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Prepare living cells for label-free imaging in a clean, instrument-compatible vessel with an optical bottom; keep them in a medium and environment that preserve viability for the entire acquisition; and avoid fixation or staining when the intrinsic optical signal is the measurement. The correct dish, coating, seeding density, medium, temperature, gas control and equilibration time depend on the cell model, objective, imaging modality and instrument protocol. There is no universal recipe.
Label-free methods obtain contrast from the specimen’s optical properties rather than a required exogenous stain. Quantitative phase imaging measures phase changes caused by the specimen and its surrounding medium, while holotomography reconstructs a three-dimensional refractive-index map. Both still require careful sample preparation: the optical path must match the objective, and living cells must remain healthy and stable during acquisition.
What label-free preparation does—and does not—mean
In a label-free experiment, the image signal comes from intrinsic optical differences in the sample. Depending on the system, that may be a two-dimensional phase image or a three-dimensional refractive-index volume. Holotomography is one label-free approach, not a synonym for every label-free method.
Removing a stain does not remove the need for culture conditions, sterility, environmental control or an optically suitable vessel. It also does not prohibit every fluorescent measurement. A fluorescence channel can be added for molecular identity in a separate or correlative acquisition when the biological question requires specificity; the label-free channel itself does not depend on that label.
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The surrounding medium is part of the optical measurement. In quantitative phase imaging, a change in medium refractive index can change the measured phase signal even when the cells have not changed. Therefore, comparisons between wells, time points or treatments should use controlled medium conditions and the instrument protocol’s specified medium.
Decide the measurement before choosing the sample vessel
Identify the optical quantity
Ask whether the system records a two-dimensional phase image, reconstructs a three-dimensional refractive-index volume, or combines label-free data with another channel. The answer determines the acceptable optical path, acquisition geometry and interpretation of contrast. A phase image and a refractive-index volume are not interchangeable outputs.
Check the instrument’s physical limits
Before seeding cells, confirm the manufacturer or microscopy facility protocol for vessel dimensions, optical-bottom thickness, bottom geometry, objective working distance and any restrictions on chamber height. A vessel that is optically clear to the eye can still place the cell layer outside the objective’s working range or introduce aberration. Treat optical-bottom thickness and geometry as compatibility specifications, not interchangeable packaging details.
Confirm the intended acquisition duration
A short snapshot and a multi-hour time series impose different demands. The medium must support the cells for the planned interval, and the vessel must limit evaporation and remain stable on the stage. Environmental control should be selected for the cell type, medium and duration rather than copied from an unrelated protocol.
Choose an optical-bottom culture vessel
For high-resolution live-cell work, a coverglass-bottom dish or plate is a common starting category. A coverslip-bottom imaging plate can provide the thin optical path required by a high-numerical-aperture objective, but only if its specification matches the instrument. A #1.5 glass-bottom dish is an example of a product category, not a universal requirement.
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| Preparation choice | What to verify | What the evidence supports |
|---|---|---|
| Coverglass-bottom dish | Bottom thickness, flatness, chamber depth, objective working distance and stage fit | Recommended category for high-resolution microscopy when accepted by the facility or instrument protocol |
| Glass-bottom multiwell plate | Well geometry, optical area, plate dimensions, sealing method and compatibility with the stage | A 96-well glass-bottom plate was used in one live-cell holotomography example; this does not establish universal compatibility |
| Other chamber format | Optical path, gas exchange, volume, mounting and evaporation control | Use only when the instrument or facility protocol explicitly accepts it |
Do not select a vessel solely by well count or visual clarity. Verify whether the optical bottom is intended for the objective you will use, whether the cell layer can be positioned within its working distance, and whether the vessel can be secured without stressing the sample. If a microscopy core supplies a validated chamber format, use that specification rather than substituting a superficially similar plate.
Culture or seed cells using the validated cell-specific procedure
Keep established handling practices
Seed and handle the cells with the sterile technique and growth procedure already validated for that cell type. Label-free imaging changes how the cells are observed; it does not automatically change their attachment, feeding or passaging requirements.
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Some models need an established coating to attach or spread, while others do not. Apply the coating, concentration and incubation conditions validated for the cell type and experiment. The available evidence does not support one coating recipe for all living cells or all label-free systems.
Do not invent a universal seeding density
Cell size, growth rate, adhesion, assay duration and the field of view all affect the useful density. Use the density in the validated cell protocol or optimize it as an explicit pilot variable. A density that gives isolated cells for morphology may be unsuitable for a confluent monolayer study, and neither can be presented as a general rule.
Allow the sample to settle into its intended state
Image cells only after they have had the time required by the cell-specific procedure to attach and recover from handling. The appropriate interval is model-dependent; the cited methods do not establish one equilibration period that applies to every cell line.
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Prepare the imaging medium as a controlled measurement variable
Prioritize viability for the acquisition window
Use a medium that supports the cells for the complete imaging period and is compatible with the instrument’s environmental control. Consider the planned duration, gas requirements, temperature and whether the vessel will be sealed or open during acquisition.
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Keep refractive index consistent for quantitative comparisons
Because phase depends on the refractive index difference between the specimen and its surroundings, changing medium can alter the signal. Record the medium identity and keep its conditions consistent across control and treatment groups whenever the goal is quantitative comparison. If the instrument protocol specifies a particular medium, follow that specification rather than choosing by color or convenience.
Understand the phenol-red-free example correctly
A 2024 Bio-protocol method for live-cell holotomography used Hep3B cells in 96-well glass-bottom plates and exchanged them into phenol-red-free medium before imaging. That detail belongs to that experimental setup. It is not evidence that every label-free experiment requires phenol-red-free medium; use it only when it fits the cell model, assay and instrument protocol.
Measure or document medium conditions when required
A 2025 holotomography study measured the refractive index of the surrounding medium and used different media for distinct preparations. This illustrates why medium selection and quantitative interpretation are method-specific. If your system requires refractive-index input or correction, obtain it using the procedure specified by the instrument or method and document the value with the sample metadata.
Equilibrate the sample and imaging environment
Match temperature and gas to the cells and medium
Maintain the temperature and gas conditions required by the cell model and its medium for the planned acquisition. The correct settings are not universal: they depend on the biology, medium formulation, vessel and environmental-control hardware.
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Let the instrument reach operating conditions
Use the facility or manufacturer procedure for warming, environmental stabilization and optical equilibration. The published holotomography example used a pre-equilibrated system. Starting acquisition before the system reaches stable operating conditions can add drift or make early frames unlike later frames.
Control evaporation and handling
For long acquisitions, use the approved method for limiting evaporation and avoid unnecessary opening, transfers or stage movement. Evaporation changes volume and medium composition, while repeated handling can disturb attachment and morphology. If a chamber requires a seal, confirm that the seal is compatible with gas exchange and the instrument’s focus geometry.
Inspect the sample immediately before acquisition
Check morphology and attachment
Look for the normal appearance expected for the cell model, consistent attachment and an absence of obvious debris or widespread damage. The acceptance criteria must be defined for the particular experiment; the available sources do not establish one universal threshold for cell shape, confluence or viability.
Remove optical hazards without disturbing the cells
Inspect the imaging area for bubbles, droplets on the optical surface, scratches, condensation and debris. Correct the problem using the approved handling procedure rather than repeatedly moving the plate or aspirating near the cell layer. Bubbles in the optical path can obscure cells or create phase artifacts.
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Record conditions that affect interpretation
Document vessel type and bottom specification, cell passage or preparation identifier, coating if used, medium identity, environmental settings, equilibration status, acquisition duration and objective. These records allow a phase or refractive-index change to be separated from a preparation change.
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Acquire with the least intervention compatible with the experiment
Once the sample is stable, acquire through the validated instrument workflow without adding a stain or fixative to the label-free measurement. Fixation and staining change the living state and can alter optical properties; they are incompatible with a measurement intended to follow viable cells in real time.
Set focus, illumination or scan parameters, field selection and time interval according to the instrument protocol and biological question. The cited sources do not define universal acquisition settings or a single quality-control cutoff. Use pilot data to determine whether the cells remain healthy and whether the signal supports the intended analysis.
How preparation choices differ by label-free modality
| Decision axis | Two-dimensional quantitative phase | Holotomography |
|---|---|---|
| Primary contrast | Optical phase produced by the specimen and surrounding medium | Intrinsic refractive-index contrast reconstructed as a three-dimensional volume |
| Vessel concern | Optical-bottom thickness, flatness and objective working distance | The same optical compatibility requirements, plus the instrument’s reconstruction and illumination geometry |
| Medium concern | Medium refractive index affects phase and must be controlled for comparisons | Surrounding-medium refractive index can affect the reconstructed interpretation and may be measured explicitly |
| Environmental concern | Viability and stability over the selected imaging interval | Viability and stability during the longer or repeated acquisition required for volumetric data |
| Molecular identity | Intrinsic contrast may not identify a specific molecule | Intrinsic refractive-index structure may not identify a specific molecule; a separate fluorescence channel can be added when required |
These are preparation and interpretation axes, not a head-to-head ranking of instruments. Select the modality that supplies the information your biological question requires, then follow its validated vessel and environmental specifications.
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The 2024 method provides a concrete example without being a universal recipe. Hep3B cells were seeded in 96-well glass-bottom plates, exchanged into phenol-red-free medium, and imaged by live-cell holotomography in a pre-equilibrated instrument under controlled environmental conditions.
- Confirm that the 96-well glass-bottom geometry and optical bottom are accepted by your holotomography system and objective.
- Seed Hep3B cells using the method’s validated handling and density rather than transferring those values to another cell line.
- Exchange into the method’s imaging medium using a procedure that preserves attachment and viability.
- Pre-equilibrate the instrument and establish the required temperature and gas conditions before loading the plate.
- Inspect the wells for normal morphology, bubbles and evaporation, then acquire without fixation or staining.
- Report any change in cell model, vessel, medium, environmental setting or acquisition duration as a method deviation.
The value of this example is its sequence and its explicit control of vessel, medium and environment. Its exact choices should not be generalized to primary cells, other cancer lines, different objectives or another label-free platform.
Troubleshooting common preparation failures
| Symptom | Likely preparation issue | Corrective action |
|---|---|---|
| Cells are out of focus or the usable field is small | Optical-bottom thickness, vessel geometry or objective working distance is mismatched | Check the instrument’s accepted vessel specification and use a compatible optical-bottom format before repeating the experiment |
| Phase or refractive-index values differ between otherwise similar wells | Medium identity, refractive index, volume or evaporation changed | Standardize medium handling, document conditions and control evaporation over the acquisition interval |
| Cells round up or detach after loading | Handling, coating, temperature or medium exchange was not suitable for the cell model | Return to the validated cell-specific attachment and exchange procedure; reduce unnecessary transfers |
| Images contain large distorted regions | Bubble, condensation, debris or an optical-surface defect | Inspect the vessel and optical path, correct it with the approved procedure and avoid disturbing the cell layer |
| Later time points drift from early frames | The instrument or environmental chamber was not equilibrated, or evaporation occurred | Allow the system to reach operating conditions and use the approved strategy for stable temperature, gas and volume |
| Intrinsic images do not answer a molecular-identification question | Label-free contrast lacks the needed molecular specificity | Add a separately validated fluorescence or other correlative channel if the research question requires it |
A pre-run checklist
- Instrument modality and output are defined: phase image, refractive-index volume or both.
- Vessel dimensions, optical-bottom thickness, geometry, stage fit and objective working distance are confirmed.
- Cell-specific coating and seeding procedure are documented, with no unsupported universal density assumption.
- Imaging medium supports viability for the planned duration and matches the instrument protocol.
- Medium conditions are consistent across samples when quantitative comparisons are intended.
- Temperature, gas control, sealing and evaporation strategy are appropriate for the cells and duration.
- The system and sample are equilibrated before acquisition.
- Cells show expected morphology and attachment, with no bubbles or avoidable debris in the field.
- No fixation or stain has been added to the label-free measurement.
- Vessel, medium, environmental and acquisition metadata are recorded for interpretation.
Key principles to carry between instruments
Use the instrument’s accepted optical vessel, not a generic clear container. Treat the medium’s refractive index as part of the quantitative measurement. Keep environmental conditions appropriate to the cell model for the full acquisition, and regard published vessel or medium choices as examples tied to their method. Finally, preserve the distinction between obtaining intrinsic optical contrast and obtaining molecular identity: label-free imaging can show structure and dynamics without a stain, but another channel may still be necessary to identify a particular molecule.
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