Accurate and quantitative classification of cell death pathways is fundamental for elucidating disease mechanisms and assessing therapeutic efficacy, as dysregulated cell death underlies a wide range of pathological conditions including cancer and therapy resistance. However, conventional imaging methods such as fluorescence and bright-field microscopy, or 2D phase imaging, often suffer from phototoxicity, labeling artifacts, or limited morphological contrast. Herein, a real-time, label-free platform for classifying cell death phenotypes-apoptosis, necroptosis, and necrosis-is introduced by combining 3D holotomography with deep learning. A convolutional neural network, trained on refractive index (RI)-based features from HeLa cells, achieves high classification accuracy (99.3%) under varying cell densities. Notably, the model detected early RI changes during necroptosis several hours before conventional fluorescence-based markers, validated by population-level comparison with flow cytometry. The framework's adaptability is demonstrated by successfully fine-tuning the model for A549 lung cancer cells. Collectively, these findings demonstrate the potential of HT-based AI as a universal, high-resolution, and label-free platform for quantitative cell death profiling and translational drug-response analysis.

Accurate classification of cell death pathways is critical in understanding disease mechanisms and evaluating therapeutic responses, as dysregulated cell death underlies a wide range of pathological conditions including cancer and therapy resistance. Conventional imaging methods such as fluorescence and bright-field microscopy, or 2D phase imaging, often suffer from phototoxicity, labeling artifacts, or limited morphological contrast. Here, we present a real-time, label-free platform for classifying cell death phenotypes—apoptosis, necroptosis, and necrosis—by combining three-dimensional holotomography with deep learning. Our convolutional neural network, trained on refractive index (RI)-based features from HeLa cells, achieved high classification accuracy (97.2 ± 2.8%) under varying cell densities. Notably, the model identified early RI changes during necroptosis several hours prior to fluorescence-based markers. These findings demonstrate the potential of holotomography-based AI for high-resolution, label-free cell death profiling. ### Competing Interest Statement Y.K.P., S.O., J.D., and J.P. have financial interests in Tomocube, a company that commercializes HT instruments. All other authors declare no competing interests. National Research Foundation of Korea, RS-2024-00442348, 2022M3H4A1A02074314

Abstract Background: Immune Cell Killing (ICK) Assay is pivotal in immunology and clinical research, uncovering the intricate connections between the immune system and diseases. Traditional ICK assays utilizing antibody staining for endpoint data generation pose challenges in capturing dynamic interactions. Moreover, antibody labeling for data quantification may compromise sensitivity, especially with low-affinity antibodies or a limited number of cells. The anticipation of label-free lymphocyte subset classification and real-time effector/target cell interaction analysis in one field of view is expected to provide more valuable insights than traditional ICK assays. Previous studies achieved 70-80% accuracy in image-based lymphocyte subset classification using digital holographic microscopy, light scattering, etc. but the cells’ condition during the cell sorting or sample preparation process may affect the lymphocyte subset status, resulting in real morphological discrepancies. Our approach focuses on directly sorting human PBMCs to enable label-free, dynamic interaction analysis of natural immune system responses in one field of view. Method: In this study, PBMC samples from 6 individuals were separated using standard-density gradient centrifugation and labeled with antibodies. Data were acquired using the Holotomographic Microscope HT-X1 (Tomocube Inc.). We employed Densenet 121 as the deep learning model architecture, considering the trade-off between memory consumption and performance. The cross-entropy loss function was utilized, and the AdamW optimizer was chosen for model parameter optimization. Input data consisted of 3D refractive index (RI) data for individual cells, each of sized (20, 54, 54). Individual cell labeling was performed using staining information. Results: An overall accuracy of 93.75% in Human PBMC subtype cell classification was achieved. In stage 1, an accuracy of 97.56% was demonstrated in classifying [CD14, CD15, Others]. Among the cells classified as "Others" in stage 2, [CD3, CD19, CD16&CD56] displayed an accuracy of 93.5%. In stage 3, classifying [CD4, CD8] within the CD3 subtype achieved an accuracy of 90.2%. This classification is presumably based on pattern recognition of the chromosomal landscape in holotomography images. Conclusion: Our research has successfully demonstrated the feasibility of real-time label-free lymphocyte subset classification. This approach enabled the classification of effector cells as live cells in ICK assay and the analysis of effector-target cell dynamic interactions. Further training in the classification of more detailed subtype cells and various cell death types will provide additional insights. The advancement of this technology is expected to play a pivotal role in disease prevention, treatment, vaccine development, medical research, and academic studies. Citation Format: Sanggeun Oh, Jaephil Do, Hyun-Seok Min, Dongmin Ryu, Wei sun Park. Advancing ICK assay: Real-time, label-free imaging of lymphocyte subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2530.

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

We present a new rapid prototyping method designed for simple fabrication of 3D microfluidics using a maskless direct writing technique on polymer substrates. The entire process is enabled by a commercial cutter plotter with 10 μm resolution precision and high speed. A CAD design of top and bottom microstructures is directly written on a polymer substrate using a cutter plotter after setting up the suitable force. The smallest channel width of 20 μm was obtained with the minimum force and 100 μm from the maximum. Also the written depth increased linearly with force from 30 to 130 μm. Several 3D microfluidic devices are demonstrated using a maskless writing technique. The entire fabrication process from CAD layout to a final 3D device can be completed in 30 min outside the clean room facilities.

We have developed a portable, simple to fabricate, and highly effective polymeric microfluidic sample concentration device to increase the speed and sensitivity of molecular diagnostics of influenza from patient respiratory samples. This method is more friendly than off-chip methods to in-line on chip processing since it obviates the need for a centrifuge. The disposable chip fabricated with xurography utilizes evaporation and interfacial dragging effects. Up to 18 times concentration for viral RNA from influenza A virions was achieved by evaporating 1 mL to 50 µL in 0.5 hour or less with an average recovery of 60.3% (+/-20.5%) from patient specimens.

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium.

We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from liquid samples using enhanced evaporation targeted at downstream detection using surface enhanced Raman spectroscopy (SERS). The device is composed of a poly(dimethylsiloxane) (PDMS) liquid sample flow layer, a reusable metal airflow layer, and a porous PTFE (Teflon™) membrane sandwiched in between the liquid and air layers. The concentration capacity of the device was successfully demonstrated with fluorescently tagged Escherichia coli (E. coli). The recovery concentration was above 85% for all initial concentrations lower than 1 × 10(4) CFU mL(-1). In the lowest initial concentration cases, 100 µL initial volumes of bacteria solution at 100 CFU mL(-1) were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min. Subsequent tests with SERS on clinically relevant Methicillin-Sensitive Staphylococcus aureus (MSSA) after concentration in this device proved more than 100-fold enhancement in SERS signal intensity compared to the signal obtained from the unconcentrated sample. The concentration device is straightforward to design and use, and as such could be used in conjunction with a number of detection technologies.

A new method for the self-assembly of a carbon nanotube (CNT) using magnetic capturing and fluidic alignment has been developed and characterized in this work. In this new method, the residual iron (Fe) catalyst positioned at one end of the CNT was utilized as a self-assembly driver to attract and position the CNT, while the assembled CNT was aligned by the shear force induced from the fluid flow through the assembly channel. The self-assembly procedures were successfully developed and the electrical properties of the assembled multi-walled carbon nanotube (MWNT) and single-walled carbon nanotube (SWNT) were fully characterized. The new assembly method developed in this work shows its feasibility for the precise self-assembly of parallel CNTs for electronic devices and nanobiosensors.

An on-site analyzer system for monitoring of heavy metals has been presented. This analyzer can automatically perform long-term continuous water sampling and on-site heavy metals measurement using an array of disposable polymer lab-on-a-chips (lab chip) and a continuous flow sensing method. The system consists of a plastic fluidic motherboard with a microchannels network, microvalves and pump, control circuits, a wireless communication module, a potentiostat, LabVIEW control, and seven disposable heavy metal lab chips. Square wave anodic stripping voltammetry was performed using a microfabricated planar bismuth electrode on the chip for detecting heavy metal (e.g., cadmium, Cd) concentrations. Sensing performance sensitivity was improved with by the continuous flow sensing method propelled by the analyzer. On-site measurement of the Cd concentration change of the soil pore and ground water samples from a lab-scale reactor was automatically performed to evaluate the performance of the analyzer with lab chips.

This paper presents an on-chip magnetic cell sorting system for the sorting of cells based on a variety of surface markers. A polymer lab on a chip integrated with an electroplated array of Ni/Fe permalloy has been designed, fabricated, and characterized for the separation of cell substitutes at a variety of flow rates and incubation times. The system sequentially labels cell substitutes with magnetic beads and sorts them, repeating this process to sort for a variety of surface markers. Flow rates and incubation times were varied to characterize the system and produce the best combination of high specific capture and low nonspecific capture. The separation system developed on polymer is selective and efficient while being low cost, portable, and fabricated in a modular structure that can be integrated with other cell handling processes.

This paper presents the development of an easy-to-handle and disposable clinical diagnostic lab-on-a-chip using fully integrated plastic microfluidic components, which has the sampling/identifying capability to make fast and reliable measurements of metabolic parameters from human whole blood. A smart and functional lab-on-a-chip cartridge, which incorporates a full on-chip auto-calibration function for in the field applications, has been developed, and then fully characterized using a portable analyzer (3 (1/4)''x 5''x 1'') with multi-analyte detection capability. In addition, several new approaches in realizing smart and functional lab-on-a-chips on polymer have been adopted, which include the pinch valve for automatic fluidic sealing, a by-pass channel as the sampling indicator, and a robust connector design for long analyzer lifetimes. Metabolic parameters such as glucose, lactate, and partial oxygen from human whole blood have been successfully measured using the functional polymer lab-on-a-chips and the portable analyzer developed in this work.

This paper presents an environmentally friendly disposable heavy metal ion sensor for in situ and online monitoring in the nature and physiological systems. The miniaturized sensor chip consists of a non-toxic microfabricated bismuth (Bi) working electrode that replaces the conventional mercury electrodes, an integrated Ag/AgCl reference electrode, a gold counter electrode, and microfluidic channels. In this work, the electrochemical behavior of the Bi working electrode was characterized in several non-deaerated buffer solutions using cyclic voltammetry. The detection and quantification of Pb (II) and Cd (II) were statically performed using anodic stripping voltammetry inside the microchannels, in the Pb (II) concentration range of 25–400ppb (R2=0.991) with limit of detection of 8ppb for 60s deposition, and in the Cd (II) concentration range of 28–280ppb (R2=0.986) with limit of detection of 9.3ppb for 90s deposition. Particularly, the applications of this sensor chip have been reported with the examples of in situ measurement of Cd (II) concentration in soil pore and ground water and online direct measurement of Cd (II) concentration in cell culture media in its native environment.

This paper presents a new polymer lab-on-a-chip for magnetic bead-based immunoassay with fully on-chip sampling and detection capabilities, which provides a smart platform of magnetic immunoassay-based lab-on-a-chip for point-of-care testing (POCT) toward biochemical hazardous agent detection, food inspection or clinical diagnostics. In this new approach, the polymer lab-on-a-chip for magnetic bead-based immunoassay consists of a magnetic bead-based separator, an interdigitated array (IDA) micro electrode, and a microfluidic system, which are fully incorporated into a lab-on-a-chip on cyclic olefin copolymer (COC). Since the polymer lab-on-a-chip was realized using low cost, high throughput polymer microfabrication techniques such as micro injection molding and hot embossing method, a disposable polymer lab-on-a-chip for the magnetic bead-based immunoassay can be successfully realized in a disposable platform. With this newly developed polymer lab-on-a-chip, an enzyme-labelled electrochemical immunoassay (ECIA) was performed using magnetic beads as the mobile solid support, and the final enzyme product produced from the ECIA was measured using chronoamperometry. A sampling and detection of as low as 16.4 ng mL(-1) of mouse IgG has been successfully performed in 35 min for the entire procedure.

Precise self-assembly of carbon nanotubes (CNTs) by magnetic attraction on a catalyst and alignment by fluidic shear forces is reported in this work. The solution containing dispersed nanotubes was flowed in a microchannel and external magnetic field was applied by a permanent magnet for attracting a metal catalyst located at the end of the CNT. The assembly procedure and electrical characterization of the assembled nanotubes are presented and results are discussed. This work can provide a potential breakthrough for creating massively parallel CNT circuits for high performance nano electronic devices or nano biosensors.

In this paper, high throughput fabrication of nanoelectrodes on polymer using nanoinjection and trench-filling techniques is developed and results are presented. The new fabrication method for producing nanoelectrodes on polymer substrates is characterized with scanning electron microscopy, surface profilometry, and atomic force microscopy. This method may be applied to mass-production of low-cost polymer biochips containing ultra-sensitive nanoelectrodes for biosensors in point-of-care medicine.

The paper presents the development of a micromachined hybrid type magnetic particle separator, for accurate positioning of magnetic beads in two-dimensional array, using a magnetic interconnection technique between permalloy microstructures and external electromagnets. The developed magnetic particle separator generates relatively large magnetic force on magnetic particles (or beads) with a simple structure and low power consumption. The developed device has no heat problems because the electromagnets are isolated from the sites where magnetic particle separation takes place. In addition accurate positioning of magnetic particles in 2-dimesional array patterns can be achieved.