Real-Time Label-Free Imaging and Quantitative Analysis of Macrophage Morphodynamics Using Optical Diffraction Tomography

Summary

This protocol describes a label-free optical diffraction tomography-based workflow for real-time imaging and quantitative analysis of macrophage morphodynamics. The method enables continuous tracking of single-cell morphology and migration, providing time-resolved measurements of projected area, perimeter, and average migration speed during long-term live-cell imaging.

Abstract

This study establishes a label-free optical diffraction tomography (ODT)-based workflow for live-cell imaging and analysis to observe time-dependent morphological changes in macrophages. The method enables continuous recording of single-cell morphology and movement over extended periods under stable environmental conditions and allows extraction of quantitative parameters, including projected area, perimeter, and average migration speed. This workflow provides a practical approach for capturing dynamic cellular behaviors at the single-cell level without exogenous labeling. Using RAW264.7 macrophages as a model, time-lapse imaging was performed under lipopolysaccharide stimulation with baicalin pretreatment to capture dynamic cellular changes under different conditions. Representative cells were selected for tracking and quantitative analysis. The results show that this workflow supports stable long-term single-cell tracking and reflects temporal changes in cell morphology and motility. This approach provides a label-free method for observing dynamic cellular behaviors in response to different stimuli and can serve as a useful complement to conventional endpoint-based assays. It may also be applicable to other adherent cell types for studies of cell morphodynamics.

Introduction

Macrophages play a central role in innate immune responses and tissue homeostasis, and their functional states can dynamically change in response to microenvironmental stimuli. Traditionally, macrophages have been described as adopting pro-inflammatory or anti-inflammatory/repair-associated phenotypes^1,2. However, increasing evidence suggests that macrophage activation is a continuous and plastic process rather than a discrete binary state3,4. Under different stimuli, macrophages undergo gradual changes over time, accompanied by alterations in cell morphology and migratory behavior⁵. Th....

Protocol

1. Macrophage culture

CAUTION: Perform all procedures in a biosafety cabinet while wearing laboratory gloves and a lab coat.

1. Prepare complete culture medium (high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin–streptomycin).
2. Thaw a vial of RAW264.7 macrophage cells in a 37 °C water bath for less than 1 min.
3. Transfer the cell suspension to high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.
4. Centrifuge at 300 × g for 5 min.
5. Discard the supernatant.
....

Results

To evaluate the performance of the ODT-based workflow, time-lapse imaging was performed for two treatment groups (LPS and BAI + LPS), with five independent samples per group. All samples were acquired under identical imaging conditions. Due to the high workload associated with long-term single-cell tracking and quantitative analysis, one representative sample from each group was selected for single-cell analysis and visualization. The results are presented descriptively and are not intended for statistical inference.

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Discussion

This study establishes a label-free ODT-based workflow for continuous imaging and quantitative analysis of macrophage morphodynamics at the single-cell level. The approach enables time-resolved measurement of projected area, perimeter, and average migration speed during long-term live-cell imaging. Cell seeding density is a key factor affecting segmentation and tracking performance. High density results in overlapping cells, whereas low density reduces the number of analyzable cells. Therefore, seeding conditions should .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ODT imaging system	Pellucid Optics	MH-PanoView	Used for label-free live-cell imaging
Acquisition software	Pellucid Optics	Lucida-Pano v2.1.0	Used for image acquisition
Analysis software	Pellucid Optics	Intellyseg v2.1.0	Used for image reconstruction, segmentation, and tracking
RAW264.7	Wuhan Zishan Biotechnology	STCC20020P	Murine macrophage cell line used for in vitro polarization and imaging experiments.
Baicalin (BAI)	Shanghai Yuanye	JB246114	Used for cell pretreatment
Lipopolysaccharide (LPS)	Solarbio	L8880	Used to stimulate macrophages
DMEM medium	Gibco	C11995500BT	Cell culture medium
Fetal bovine serum	Procell	164210	Supplement for cell culture
Penicillin–Streptomycin (100x)	Procell	PB180120	Antibiotics for cell culture
60 mm culture dish	Corning	CLS430166	Used for cell culture
35 mm glass-bottom dish	Cellvis	D35-10-1.5-N	Used for ODT imaging
Inverted microscope	OLYMPUS	CKX53	Used for routine cell observation and monitoring during culture.
Cell counter	RWD Life Science	C100	Used for cell density determination