strongly dependent on the size, morphology, mechanical properties, and material properties of the flowing particles; these parameters can be extracted by analyzing unique features in the power spectrum of the signals. Frequencies less than 100 MHz do not have these unique spectral signatures. We show that we can reliably distinguish between different particles in a sample using the acoustic-based flow cytometer. This technique, when extended to biomedical applications, allows us to rapidly analyze the spectral signatures from individual single cells of a large cell population, with applications towards label-free detection and characterization of healthy and diseased cells.
Hydrodynamic 3D flow-focusing techniques in microfluidics are categorized as (a) sheathless techniques which require high flow rates and long channels, resulting in high operating cost and high flow rates which are inappropriate for applications with flow rate limitations, and (b) sheath-flow based techniques which usually require excessive sheath flow rate to achieve hydrodynamic 3D flow-focusing. Many devices based on these principles use complicated fabrication methods to create multi-layer microchannels. We have developed a sheath-flow based microfluidic device that is capable of hydrodynamic 3D self-flow-focusing. In this device the main flow (black ink) in a low speed, and a sheath flow, enter through two inlets and enter a 180 degree curved channel (300 × 300 μm cross-section). Main flow migrates outwards into the sheath-flow due to centrifugal effects and consequently, vertical focusing is achieved at the end of the curved channel. Then, two other sheath flows horizontally confine the main flow to achieve horizontal focusing. Thus, the core flow is three-dimensionally focused at the center of the channel at the downstream. Using centrifugal force for 3D flow-focusing in a single-layer fabricated microchannel has been previously investigated by few groups. However, their demonstrated designs required high flow speed (>1 m/s) which is not suitable for many applications that live biomedical specie are involved. Here, we introduce a new design which is operational in low flow speed (<0.05 m/s) and is suitable for applications involving live cells. This microfluidic device can be used in detecting, counting and isolating cells in many biomedical applications.
A flow cytometer that uses sound waves to determine the size of biological cells is presented. In this system, a microfluidic device made of polydimethylsiloxane (PDMS) was developed to hydrodynamically flow focus cells in a single file through a target area. Integrated into the microfluidic device was an ultrasound transducer with a 375 MHz center frequency, aligned opposite the transducer was a pulsed 532 nm laser focused into the device by a 10x objective. Each passing cell was insonfied with a high frequency ultrasound pulse, and irradiated with the laser. The resulting ultrasound and photoacoustic waves from each cell were analyzed using signal processing methods, where features in the power spectra were compared to theoretical models to calculate the cell size. Two cell lines with different size distributions were used to test the system: acute myeloid leukemia cells (AML) and melanoma cells. Over 200 cells were measured using this system. The average calculated diameter of the AML cells was 10.4 ± 2.5 μm using ultrasound, and 11.4 ± 2.3 μm using photoacoustics. The average diameter of the melanoma cells was 16.2 ± 2.9 μm using ultrasound, and 18.9 ± 3.5 μm using photoacoustics. The cell sizes calculated using ultrasound and photoacoustic methods agreed with measurements using a Coulter Counter, where the AML cells were 9.8 ± 1.8 μm and the melanoma cells were 16.0 ± 2.5 μm. These results demonstrate a high speed method of assessing cell size using sound waves, which is an alternative method to traditional flow cytometry techniques.
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