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 4, 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 is both the science and technology that precisely control fluids at the microscale and manufacture microminiaturized devices. The recent advances in microfluidics have enabled great progress in diagnostics, material synthesis, and cellular studies. Thus, there is an increasing need for accurate, high-resolution imaging and quantitative analysis. Visualization of microfluidic processes is crucial for comprehending fluid dynamics, particle transport, and biochemical interactions in these systems.
This review gives an overview of the main microfluidic imaging techniques applied in research. It starts with optical methods, including bright-field microscopy, chemiluminescence detection, spectroscopy, and fluorescence microscopy, then it addresses tomography methods such as X-ray and neutron imaging, followed by electron microscopy techniques. A summary of each imaging principle, limitations, and applicability to microfluidic systems is given in 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 techniques are the most commonly used imaging approaches in microfluidics. They are non-invasive and compatible with live, real-time monitoring. Moreover, they enable the direct visualization of flow profiles, cellular dynamics, chemical gradients, and molecular interactions. Optical techniques can have different resolutions and sensitivities, and include bright-field microscopy, fluorescence, and spectroscopy.
Bright-field microscopy
Bright-field microscopy is a standard technique in imaging that is based on light transmission, providing direct sample illumination. It is often used for initial system characterization and real-time tracking of droplets, as well as visualization of microchannels.
Kim et al. used this imaging technique to track the motility of Chlamydomonas reinhardtii within an acoustofluidic device. The researchers integrated algae as dynamic tracers to map the acoustic pressure field. The results were as accurate as with conventional methods [3]. Advanced imaging techniques are being developed, including bright-field imaging and complementary techniques.
Chemiluminescence
Chemiluminescence imaging techniques are based on light emission generated during chemical reactions; thus, they do not require excitation sources or optical filters. Its simplicity and low instrumentation requirements make it suitable for lab-on-chip platforms.
Neumair et al. implemented an automated chemiluminescence-based microarray to screen binding interactions of proteins, antibiotics, and lectins with bacteria. Detection relied on luminol-Horseradish Peroxidase (HRP) reaction to quantify bound bacteria (Figure 2). The authors were able to identify novel binding behaviors, such as polymyxin B interactions with Enterococcus faecalis, and effective elution agents for bacterial release [4].
Spectroscopy
Spectroscopy imaging techniques are based on light interactions such as absorption, emission, and Raman scattering to characterize molecular and chemical properties in situ. They are suitable for monitoring concentration gradients, reaction kinetics, and biochemical pathways within microfluidic channels.
Raman microscopy
Raman microscopy works by detecting inelastic scattering of light. This reveals molecular vibrations and allows high-specificity chemical imaging without labels.
Poonoosamy et al. combined this imaging technique with a flow-through microreactor. They studied mineral dissolution and crystal precipitation under flowing conditions. This setup allowed direct 3D observation of surface changes in porous media [5].
Ozeki et al. advanced the method by developing a multicolor stimulated Raman scattering (SRS) system with tunable fiber lasers. This system enabled fast, multiplexed imaging of live samples without labeling (Figure 3) [6].
Surface plasmon resonance (SPR)
Surface plasmon resonance (SPR) occurs when a photon of incident light hits a metal surface and measures the changes in the reflected light. By removing the need for labels, this technique allows real-time microfluidic imaging and the analysis of binding kinetics.
Xiao et al. integrated a 3D-printed microfluidic channel onto a SPR sensing surface (Figure 4), achieving reliable sensing under high flow, while a smartphone camera recorded signals. The platform enabled real-time monitoring of protein interactions at low cost and footprint, making it suitable for portable biosensing applications [7].
Fluorescence microscopy
Fluorescence microscopy is the most used optical imaging technique in microfluidics due to its sensitivity and specificity. It enables quantitative detection of low-concentration samples and the dynamic visualization of cellular or molecular events.
Aldridge et al. applied time-lapse fluorescence microscopy within microfluidic chambers to track growth, division, and morphological changes in single Mycobacterium cells under controlled antibiotic exposure (Figure 5). In addition, phase-contrast imaging provided structural visualization [8]. This setup can be used to investigate the heterogeneity of drug responses within bacterial populations.
Confocal microscopy
A confocal microscope uses focused illumination and detection at the same spot, capturing one point at a time during a scan. This 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 the nucleation of calcium carbonate inside giant unilamellar vesicles formed by droplet microfluidics. Fluorescent labeling defined vesicle boundaries, while confocal imaging captured crystal growth and lipid-crystal interactions [9].
Light-sheet fluorescence microscopy (LSFM)
The light sheet fluorescence microscopy (LSFM) illuminates samples with a thin light sheet perpendicular to the sample. It reduces photobleaching and enables rapid volumetric imaging, which is suitable for live-cell imaging.
Memeo et al. used LSFM to image Drosophila embryos in a microfluidic chamber under dynamic flow. It used dual-sided waveguide illumination to minimize light scattering and motion blur, and improve resolution [10].
An Airy-beam LSFM system integrated with quantitative phase imaging (QPI) (Figure 7) allowed a wider field of view while maintaining low phototoxicity. It acquired structural (fluorescent) and biophysical (phase-based) data from flowing cells, which can be used to study metabolic localization influenced by cellular noise [11].
Imaging flow cytometry
Flow cytometry imaging techniques couple flow cytometry with microscopy for high-throughput single-cell analysis.
Yuan et al. developed a microfluidic imaging flow cytometry platform combining high-speed fluorescence and bright-field microscopy (Figure 8). The authors used a method that generates elasto-inertial 3D blur-free images of objects moving at high linear velocities. The system enabled subcellular localization down to 500 nm, with throughputs exceeding 60,000 and 400,000 cells/sec 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 a large number of projections while the sample is rotated. 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 (XMPI) captures multiple X-ray images from different angles at the same time, providing high-resolution, real-time 3D visualization of complex flow structures.
An example that highlights these advantages is the study by Rosén et al., who split the synchrotron beam into angular projections and captured them simultaneously, achieving rotation-free 4D imaging (Figure 9). The result was rotation-free 3D imaging with millisecond and micrometer precision, all 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. used high-speed neutron radiography to study the cavitation in fast liquid flowing within microchannels (Figure 10). Given neutrons’ properties to pass through opaque materials and being sensitive to hydrogen, the technique was able to capture vapor bubble formation and movement. The method worked in flow conditions where normal optical and X-ray imaging could not [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 require high-vacuum conditions and specific sample preparation to resist exposure to the electron beam.
Scanning electron microscopy (SEM)
In scanning electron microscopy (SEM), an electron beam is focused on a specimen across a surface, emitting electron signals that reveal morphology and material composition. This technique is suitable for the structural characterization of microfluidic surfaces and materials.
Ling et al. studied the dissolution of natural rocks in microfluidic devices. Time-lapse optical microscopy tracked grain erosion during reactive flows, followed by SEM and energy-dispersive X-ray spectroscopy (EDX) to map surface structure and chemistry (Figure 11). EDX relies on the capacity of X-rays to eject ‘core’ electrons from the sample’s atom. Based on the frequency of light released, the atomic number of the atom can be determined.
The whole setup helped to understand how mineral dissolution is controlled by chemical composition and physicochemical heterogeneity at a specific flow rate [15].
Transmission electron microscopy (TEM)
Transmission electron microscopy (TEM) transmits a high-energy electron beam through ultra-thin samples to visualize 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). SAXS is based on measuring the intensity of the scattered X-ray beam downstream as a function of the angle.
The setup was able to show how lipid concentration and flow rates influenced liposome morphology. The link between nanoscale structure and functional performance can provide a better understanding of liposome behavior in drug delivery [16].
Conclusion- Towards merging imaging techniques with automation
Integrating imaging with microfluidics has improved the visualization of processes at the microscale. Optical methods provide accessible, real-time monitoring of live samples, while Raman microscopy, SPR, and confocal imaging enhance molecular sensitivity and spatial resolution. Light-sheet microscopy supports fast 3D imaging with minimal damage, and imaging flow cytometry enables high-throughput single-cell analysis, although it is limited to 2D. Tomography techniques reveal internal geometries of opaque materials that are inaccessible to single-angle imaging, while electron techniques can reach the nanoscale structure, but require sample preparation. There is no conventional method for microfluidic imaging; however, combining several imaging approaches with automated analysis is promising 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
