Visualizing microfluidics
Matching imaging techniques to research needs
Writer
Celeste Chidiac, PhD
Keywords
Microfluidic Devices, Intelligent Microfluidics, Artificial Intelligence, Machine Learning
Author
Celeste Chidiac, PhD
Publication Date
August 1, 2025
Keywords
Intelligent Microfluidics
Deep Learning
Microfluidic Devices
Artificial Intelligence
Machine Learning
Imaging techniques
Optical imaging
Tomography techniques
Electron microscopy methods
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Introduction to imaging techniques
Microfluidics involves the controlled manipulation of fluids within channels with dimensions from tens to hundreds of micrometers. It has enabled advances in diagnostics, materials synthesis, and cellular studies through precise control of microscale environments. As microfluidic systems have increased in complexity, so has the need for accurate, high-resolution imaging and quantitative analysis. Visualization of microfluidic processes is central to understanding fluid dynamics, particle transport, and biochemical interactions in these systems.
This review examines the primary microfluidic imaging techniques applied in research. It focuses on optical methods, including bright-field microscopy, chemiluminescence detection, spectroscopy, fluorescence microscopy, and imaging flow cytometry. It also addresses tomography methods such as X-ray and neutron imaging, along with electron microscopy techniques. Each method is discussed in terms of its underlying principle, limitations, and applicability to microfluidic systems (Table 1).
Table 1: Features, limitations, and applications of each imaging technique [2].
Imaging techniques | Image dimensions | Image resolution | Features | Limitations | Selected applications |
Bright-field microscopy | 2D | 200-500 nm | Simple, fast, label-free | Low contrast, no molecular info | Morphology, droplet tracking, microchannel visualization |
Chemiluminescence imaging | 2D | Low (pixel-limited) | No excitation source, low background noise | No spatial detail, limited real-time capability | On-chip enzymatic assays, immunoassays |
Raman microscopy | 3D | 0.5-1 µm | Label-free, chemical specificity | Weak signal, slow, laser heating risk | Molecular identification, Raman mapping |
Surface plasmon resonance (SPR) | 2D | 1-10 µm | Label-free, real-time biomolecular interaction | Low spatial resolution | Biosensing, affinity measurement, diagnostics |
Confocal microscopy | 3D | 200 nm lateral / 500 nm axial | Optical sectioning, high resolution | Slow, phototoxic, expensive | Subcellular imaging, tissue-on-chip, biomarker mapping |
Light-sheet microscopy | 3D | 300-500 nm | Fast, low phototoxicity, volumetric imaging | Complex alignment, orientation-sensitive | Organ-on-a-chip, live cell imaging |
Imaging flow cytometry | 2D | 300-700 nm | High-throughput imaging and quantification | Data-heavy, high-speed optics needed | Cell classification, biomarker analysis in flow |
X-ray tomography | 3D | 1-50 µm (synchrotron), 50-500 µm (industrial & medical) | Internal imaging, high-density contrast | Operator dependency, imaging artefacts | Pore structure, multiphase flow |
Neutron tomography | 3D | 16-100 µm | High penetration depth, large samples | Longer acquisition, lower resolution, limited access | Fluid distribution in porous media, phase separation |
Scanning electron microscopy (SEM) | 3D | 1-10 nm | High-resolution surface detail | Not appropriate for heterogeneous samples | Microfluidic surface, material analysis |
Transmission electron microscopy (TEM) | 2D | 0.1-1 nm | Ultrastructure, internal morphology | Complex prep, small field of view | Nanoparticles, membrane structure, liposomes |
Optical imaging techniques
Optical imaging remains the most widely employed approach in microfluidics due to its non-invasive nature and compatibility with live, real-time monitoring. It enables direct visualization of flow profiles, cellular dynamics, chemical gradients, and molecular interactions. Optical techniques encompass a range of modalities with varying resolution and sensitivity, from basic bright-field microscopy to advanced fluorescence and spectroscopy-based platforms.
Bright-field microscopy
Bright-field microscopy is a standard technique that provides direct sample illumination and image formation based on light transmission. It is often used for initial system characterization and real-time tracking of micro-scale entities.
Kim et al. used this imaging technique to track the motility of Chlamydomonas reinhardtii within an acoustofluidic device. The algae served as dynamic tracers for acoustic pressure field mapping with accuracy comparable to conventional methods [3]. Bright-field imaging is also frequently integrated with complementary techniques to expand its analytical capacity.
Chemiluminescence
Chemiluminescence imaging techniques involve light emission generated during chemical reactions, eliminating the need for excitation sources or optical filters. Its simplicity and low instrumentation requirements make it suitable for integrated lab-on-chip platforms.
Neumair et al. implemented an automated chemiluminescence-based microarray for screening binding interactions of proteins, antibiotics, and lectins with bacterial targets. Detection relied on a luminol-HRP reaction to quantify captured bacteria (Figure 2). The platform identified novel binding behaviors, such as polymyxin B interactions with Enterococcus faecalis, and effective elution agents for bacterial release [4].
Spectroscopy
Spectroscopy imaging techniques analyze light interactions such as absorption, scattering, emission, and Raman scattering to characterize molecular and chemical properties in situ. They are well-suited for monitoring concentration gradients, reaction kinetics, and biochemical pathways within microfluidic channels.
Raman microscopy
Raman microscopy uses inelastic scattering of monochromatic light to probe molecular vibrations. It allows label-free chemical imaging with high specificity.
Poonoosamy et al. combined this imaging technique with a flow-through microreactor to study mineral dissolution and secondary precipitation under dynamic flow. This integration enabled direct, 3D observation of surface changes in porous and fractured media [5]. Ozeki et al. further advanced the method by developing a multicolor stimulated Raman scattering (SRS) system using tunable fiber lasers, enabling rapid, multiplexed imaging of live samples without labeling (Figure 3) [6].
Surface plasmon resonance (SPR)
Surface plasmon resonance (SPR) imaging technique detects refractive index changes at metal surfaces caused by molecular adsorption. It enables microfluidic imaging in real-time, with label-free detection capabilities, which are valuable for binding kinetics analysis.
Xiao et al. developed an innovative miniaturized biosensor by integrating a 3D-printed unibody microfluidic channel directly onto a SPR sensing surface (Figure 4). This platform achieved robust sealing and reliable sensing under high flow, with readout performed using a smartphone camera. It demonstrated real-time monitoring of protein interactions while reducing both cost and footprint, highlighting its suitability for portable biosensing applications [7].
Fluorescence microscopy
Fluorescence microscopy remains the dominant optical imaging technique in microfluidics due to its sensitivity and specificity. It enables quantitative detection of low-concentration samples and dynamic visualization of cellular or molecular events.
Aldridge et al. applied time-lapse fluorescence microscopy within microfluidic chambers to track single-cell responses of Mycobacterium species to antibiotics. Fluorescence imaging enabled the real-time tracking of growth, division, and morphological changes in single cells under controlled antibiotic exposure (Figure 5), providing insights into the heterogeneity of drug responses within bacterial populations. Phase-contrast imaging provided structural visualization [8].
Confocal microscopy
A confocal microscope uses focused illumination and detection at the same diffraction-limited spot, capturing one point at a time during a scan. This point-by-point imaging technique enables optical sectioning, allowing for high-resolution 3D reconstruction of the sample.
Witt et al. combined confocal laser scanning microscopy (Figure 6) with bright-field imaging to examine calcium carbonate nucleation inside giant unilamellar vesicles formed by droplet microfluidics. Fluorescent labeling defined vesicle boundaries, while confocal imaging captured crystal growth and lipid-crystal interactions, confirmed by atomic force microscopy [9].
Light-sheet fluorescence microscopy (LSFM)
The light sheet fluorescence microscopy (LSFM) illuminates samples with a thin light sheet orthogonal to detection, reducing photobleaching and enabling rapid volumetric microfluidic imaging, making it ideal for live-cell imaging.
Memeo et al. used this imaging technique to dynamically view Drosophila embryos in flow, combining dual-sided waveguide illumination with a microfluidic chamber to minimize light scattering and motion blur and improve resolution [10].
An Airy-beam LSFM system integrated with quantitative phase imaging (QPI) (Figure 7) extended the field of view while maintaining low phototoxicity. It allowed acquisition of structural (fluorescent) and biophysical (phase-based) data from flowing cells, providing insights into subcellular processes such as metabolic localization influenced by cellular noise [11].
Imaging flow cytometry
Flow cytometry imaging techniques couple flow cytometry with microscopy for high-throughput, high-content single-cell analysis.
Yuan et al. developed a sheathless microfluidic imaging flow cytometry platform integrating stroboscopic illumination with high-speed fluorescence and bright-field microscopy (Figure 8). The system enables blur-free imaging, fluorescence quantification, and subcellular localization down to 500 nm, with throughputs exceeding 60,000 and 400,000 cells per second for fluorescence and bright-field detection, respectively. The system supported detailed analysis of structures such as P-bodies and stress granules while remaining compact and cost-efficient [12].
Tomography imaging techniques
Tomography provides 3D microfluidic imaging by reconstructing cross-sectional images. It allows detailed observation of internal geometries and multiphase flows that are inaccessible to single-angle imaging.
X-ray
Synchrotron-based X-ray multi-projection imaging technique (XMPI) enables high-resolution, real-time 3D visualization of complex flow structures.
Rosén et al. used XMPI to observe multiphase flows in real time. By splitting the synchrotron beam into angularly spaced projections and capturing them simultaneously, they achieved rotation-free 4D imaging (Figure 9). This approach tracked 3D particle positions and trajectories with millisecond and micrometer precision without disturbing flow dynamics [13].
Neutron tomography
Neutron tomography imaging techniques use neutron beams to generate images based on the material’s neutron absorption. By capturing multiple 2D projections at different angles, it reconstructs a detailed 3D view of the object’s internal structure.
Zhang et al. employed high-speed neutron radiography to measure the occurrence of cavitation in fast-moving liquid flows within nozzles and microchannels (Figure 10). Because neutrons can pass through opaque materials and are sensitive to hydrogen, the technique captured vapor bubble formation and movement in real time under flow conditions where optical and X-ray methods fail [14].
Electron microscopy imaging techniques
Electron microscopy provides imaging at the nanometer scale, allowing detailed examination of microfluidic channel structures, surface morphologies, and nanoparticle behavior. These techniques typically require high-vacuum conditions and specific sample preparation to withstand electron beam exposure.
Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) scans a focused electron beam across a surface, producing signals that reveal morphology and composition. Backscattered electrons provide fine topographic detail, while secondary electrons highlight material compositional contrast.
Ling et al. combined SEM with an optical imaging technique to study mineral dissolution in microfluidic devices. Time-lapse optical microscopy tracked grain erosion during reactive flows, followed by SEM (Figure 11) and energy-dispersive spectroscopy (EDS) to map surface structure and chemistry. This approach revealed how microstructural and chemical heterogeneities influence mineral dissolution in confined environments [15].
Transmission electron microscopy (TEM)
Transmission electron microscopy (TEM) imaging technique transmits a high-energy electron beam through ultra-thin samples for microfluidic imaging of internal features and nanostructures at near-atomic resolution.
Yuka et al. used TEM with small-angle X-ray scattering (SAXS) to study liposome formation under varying flow rates in microfluidic systems (Figure 12). TEM showed how lipid concentration and flow rates shaped liposome morphology, linking nanoscale structure to functional performance, contributing to a deeper understanding of liposome behavior in drug delivery [16].
Conclusion- Towards merging imaging techniques with automation
Integrating imaging with microfluidics has improved the visualization of microscale processes. Optical methods provide accessible, real-time monitoring of live samples, while Raman microscopy, SPR, and confocal imaging extend molecular sensitivity and spatial resolution. Light-sheet microscopy supports fast 3D imaging with minimal photodamage, and imaging flow cytometry enables high-throughput single-cell analysis, albeit limited to 2D. Tomography techniques reveal internal structures in opaque materials, while SEM and TEM deliver nanoscale insights into surfaces and materials, but require sample preparation. No single method is universal for microfluidic imaging, yet combining complementary approaches with automated analysis offers powerful solutions for studying complex systems in microfluidic environments.
References
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Funding and Support
This review was written under the European Union’s Horizon research and innovation program under the Marie Skłodowska-Curie grant agreement no. 101119729 (NEXTSCREEN),
and the French Agence Nationale de la Recherche (ANR) with project ID: ANR-21-CE15-0045 (TREATABLE).
This review was written by Celeste Chidiac, PhD, and Yuwei Liu, PhD candidate
Published in August 2025.
Contact: Partnership[at]microfluidic.fr
