Name: Field-Deployable Lens-Free Imaging Platform for Rapid Label-Free Analysis of Natural Killer Cell Activation (Video) | JoVE Journal
Uploaded: 2025-08-08T14:40:00.000+00:00
Duration: 8 min 34 s
Description: Discover lens-free shadow imaging technology for rapid label-free analysis of natural killer cell activation. This computational method enables real-time immune monitoring without staining or costly flow cytometry.

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This study was supported by the Basic Science Research Programs of the National Research Foundation (NRF) of Korea (Grant#: RS-2024-00353675, Grant#: 2021R1I1A3056109), the ITRC (Information Technology Research Center) support program supervised by the IITP (Institute for Information and Communications Technology Planning and Evaluation) and the Commercialization Promotion Agency for R&#38;D Outcomes (COMPA) funded by the Ministry of Science and ICT (MSIT), Korea (Grant#: IITP-2025-RS-2023-00258971(50%), Grant#: 2710084652), the Korea Institute of Marine Science and Technology Promotion (KIMST) support program funded by the Ministry of Oceans and Fisheries, Korea (Grant#: 20210660), and the Korea University Grant. The authors acknowledge Metaimmunetech Inc. for their collaboration, based on the technology transferred from Korea University, in developing the Cellytics NK device.

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.
<ol>
	<li>Collect peripheral whole blood from the patient in a sterile heparin tube aseptically.</li>
	<li>Gently invert the tube 8-10 times to mix the anticoagulant with the blood.</li>
	<li>Analyze the blood sample immediately after collection. If analysis is delayed, store the sample in a heparinized tube at 2-8 &#176;C and use within 48 h. 
	NOTE: Do not freeze whole blood. Use fresh samples to preserve cellular integrity.</li>
</ol>
2. NK cell isolation and activation
NOTE: This protocol presents an optimized and comprehensive workflow for the isolation and activation of NK cells directly from human whole blood using the NK Prep Kit, which consists of the NK Sep Kit for cell isolation and the NK Activation Kit for stimulation. This kit integrates magnetic bead-based negative selection and stimulation to enable rapid phenotypic assessment via a lens-free imaging cytometry platform.
<ol>
	<li>Pre-test setup
	<ol>
		<li>Take out the kits from the refrigerator and bring them to room temperature (15-28 &#176;C). 
		NOTE: The NK Sep kit includes the following components: 
		(a) Green lid: Reaction tube containing the antibody cocktail. 
		(b) Red lid: Separation tube 1 containing magnetic particles in the dilution media (1.5 mL). 
		(c) Purple lid: Separation tube 2 containing the magnetic particles. 
		(d) Gray lid: Recovery tube (clean and empty). 
		The NK Activation kit includes the following components: 
		(a) Vehicle: Eight transparent tubes in a sealed aluminum bag. 
		(b) ASC: Eight yellow tubes in a sealed aluminum bag. 
		Remove only the required tubes from the sealed bags. Reseal and refrigerate any unused components.</li>
		<li>Prepare the necessary equipment: magnetic separator, pipettes with sterile tips, and a calibrated incubator or heating block set at 37 &#176;C.</li>
	</ol>
	</li>
	<li>NK cell isolation procedure
	<ol>
		<li>Add 0.5 mL of whole blood to the reaction tube with the green cap containing the antibody cocktail. Mix gently by inverting the tube 2-3 times and incubate for 5 min.</li>
		<li>Transfer the entire contents into the separation tube 1 with a red cap. Mix gently 2-3 times.</li>
		<li>Place the tube on a magnetic separator and incubate for 10 min at room temperature.</li>
		<li>With the magnet in place, transfer approximately 1.5 mL of the supernatant to the purple-capped separator tube 2. Mix gently 2-3 times.</li>
		<li>Place the separation tube two on the magnetic stand and incubate for 10 min.</li>
		<li>With the magnet still attached, transfer approximately 1.0 mL of the supernatant into a recovery tube with a gray lid. Mix carefully.</li>
	</ol>
	</li>
	<li>NK cell activation
	<ol>
		<li>Add 100 &#181;L of the isolated NK cell suspension to the Vehicle tube and another 100 &#181;L to the ASC tube</li>
		<li>Carefully mix the contents of the two tubes.</li>
		<li>Incubate the tubes at 37 &#176;C for 1 h. 
		NOTE: Use a calibrated heating block to maintain a constant incubation temperature.</li>
	</ol>
	</li>
</ol>
3. Imaging and analysis with the LSIT platform
NOTE: Cells should be analyzed immediately after incubation. If immediate analysis is not possible, store the incubated cells at 2-8 &#176;C and analyze them within 6 h. The LSIT platform evaluates cell morphology and count in human blood samples using DIH. To analyze immune activity, both unstimulated and ASC-stimulated NK cells were loaded onto a special slide, inserted into the device, and automatically imaged.
<ol>
	<li>Device setup and calibration 
	NOTE: Always perform background calibration before analyzing a sample.
	<ol>
		<li>Switch on the LSIT platform. Ensure that the LED, fan, and camera are functioning.</li>
		<li>Start the LSIT Capture software. Log in by entering your ID and password.</li>
		<li>Go to Settings and select Calibrate.</li>
		<li>Press the Open button on the touchscreen or use the physical sliding door button to open the door. When the drawer opens, remove the calibration slide and store it.</li>
		<li>Ensure that no test slide is present. Close the drawer. 
		NOTE: Use only optical wipes to clean the surfaces of the slides.</li>
		<li>Click on Set background to calibrate the optical intensity.</li>
		<li>Replace the calibration slide and close the drawer.</li>
		<li>Click on Start calibration. 
		NOTE: If liquid is spilled on the device, switch it off immediately. Wipe it clean. Do not disassemble the device.</li>
	</ol>
	</li>
	<li>Assay slide preparation and loading
	<ol>
		<li>Remove an assay slide from its pouch, label it with the sample data, and place it on a clean, flat surface.</li>
		<li>Pipette 10 &#181;L of the vehicle (unstimulated) sample into channels A and B.</li>
		<li>Pipette 10 &#181;L of the ASC-stimulated sample into channels C and D. 
		&#8203;NOTE: Avoid bubble formation and overfilling.</li>
		<li>Press Open to eject the drawer, remove the calibration slide, and place it in a storage box.</li>
		<li>Insert the prepared assay slide and press Close.</li>
	</ol>
	</li>
	<li>Image acquisition: Test Menu 1 - NK cell activity 
	NOTE: If image acquisition fails, check the illumination and sample volume. Recalibrate if necessary.
	<ol>
		<li>Log in to the LSIT system and select the NK Cell Activity Test on the main screen.</li>
		<li>Enter the sample ID or use the barcode scanner. 
		NOTE: To retrieve the sample ID from the archived list, use the Retrieve button.</li>
		<li>Press Capture to start the recording.&#160;</li>
		<li>After capturing the image, click on Analyze</li>
		<li>Check the NK cell activity (%) and cell count displayed on the screen. 
		NOTE: Valid results are displayed numerically. If I3 is below the range, &#34;&#8804;100%&#34; is displayed. Above the range, &#34;&#62;300%&#34; is displayed.</li>
		<li>Download the results to a USB or print them using the touchscreen menu.</li>
	</ol>
	</li>
	<li>Image acquisition: Test Menu 2 - cell counting
	<ol>
		<li>Select the Cell counting option under Test menu 2 on the main interface.</li>
		<li>Load 10 &#181;L of the sample onto a new assay slide channel.</li>
		<li>Insert the slide, close the drawer, and enter the sample ID.</li>
		<li>Enter the dilution factor if applicable.</li>
		<li>Press Count. 
		NOTE: If the result is within the test range, the number of NK cells is displayed as a numerical value on the system screen. For results below the test range, &#34;&#8804; 10 cells/&#181;L&#34; is displayed, for results above &#34;&#62; 1,000 cells/&#181;L.&#34;</li>
		<li>Display results as cells/&#181;L. 
		NOTE: Toggle the black result field to view the actual cell count in images.</li>
		<li>Review the results and download or print them. 
		NOTE: Ensure that the correct dilution factor is entered to obtain accurate results.</li>
	</ol>
	</li>
</ol>
4. Post-analysis clean-up
<ol>
	<li>Open the drawer using the Open button or the physical toggle switch.</li>
	<li>Remove the used slide.</li>
	<li>Place the calibration slide back in the drawer to protect the sensor.</li>
	<li>Close the drawer.</li>
	<li>Dispose of used tubes, tips, and slides in accordance with the regulations for laboratory waste.</li>
</ol>

I.L., S.H., H.S.J., and S.S. are affiliated with Metaimmunetech Inc., a company involved in the development and commercialization of the LSIT platform presented in this study. Specifically, H.S.J. is the Founder and CEO, S.H. is the CEO, and S.S. is the CTO of the company. However, the authors affirm that the results and interpretations in this manuscript are reported objectively and are not influenced by commercial interests of any kind. All the other authors declare no conflicts of interest.

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-...

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 disease2,3,4,5,6.
Among these crucial cells are natural killer (NK) cells, which are a central component of the innate immune system7,8. They eliminate virus-infected and cancerous cells and regulate the adaptive immune response through cytokine release9,10,11. Cancer, viral infections, and immunodeficiencies often reduce the number of NK cells and impair their functions12,13,14. Therefore, NK cell activation serves as an important biomarker and provides information about the overall health of the immune system. Consequently, rapid single-cell assessment of NK cell activation is essential for prognosis, diagnosis, disease monitoring, and patient stratification15,16,17,18.
Currently, NK cell activity is assessed based on their cytotoxic capacity (ability to kill target cells) and cytokine production19,20,21,22. Conventional methods for measuring cell-mediated cytotoxicity23, such as chromium-51 (51Cr) release assays24,25, flow cytometric assays26,27, and enzyme-linked immunosorbent assays (ELISA) to quantify interferon-gamma (IFN-&#947;) secreted by NK cells after stimulation28,29,30 have several limitations. These include the lack of standardization, lengthy protocols (e.g., overnight ELISA incubation), dependence on cell labeling, and high equipment costs31,32,33. These challenges highlight the need for innovative methods that offer rapid results, cost-effectiveness, label-free analysis, and high throughput. In addition, the assessment of NK cell activation at the single-cell level provides detailed insights into population heterogeneity and individual cellular responses to stimulation.
Among the emerging technologies aimed at overcoming these limitations, lens-free shadow imaging technology (LSIT) is a particularly effective solution34,35,36. LSIT, a simplified digital inline holography (DIH), enables rapid, label-free, high-throughput analysis of NK cell activation at single-cell resolution37,38,39. The DIH captures interference patterns between scattered laser light and a collinear reference beam on a digital sensor to reconstruct three-dimensional (3D) information about microscopic objects40,41. The LSIT simplifies this approach by using a partially coherent LED, micropinhole, and CMOS sensor to capture the diffraction patterns (i.e., shadow images) of cells42,43,44,45. The rationale for this technique is based on the observation that the activation of immune cells often causes morphological changes, including variations in cell size, shape, cytoplasmic granularity, and nuclear structure46,47,48,49,50. The LSIT is highly sensitive to these changes, which have a direct effect on the diffraction patterns51,52,53.
Building on the sensitivity of LSIT to these morphological changes and their effects on diffraction patterns, this protocol presents a novel method for the rapid and accurate quantification of NK cell count and functional activity at the single-cell level using the Cellytics NK platform, hereafter referred to as the LSIT platform54. As shown in Figure 1A, the LSIT platform integrates sample processing, activation, and shadow imaging into a compact multichannel workflow. NK cells were isolated from whole blood, stimulated with a specially formulated activation stimulator cocktail (ASC), and loaded onto a dedicated assay chip. ASC induces morphological changes in NK cells within 1 h of exposure, such as increased cell size and internal complexity, which can be precisely detected using the LSIT platform55,56.
The LSIT platform uses an LED light source and a CMOS sensor to capture the DIH patterns (shadows) of individual cells (Figure 1B). A special algorithm then analyzes these shadow patterns and extracts key parameters, such as the peak-to-peak distance (PPD), which correlates with cell size, and the standard deviation of the width of the secondary maximum (WSM-SD), which correlates with the complexity and irregularity of the cytoplasm (Figure 1C,D). By comparing the values before and after stimulation, the platform calculates composite indices: the combined shadow parameter (CSP = PPD &#215; WSM-SD) and the innate immunity index (I&#179;), which reflect the percentage change in CSP after activation. The reason for introducing CSP is that immune activation involves both cell enlargement and increased cytoplasmic complexity. Relying on only one of the two criteria can miss borderline activation or be affected by noise, such as debris or irregular illumination. The CSP improves the classification by requiring consistent shifts in both metrics. These indices classify the activation states of different cell populations without fluorescent labeling or complex sample processing.
This protocol is particularly useful for researchers and clinicians who require a rapid, quantitative, and label-free method for monitoring activation-induced morphological changes in immune cells. It is suitable for basic immunological research, preclinical drug screening, and evaluation of immunotherapies, and can be used as a diagnostic or prognostic tool in clinical settings where rapid assessment of immune status is essential. This method requires isolated cell populations, access to the LSIT platform, and a specific NK cell activation cocktail.

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

field-deployable lens-free imaging platform for rapid label-free analysis of natural killer cell activation

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

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.

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...

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.

Natural killer (NK) cells are important effectors of innate immunity that eliminate virus-infected and malignant cells. Monitoring NK cell activity is ...

Our goal is to rapidly assess immune function by quantifying NK cell activation in real time, using label-free, lens-free imaging to enhance immune monitoring. We have shown that the innate immunity index, or I3, reliably distinguishes between the NK cell activity of healthy and immunocompromised donors and correlates strongly with the cytokines and flow cytometry markers. The existing NK cell assays requires labels, bulky instruments, and long incubation times.Our LSIT platform fill this gap by enabling fast, affordable, and label-free profiling using simple optoelectronics. Our label-free LSIT platform differentiates NK cell activation at a single cell level within 30 seconds using shadow parameters, eliminating the need for staining and costly flow cytometry. We will extend LSIT to monitor T and B cell activation and integrate deep learning algorithms for high-throughput classification of different immune cell states.To begin, remove the kits for natural killer, or NK, cell isolation from the refrigerator and allow them to come to room temperature, between 15 and 28 degrees Celsius. Prepare the required equipment by placing a magnetic separator, pipettes with sterile tips, and set an incubator or heating block to 37 degrees Celsius. Using a pipette, add 0.5 milliliters of whole blood to the green-capped reaction tube containing the antibody cocktail.Gently mix the contents by pipetting up and down the tube five to six times. Then, incubate the tube for five minutes. Transfer the entire contents of the green-capped tube into the red-capped separation tube one using a pipette, and gently mix by pipetting the tube five to six times.Place the red-capped tube on the magnetic separator and incubate at room temperature for 10 minutes. While keeping the magnet in place, use a pipette to transfer approximately 1.5 milliliters of the supernatant into the purple-capped separation tube two. Gently mix the contents by pipetting the tube five to six times.Now, place the purple-capped tube on the magnetic separator and incubate for 10 minutes. While the magnet is still in place, transfer approximately 1 milliliter of the supernatant into a recovery tube with a gray lid using a pipette, and carefully mix the contents by pipetting the tube. Next, add 100 microliters of the isolated natural killer cell suspension into the vehicle tube and another 100 microliters into the activation stimulator cocktail, or ASC, tube.Gently mix the contents of both tubes using a vortex mixer to ensure even distribution. Incubate the tubes in the heating block or incubator set at 37 degrees Celsius for one hour. Switch on the LSIT platform.Launch the LSIT capture software. Log in by entering your ID and password, then select Calibrate to initiate calibration mode. Press the Open button on the touchscreen or use the physical sliding door button to open the drawer.When the drawer opens, remove the calibration slide and store it. Now, click Set Background to calibrate the optical intensity. Replace the calibration slide in the drawer and close the drawer.Then, click Start Calibration to complete the process. Remove an assay slide from its pouch. Label it with the sample data before placing it on a clean, flat surface.Pipette 10 microliters of the vehicle sample into channels A and B, and then pipette 10 microliters of the ASC-stimulated sample into channels C and D.Now, press Open to eject the drawer. Remove the calibration slide and place it in a storage box. Insert the prepared assay slide into the drawer and press Close to secure it.Go back to the main menu and select the NK Cell Activity and Test from the main screen. Enter the sample ID or scan the barcode to load the sample profile. Then, press Capture to begin image recording.After capturing the image, click on Analyze to begin processing. Review the NK cell activity percentage and cell count displayed on the screen. Download the results to a USB drive or print them using the touchscreen menu.Select Cell Counting and Test on the LSIT main interface. Load 10 microliters of the new sample onto a fresh assay slide channel using a pipette. Insert the slide into the drawer.Close the drawer using the touchscreen or button and enter the sample ID on the system. Enter the dilution factor on the touchscreen, if applicable, and press Counting to begin cell quantification. View the results displayed as cells per microliter on the screen.After analysis, open the drawer by pressing the Open button or toggling the physical switch. Remove the used slide from the drawer using gloved hands. Finally, place the calibration slide back into the drawer to protect the sensor and close the drawer using the touchscreen or the physical toggle switch.The LSIT platform with integrated software quantitatively assessed NK cell activation by comparing the CSP values of activated and unstimulated control cells. It provided the immunity report with a summarized statistical table. ASC stimulation induced clear morphological changes in NK cells, including increased size and cytoplasmic complexity, as seen in Hema 3-stained Cytospins after 30 minutes and two hours.Shadow images showed ASC-stimulated cells had visibly more complex diffraction patterns compared to vehicle controls, with extracted parameters indicating increased peak-to-peak distance, cytoplasmic granularity, and combined shadow parameter. Combined shadow parameter values allowed clear classification between healthy donors and cancer patients, with minimal overlap in single cell distributions of activated and nonactivated NK cells. Healthy donors showed significantly higher innate immunity index values than cancer patients following ASC stimulation.

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Discover lens-free shadow imaging technology for rapid label-free analysis of natural killer cell activation. This computational method enables real-time immune monitoring without staining or costly flow cytometry.

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

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