Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

Microfluidic Separation and Enrichment of Escherichia coli by Size Using Viscoelastic Flows.

Here, we achieve the separation and enrichment of Escherichia coli clusters from its singlets in a viscoelastic microfluidic device. E. coli, an important prokaryotic model organism and a widely used microbial factory, can aggregate in clusters, leading to biofilm development that can be detrimental to human health and industrial processes. The ability to obtain high-purity populations of E. coli clusters is of significance for biological, biomedical, and industrial applications. In this study, polystyrene particles of two different sizes, 1 and 4.8 µm, are used to mimic E. coli singlets and clusters, respectively. Experimental results show that particles migrate toward the channel center in a size-dependent manner, due to the combined effects of inertial and elastic forces; 4.8 and 1 µm particles are found to have lateral equilibrium positions closer to the channel centerline and sidewalls, respectively. The size-dependent separation performance of the microdevice is demonstrated to be affected by three main factors: channel length, the ratio of sheath to sample flow rate, and poly(ethylene oxide) (PEO) concentration. Further, the separation of E. coli singlets and clusters is achieved at the outlets, and the separation efficiency is evaluated in terms of purity and enrichment factor.

Inertial Separation of Particles Assisted by Symmetrical Sheath Flows in a Straight Microchannel.

Over the past two decades, inertial microfluidics, which works at an intermediate range of Reynolds number (∼1 < Re < ∼100), has been widely used for particle separation due to its high-throughput and label-free features. This work proposes a novel method for continuous separation of particles by size using inertial microfluidics, with the assistance of symmetrical sheath flows in a straight microchannel. Here, larger particles (>3 µm) are arranged close to the channel sidewalls, while smaller particles (<2 µm) remain flowing along the channel centerline. This conclusion is supported by experimental data with particles of different sizes ranging from 0.79 to 10.5 µm. Symmetrical Newtonian sheath flows are injected on both sides of particle mixtures into a straight rectangular microchannel with an aspect ratio (AR = height/width) of 2.5. Results show that the separation performance of the developed microfluidic device is affected by three main factors: channel length, total flow rate, and flow rate ratio of sheath to sample. Besides, separation of platelets from whole blood is demonstrated. The developed microfluidic platform owns the advantages of low fabrication cost, simple experiment setup, versatile selections of particle candidates, and stable operations. This systematic study provides a new perspective for particle separation, which is expected to find applications across various fields spanning physics, biology, biomedicine, and industry.

Trihelix transcription factors GTL1 and DF1 prevent aberrant root hair formation in an excess nutrient condition.

Root hair growth is tuned in response to the environment surrounding plants. While most previous studies focused on the enhancement of root hair growth during nutrient starvation, few studies investigated the root hair response in the presence of excess nutrients. We report that the post-embryonic growth of wild-type Arabidopsis plants is strongly suppressed with increasing nutrient availability, particularly in the case of root hair growth. We further used gene expression profiling to analyze how excess nutrient availability affects root hair growth, and found that RHD6 subfamily genes, which are positive regulators of root hair growth, are downregulated in this condition. However, defects in GTL1 and DF1, which are negative regulators of root hair growth, cause frail and swollen root hairs to form when excess nutrients are supplied. Additionally, we observed that the RHD6 subfamily genes are mis-expressed in gtl1-1 df1-1. Furthermore, overexpression of RSL4, an RHD6 subfamily gene, induces swollen root hairs in the face of a nutrient overload, while mutation of RSL4 in gtl1-1 df1-1 restore root hair swelling phenotype. In conclusion, our data suggest that GTL1 and DF1 prevent unnecessary root hair formation by repressing RSL4 under excess nutrient conditions.

Identification of Single Yeast Budding Using Impedance Cytometry with a Narrow Electrode Span.

Impedance cytometry is wildly used in single-cell detection, and its sensitivity is essential for determining the status of single cells. In this work, we focus on the effect of electrode gap on detection sensitivity. Through comparing the electrode span of 1 µm and 5 µm, our work shows that narrowing the electrode span could greatly improve detection sensitivity. The mechanism underlying the sensitivity improvement was analyzed via numerical simulation. The small electrode gap (1 µm) allows the electric field to concentrate near the detection area, resulting in a high sensitivity for tiny particles. This finding is also verified with the mixture suspension of 1 µm and 3 µm polystyrene beads. As a result, the electrodes with 1 µm gap can detect more 1 µm beads in the suspension than electrodes with 5 µm gap. Additionally, for single yeast cells analysis, it is found that impedance cytometry with 1 µm electrodes gap can easily distinguish budding yeast cells, which cannot be realized by the impedance cytometry with 5 µm electrodes gap. All experimental results support that narrowing the electrode gap is necessary for tiny particle detection, which is an important step in the development of submicron and nanoscale impedance cytometry.

Mechanosensitive axon outgrowth mediated by L1-laminin clutch interface.

Mechanical properties of the extracellular environment modulate axon outgrowth. Growth cones at the tip of extending axons generate traction force for axon outgrowth by transmitting the force of actin filament retrograde flow, produced by actomyosin contraction and F-actin polymerization, to adhesive substrates through clutch and cell adhesion molecules. A molecular clutch between the actin filament flow and substrate is proposed to contribute to cellular mechanosensing. However, the molecular identity of the clutch interface responsible for mechanosensitive growth cone advance is unknown. We previously reported that mechanical coupling between actin filament retrograde flow and adhesive substrates through the clutch molecule shootin1a and the cell adhesion molecule L1 generates traction force for axon outgrowth and guidance. Here, we show that cultured mouse hippocampal neurons extend longer axons on stiffer substrates under elastic conditions that correspond to the soft brain environments. We demonstrate that this stiffness-dependent axon outgrowth requires actin-adhesion coupling mediated by shootin1a, L1, and laminin on the substrate. Speckle imaging analyses showed that L1 at the growth cone membrane switches between two adhesive states: L1 that is immobilized and that undergoes retrograde movement on the substrate. The duration of the immobilized phase was longer on stiffer substrates; this was accompanied by increases in actin-adhesion coupling and in the traction force exerted on the substrate. These data suggest that the interaction between L1 and laminin is enhanced on stiffer substrates, thereby promoting force generation for axon outgrowth.

Relationship between surrounding tissue morphology and directional collective migration of the posterior lateral line primordium in zebrafish.

Collective cell migration, in which cells assemble and move together, is an essential process in embryonic development, wound healing and cancer metastasis. Chemokine signaling guides cell assemblies to their destinations. In zebrafish posterior lateral line primordium (PLLP), a model system for collective cell migration, it has been proposed that the chemokine ligand Cxcl12a secreted from muscle pioneer cells (MPs) and muscle fast fibers (MFFs), which are distributed along with the horizontal midline, binds to the receptor Cxcr4b in PLLP and that Cxcl12a-Cxcr4b signaling guides the anterior-to-posterior migration of PLLP along the horizontal midline. However, how the surrounding tissues affect PLLP migration remains to be elucidated. Here, we investigated the relationship between the PLLP and the surrounding tissues and found that a furrow between the dorsal and ventral myotomes is generated by Sonic hedgehog (Shh) signaling-dependent MP and MFF differentiation and that the PLLP migrates in this furrow. When transient inhibition of Shh signaling impaired both the furrow formation and differentiation of cxcl12a-expressing MPs/MFFs, directional PLLP migration was severely perturbed. Furthermore, when differentiated MPs and MFFs were ablated by femtosecond laser irradiations, the furrow remained and PLLP migration was relatively unaffected. These results suggest that the furrow formation between the dorsal and ventral myotomes is associated with the migratory behavior of PLLP.

Super-resolution for a dispersive spectrometer using a tilted area sensor and spectrally varying blur kernel interpolation.

The grating, lens, and linear sensor determine a spectrometer's wavelength resolution and measurement range. While conventional methods have tried to improve the optical design to obtain a better resolution, they have a limitation caused by the physical property. To improve the resolution, we introduce a super-resolution method from the computer vision field. We propose tilting an area sensor to realize accurate subpixel shifting and recover a high-resolution spectrum using interpolated spectrally varying kernels. We experimentally validate that the proposed method achieved a high spectral resolution of 0.141nm in 400-800nm by just tilting the sensor in the spectrometer.

Effects of Flow-Induced Microfluidic Chip Wall Deformation on Imaging Flow Cytometry.

Imaging flow cytometry is a powerful tool by virtue of its capability for high-throughput cell analysis. The advent of high-speed optical imaging methods on a microfluidic platform has significantly improved cell throughput and brought many degrees of freedom to instrumentation and applications over the last decade, but it also poses a predicament on microfluidic chips. Specifically, as the throughput increases, the flow speed also increases (currently reaching 10 m/s): consequently, the increased hydrodynamic pressure on the microfluidic chip deforms the wall of the microchannel and produces detrimental effects lead to defocused and blur image. Here, we present a comprehensive study of the effects of flow-induced microfluidic chip wall deformation on imaging flow cytometry. We fabricated three types of microfluidic chips with the same geometry and different degrees of stiffness made of polydimethylsiloxane (PDMS) and glass to investigate material influence on image quality. First, we found the maximum deformation of a PDMS microchannel was >60 µm at a pressure of 0.6 MPa, while no appreciable deformation was identified in a glass microchannel at the same pressure. Second, we found the deviation of lag time that indicating velocity difference of migrating microbeads due to the deformation of the microchannel was 29.3 ms in a PDMS microchannel and 14.9 ms in a glass microchannel. Third, the glass microchannel focused cells into a slightly narrower stream in the X-Y plane and a significantly narrower stream in the Z-axis direction (focusing percentages were increased 30%, 32%, and 5.7% in the glass channel at flow velocities of 0.5, 1.5, and 3 m/s, respectively), and the glass microchannel showed stabler equilibrium positions of focused cells regardless of flow velocity. Finally, we achieved the world's fastest imaging flow cytometry by combining a glass microfluidic device with an optofluidic time-stretch microscopy imaging technique at a flow velocity of 25 m/s. © 2019 International Society for Advancement of Cytometry.

Efficacy of femtosecond lasers for application of acupuncture therapy.

Acupuncture treatment utilizes the stimulation of metal acupuncture needles that are manually inserted into a living body. In the last decades, laser light has been used as an alternative to needles to stimulate acupuncture points. We previously reported suppression of myostatin (Mstn) gene expression in skeletal muscle by means of femtosecond laser (FL) irradiation, after electroacupuncture, in which acupuncture needles are stimulated with a low-frequency microcurrent. The purpose of the study here was to investigate the efficacy of FL irradiation in mouse skeletal muscle with regard to protein synthesis. After irradiation of the hindlimbs, we first analyzed Mstn gene expression and Mstn protein level in the skeletal muscle. We then evaluated phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream target 70-kDa ribosomal protein S6 kinase (p70S6K). The results showed that FL irradiation significantly reduced the amount of Mstn protein and enhanced the phosphorylation of p70S6K in of the mTOR/S6K signaling pathway. We suggest that FL irradiation activated the protein synthetic pathway in the skeletal muscle. In conclusion, we determined that FL irradiation can serve as an alternative for acupuncture needles and has the potential of being a new non-invasive acupuncture treatment of skeletal muscle.

Time-Course Statistical Evaluation of Intercellular Adhesion Maturation by Femtosecond Laser Impulse.

The maturation of intercellular adhesion is an essential process for establishing the signal transduction network in living cells. Although the maturation is naturally considered to enhance the signal transduction, the relationship between the signal transduction and the maturation process has not been revealed in detail using time-course data. Here, using a coculture of mast cells and neurites, differences in maturation between individual cells were estimated as a function of the adhesion strength by our original single-cell measurement method utilizing a laser-induced impulsive force. When an intense femtosecond laser is focused into a culture medium under a microscope, shock and stress waves are generated at the laser focal point that exert an impulsive force on individual cells. In our method, this impulse is used to break the adhesion between a mast cell and a neurite. The magnitude of the impulse is then quantified by a local force-measurement system utilizing an atomic force microscope, and the adhesion strength is estimated from the threshold of the impulse required to break the adhesion. The measurement is conducted within 1 min/cell, and thus, data on the individual differences of the adhesion strength can be obtained within only a few hours. Coculturing of neurites and mast cells for 4 h resulted in a specific adhesion that was stronger than the nonspecific adhesions between the substrate and mast cells. In the time-course investigation, we identified two distinct temporal patterns of adhesion: 1) the strength at 24 h was the same as the initial strength; and 2) the strength increased threefold from baseline and became saturated within 24 h. Based on these results, the distribution of CADM1 adhesion molecules in the neurites was suggested to be inhomogeneous, and the relationship between adhesion maturation and the signal-transduction process was considered.

Dynamics of the Light-Dependent Transition of Plant Peroxisomes.

Peroxisomes are present in almost all plant cells. These organelles are involved in various metabolic processes, such as lipid catabolism and photorespiration. A notable feature of plant peroxisomes is their flexible adaptive responses to environmental conditions such as light. When plants shift from heterotrophic to autotrophic growth during the post-germinative stage, peroxisomes undergo a dynamic response, i.e. enzymes involved in lipid catabolism are replaced with photorespiratory enzymes. Although the detailed molecular mechanisms underlying the functional transition of peroxisomes have previously been unclear, recent analyses at the cellular level have enabled this detailed machinery to be characterized. During the functional transition, obsolete enzymes are degraded inside peroxisomes by Lon protease, while newly synthesized enzymes are transported into peroxisomes. In parallel, mature and oxidized peroxisomes are eliminated via autophagy; this functional transition occurs in an efficient manner. Moreover, it has become clear that quality control mechanisms are important for the peroxisomal response to environmental stimuli. In this review, we highlight recent advances in elucidating the molecular mechanisms required for the regulation of peroxisomal roles in response to changes in environmental conditions.

Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy.

When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (O(f)) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between O(f) and the cantilever, the fsLP-IF generated at O(f) was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte-endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O(f) and the adhesive interface, were on the order of 10(-13) and 10(-12) N-s, respectively. When the total impulse at O(f) is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.

Passive microfluidic devices for cell separation.

The separation of specific cell populations is instrumental in gaining insights into cellular processes, elucidating disease mechanisms, and advancing applications in tissue engineering, regenerative medicine, diagnostics, and cell therapies. Microfluidic methods for cell separation have propelled the field forward, benefitting from miniaturization, advanced fabrication technologies, a profound understanding of fluid dynamics governing particle separation mechanisms, and a surge in interdisciplinary investigations focused on diverse applications. Cell separation methodologies can be categorized according to their underlying separation mechanisms. Passive microfluidic separation systems rely on channel structures and fluidic rheology, obviating the necessity for external force fields to facilitate label-free cell separation. These passive approaches offer a compelling combination of cost-effectiveness and scalability when compared to active methods that depend on external fields to manipulate cells. This review delves into the extensive utilization of passive microfluidic techniques for cell separation, encompassing various strategies such as filtration, sedimentation, adhesion-based techniques, pinched flow fractionation (PFF), deterministic lateral displacement (DLD), inertial microfluidics, hydrophoresis, viscoelastic microfluidics, and hybrid microfluidics. Besides, the review provides an in-depth discussion concerning cell types, separation markers, and the commercialization of these technologies. Subsequently, it outlines the current challenges faced in the field and presents a forward-looking perspective on potential future developments. This work hopes to aid in facilitating the dissemination of knowledge in cell separation, guiding future research, and informing practical applications across diverse scientific disciplines.

Enhanced nerve-mast cell interaction by a neuronal short isoform of cell adhesion molecule-1.

Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve-mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a-d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve-mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and crosslinking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca(2+) concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve-mast cell interaction, and they suggest that nerve-mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant.

Machine learning implementation strategy in imaging and impedance flow cytometry.

Imaging and impedance flow cytometry is a label-free technique that has shown promise as a potential replacement for standard flow cytometry. This is due to its ability to provide rich information and archive high-throughput analysis. Recently, significant efforts have been made to leverage machine learning for processing the abundant data generated by those techniques, enabling rapid and accurate analysis. Harnessing the power of machine learning, imaging and impedance flow cytometry has demonstrated its capability to address various complex phenotyping scenarios. Herein, we present a comprehensive overview of the detailed strategies for implementing machine learning in imaging and impedance flow cytometry. We initiate the discussion by outlining the commonly employed setup to acquire the data (i.e., image or signal) from the cell. Subsequently, we delve into the necessary processes for extracting features from the acquired image or signal data. Finally, we discuss how these features can be utilized for cell phenotyping through the application of machine learning algorithms. Furthermore, we discuss the existing challenges and provide insights for future perspectives of intelligent imaging and impedance flow cytometry.

Microsecond cell triple-sorting enabled by multiple pulse irradiation of femtosecond laser.

Femtosecond-laser-assisted cell manipulation, as one of the high throughput cell sorting techniques, is tailored for single-step multiple sorting based on controllable impulsive force. In this paper, femtosecond laser pulses are focused within a pocket structure and they induce an impulse force acting on the flowing objects. The impulsive force is shown to be controllable by a new method to adjust the femtosecond pulse properties. This allows precise streamline manipulation of objects having various physical qualities (e.g., weight and volume). The pulse energy, pulse number, and pulse interval of the femtosecond laser are altered to determine the impulsive force strength. The method is validated in single cell or bead triple-sorting experiments and its capability to perform streamline manipulation in as little as 10 µs is shown. The shift profiles of the beads acting under the impulsive force are studied in order to better understand the sorting mechanism. Additionally, beads and cells with different fluorescence intensities are successfully detected and directed into different microchannels, with maximum success rates of 90% and 64.5%, respectively. To sum up, all results suggest that this method has the potential to sort arbitrary subpopulations by altering the number of femtosecond pulses and that it takes the first step toward developing a single-step multi-selective system.

A review on intelligent impedance cytometry systems: Development, applications and advances.

Impedance cytometry is a well-established technique for counting and analyzing single cells, with several advantages, such as convenience, high throughput, and no labeling required. A typical experiment consists of the following steps: single-cell measurement, signal processing, data calibration, and particle subtype identification. At the beginning of this article, we compared commercial and self-developed options extensively and provided references for developing reliable detection systems, which are necessary for cell measurement. Then, a number of typical impedance metrics and their relationships to biophysical properties of cells were analyzed with respect to the impedance signal analysis. Given the rapid advances of intelligent impedance cytometry in the past decade, this article also discussed the development of representative machine learning-based approaches and systems, and their applications in data calibration and particle identification. Finally, the remaining challenges facing the field were summarized, and potential future directions for each step of impedance detection were discussed.

Patterned proliferation orients tissue-wide stress to control root vascular symmetry in Arabidopsis.

Symmetric tissue alignment is pivotal to the functions of plant vascular tissue, such as long-distance molecular transport and lateral organ formation. During the vascular development of the Arabidopsis roots, cytokinins initially determine cell-type boundaries among vascular stem cells and subsequently promote cell proliferation to establish vascular tissue symmetry. Although it is unknown whether and how the symmetry of initially defined boundaries is progressively refined under tissue growth in plants, such boundary shapes in animal tissues are regulated by cell fluidity, e.g., cell migration and intercalation, lacking in plant tissues. Here, we uncover that cell proliferation during vascular development produces anisotropic compressive stress, smoothing, and symmetrizing cell arrangement of the vascular-cell-type boundary. Mechanistically, the GATA transcription factor HANABA-TARANU cooperates with the type-B Arabidopsis response regulators to form an incoherent feedforward loop in cytokinin signaling. The incoherent feedforward loop fine-tunes the position and frequency of vascular cell proliferation, which in turn restricts the source of mechanical stress to the position distal and symmetric to the boundary. By combinatorial analyses of mechanical simulations and laser cell ablation, we show that the spatially constrained environment of vascular tissue efficiently entrains the stress orientation among the cells to produce a tissue-wide stress field. Together, our data indicate that the localized proliferation regulated by the cytokinin signaling circuit is decoded into a globally oriented mechanical stress to shape the vascular tissue symmetry, representing a reasonable mechanism controlling the boundary alignment and symmetry in tissue lacking cell fluidity.

An optimized PDMS microfluidic device for ultra-fast and high-throughput imaging flow cytometry.

Imaging flow cytometry (IFC) is a powerful tool for cell detection and analysis due to its high throughput and compatibility in image acquisition. Optical time-stretch (OTS) imaging is considered as one of the most promising imaging techniques for IFC because it can realize cell imaging at a flow speed of around 60 m s-1. However, existing PDMS-based microchannels cannot function at flow velocities higher than 10 m s-1; thus the capability of OTS-based IFC is significantly limited. To overcome the velocity barrier for PDMS-based microchannels, we proposed an optimized design of PDMS-based microchannels with reduced hydraulic resistance and 3D hydrodynamic focusing capability, which can drive fluids at an ultra-high flow velocity (of up to 40 m s-1) by using common syringe pumps. To verify the feasibility of our design, we fabricated and installed the microchannel in an OTS IFC system. The experimental results first proved that the proposed microchannel can support a stable flow velocity of up to 40 m s-1 without any leakage or damage. Then, we demonstrated that the OTS IFC is capable of imaging cells at a velocity of up to 40 m s-1 with good quality. To the best of our knowledge, it is the first time that IFC has achieved such a high flow velocity just by using a PDMS-glass chip. Moreover, high velocity can enhance the focusing of cells on the optical focal plane, increasing the number of detected cells and the throughput. This work provides a promising solution for IFC to fully release its capability of advanced imaging techniques by operating at an extremely high screening throughput.