Field-Deployable Lens-Free Imaging Platform for Rapid Label-Free Analysis of Natural Killer Cell Activation

Summary

This protocol describes a label-free method for the rapid single-cell analysis of natural killer (NK) cell count and activity using lens-free shadow imaging technology. This method quantifies morphological changes in individual NK cells by computerized analysis of their diffraction patterns.

Abstract

Natural killer (NK) cells are important effectors of innate immunity that eliminate virus-infected and malignant cells. Monitoring NK cell activity is essential for assessing immune function. However, conventional methods, such as flow cytometry, are labor-intensive and time-consuming. This protocol describes a label-free technique for the rapid analysis of natural killer (NK) cell activity using lens-free shadow imaging technology (LSIT). NK cells isolated from peripheral blood and stimulated with a proprietary activator were analyzed using the LSIT platform within 5 min. The LSIT platform captures holographic images, known as shadow images, created by the interference of light with NK cells, and monitors their activation status by analyzing changes in shadow parameters. Key parameters derived from the shadow images, including the peak-to-peak distance (PPD) and standard deviation of the secondary maxima width (WSM-SD), quantitatively reflected the cell morphology and internal complexity. These parameters were integrated into the combined shadow parameter (CSP) and innate immunity index (I³) to provide a comprehensive assessment and quantification of NK cell activity based on morphological changes in the cells. I³, calculated as the percentage change in CSP after activation, served as a direct measure of NK cell activity. NK cells from healthy individuals consistently showed significantly higher I³ values than those from immunocompromised individuals. The LSIT platform enables fast and cost-effective immune profiling that is suitable for point-of-care diagnostics.

Introduction

Cell morphology is emerging as a key indicator of the molecular mechanisms. It not only provides a visual representation of the cell structure but also offers valuable insights into the underlying processes and functions of individual cells¹. Analyzing the shape, size, and activity of immune cells at the single-cell level provides crucial insights into their behavior, heterogeneity, and roles in health and disease^2,3,4,5,6.

Among these crucial cells are na....

Protocol

All research followed institutional guidelines and was approved by the Institutional Review Board of Korea University Anam Hospital (approval number: 2021AN0040). The reagents and the equipment used are listed in the Table of Materials.

1. Sample collection and handling

NOTE: Handle all human blood samples using standard biosafety precautions and the appropriate personal protective equipment.

1. Collect peripheral whole blood from the patient in a sterile heparin tube aseptically.
2. Gently invert the tube 8-10 times to mix the anticoagulant with the blood.
....

Results

Following this protocol, the LSIT platform enables a quantitative, label-free assessment of NK cell activation based on morphological changes in the cells. Figure 2A shows Hema-3-stained cytospin preparations of primary NK cells from healthy donors after 2-h incubation with or without ASC. Activation by ASC results in marked changes, primarily cell enlargement and increased internal complexity or granularity, compared to vehicle (unstimulated) controls. These changes are captured in the form.......

Discussion

This protocol presents a standardized, label-free method for quantifying NK cell activation that combines cell isolation and activation using the NK Prep Kit, with analysis via the LSIT platform. It offers a rapid and quantitative alternative to conventional assays such as ⁵¹Cr release, flow cytometry, and ELISA. The protocol was developed for laboratories equipped with the LSIT platform and provides simple results based on shadow image analysis of the number of NK cells and I³. Its single-.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.7 mL Microcentrifuge Tubes	AXYGEN	MCT-175-C	Sterile, DNase/RNase-free
Assay Slide	Metaimmunetech	AS050-G	Dedicated slide for Cellytics system 50 slides in a box
Cellytics system with software	Metaimmunetech	CS001	Lens-free shadow imaging platform
Centrifuge (swing-bucket rotor)	LABOGENE	1580R	300–500 × g range
CMOS Image Sensor	Included with Cellytics Device	Integrated	Used for diffraction imaging
Cytospin Centrifuge	Nasco korea	TXT3 Cyto Centrifuge	For cytospin staining
Digital Heating Block (37 °C)	Daihan Scientific	DH.WHB00349	For cell incubation
ELISA Kit (IFN-γ)	R&D system	DIF50C	Human IFN-γ ELISA kit
Guava easyCyte Flow Cytometer	Millipore	0500-5005	For CD107a expression analysis
Hema 3 Stat Pack	ThermoFisher	122-911	For cytospin staining
IL-12 (Recombinant Human)	R&D system		Component of ASC
IL-2 (Recombinant Human)	Peprotech	200-02	Component of ASC
Magnetic Separation Rack	Metaimmunetech	M13R8	For bead separation
Microscope Slides	Marienfeld	1000612	For cytospin preparations
NK activation kit	Metaimmunetech	MIT2301V, MIT2301A	For NK cell activation
NK Sep Kit	Metaimmunetech	MIT2301-500	For NK cell isolation
PE Mouse Anti-Human CD107a, 100test	BD biosciences	555801	For CD107a expression analysis
Pinhole Aperture	Included with Cellytics Device	Integrated	Used in LSIT illumination path
Pipettes (2–20 µL, 20–200 µL)	AXYGEN	T-200-Y	Adjustable volume
Shadow Calibration Slide	Metaimmunetech	AS050-G	For device calibration. 1 Calibration slide in 1 Assay slide box
Vortex Mixer	Merck	SI-0246A	For resuspension