High throughput imaging cytometer with acoustic focussing

Zmijan, Robert; Jonnalagadda, Umesh Sai; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn; Glynne‐Jones, Peter

doi:10.1039/c5ra19497k

Cited by 28 publications

(30 citation statements)

References 54 publications

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“…Two additional yet essential elements of VIFFI flow cytometry that maximize the effect of virtual freezing are a light-sheet excitation beam scanner that scans over the entire field of view (FOV) during the exposure time of the image sensor and the precise synchronization of the timings of the image sensor's exposure and the excitation beam's illumination and localization with respect to the rotation angle of the polygon scanner. As a result of combining these elements, our virtual-freezing strategy effectively enables 1000 times longer signal integration time on the image sensor, far surpassing previous techniques 23,24 , and hence achieves microscopy-grade fluorescence imaging of cells at a high flow speed of 1 m s −1 .…”

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confidence: 99%

Virtual-freezing fluorescence imaging flow cytometry

et al. 2020

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By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually freezing the motion of flowing cells on the image sensor to effectively achieve 1000 times longer exposure time for microscopygrade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells s −1 without sacrificing sensitivity and spatial resolution. The availability of numerous information-rich fluorescence cell images allows high-dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology.

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confidence: 99%

Virtual-freezing fluorescence imaging flow cytometry

et al. 2020

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“…25,26 Rather, we are simply using the sCMOS detector as an approximate 1D array that simplifies direct collection of data across a wide field of view.…”

Section: Methodsmentioning

confidence: 99%

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry

et al. 2017

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Flow cytometry provides highly sensitive multi-parameter analysis of cells and particles, but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ~50K particles/s and the volumetric rate to ~250 μl/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g. circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g. water quality, urine analysis), or high throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3-mm wide optical flow cell. A line focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line focused laser, particle focusing using multi-node acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple and cost effective platform. Such instruments will be of great value to applications in need of high throughput yet sensitive flow cytometry analysis.

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“…Streaming flow velocities were measured using particle image velocimetry (PIV) as described in Zmijan et al 70 1 μm fluores-cent polystyrene beads were used as tracers, imaging with a 10× objective at 400 ms frame intervals. Image data was analyzed via the MATLAB package MPIV 70 to quantify bead displacement and mean particle velocity between sequential frames. Following particle analysis, the median of each frame's particle velocities was established.…”

Section: Acoustic Streaming Characterizationmentioning

confidence: 99%

Acoustically modulated biomechanical stimulation for human cartilage tissue engineering

et al. 2018

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Bioacoustofluidics can be used to trap and levitate cells within a fluid channel, thereby facilitating scaffold-free tissue engineering in a 3D environment. In the present study, we have designed and characterised an acoustofluidic bioreactor platform, which applies acoustic forces to mechanically stimulate aggregates of human articular chondrocytes in long-term levitated culture. By varying the acoustic parameters (amplitude, frequency sweep, and sweep repetition rate), cells were stimulated by oscillatory fluid shear stresses, which were dynamically modulated at different sweep repetition rates (1-50 Hz). Furthermore, in combination with appropriate biochemical cues, the acoustic stimulation was tuned to engineer human cartilage constructs with structural and mechanical properties comparable to those of native human cartilage, as assessed by immunohistology and nano-indentation, respectively. The findings of this study demonstrate the capability of acoustofluidics to provide a tuneable biomechanical force for the culture and development of hyaline-like human cartilage constructs in vitro.

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High throughput imaging cytometer with acoustic focussing

Abstract: Acoustic plane focussing and a scanning mirror enhances throughput of an imaging cytometer.

Cited by 28 publications

References 54 publications

Virtual-freezing fluorescence imaging flow cytometry

Virtual-freezing fluorescence imaging flow cytometry

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry

Acoustically modulated biomechanical stimulation for human cartilage tissue engineering

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