Prepare a living-cell sample for label-free imaging by matching an optical-bottom vessel to the objective and instrument, maintaining cells in a validated imaging medium and controlled environment, and acquiring the intrinsic optical signal without fixation or staining. The correct coating, seeding density, medium, equilibration time and vessel are cell- and instrument-specific; there is no universal recipe.
What label-free imaging actually measures
Label-free methods obtain contrast from properties already present in the specimen rather than from a dye or antibody added for visualization. Quantitative phase imaging measures how the sample changes the phase of transmitted light. Holotomography extends that principle by reconstructing a three-dimensional refractive-index map, allowing live-cell structures to be examined as a volume rather than only as a two-dimensional intensity image.
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Removing a stain does not remove the experimental constraints of live-cell work. Cells still need an appropriate medium, temperature and gas environment for the planned acquisition. The vessel must present an optical path that the objective can use, and the sample must remain stable for the entire imaging interval. Label-free also does not mean fluorescence can never be used: a separate fluorescence or correlative channel may still be appropriate when molecular identity is part of the question.
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Preparation workflow
1. Confirm the measurement and the instrument’s vessel requirements
Start with the imaging system’s operating protocol, not with a dish selected from a catalogue. Establish whether the experiment is producing a two-dimensional phase image, a three-dimensional refractive-index volume, or another form of label-free contrast. The answer determines how the optical path, working distance and sample geometry will be evaluated.
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Ask the facility or instrument documentation to specify:
- Permitted vessel formats, such as a dish, plate or chamber.
- Usable well or chamber dimensions and the region that can be reached by the objective.
- Optical-bottom material and thickness limits.
- Objective working distance and any clearance needed for the vessel wall or bottom.
- Whether environmental-control hardware is designed for the selected format.
Do this check before seeding. A culture vessel that is excellent for routine cell culture can be unusable for high-resolution imaging if its bottom is too thick, its geometry prevents focus, or it cannot fit the stage or environmental enclosure.
2. Select an optical-bottom culture vessel
For high-resolution live-cell microscopy, a coverglass-bottom dish or plate is a common starting category. Live-cell-compatible chamber formats can also be suitable when the instrument is designed for them. One published live-cell holotomography example used a 96-well glass-bottom plate, but that setup demonstrates compatibility for that experiment only; it does not make every 96-well plate suitable for every system.
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Evaluate the vessel on four separate dimensions:
| Selection question | Why it matters | What to verify |
|---|---|---|
| Is the bottom optically suitable? | The objective must image through the bottom without unacceptable aberration or attenuation. | Material, optical-bottom thickness and the instrument’s approved range. |
| Can the objective reach the cells? | Working distance and vessel geometry limit focus and usable field of view. | Objective working distance, wall height and any stage-clearance restrictions. |
| Does the format support the experiment? | Well count and area affect throughput, controls and the number of conditions that can be compared. | Dish, plate or chamber dimensions and the number of wells required. |
| Can the vessel remain stable during imaging? | Movement, evaporation or poor environmental fit can compromise a time series. | Stage retention, lid or enclosure compatibility and environmental-control interface. |
A “#1.5” glass-bottom dish or coverslip-bottom imaging plate may appear in facility recommendations, but the label alone is not a guarantee. Confirm the exact bottom specification and approved vessel list for your objective and instrument.
3. Culture or seed cells using the validated cell-specific procedure
Handle the cells with the same sterile technique and culture practice that has already been validated for that cell model. Label-free acquisition does not create a universal seeding density. The appropriate density depends on cell size, growth behavior, the time between seeding and imaging, and whether the analysis requires isolated cells, a monolayer or a denser population.
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Use a coating only when the cell type or experimental protocol requires one. The coating must promote the intended attachment without changing the biological question or creating an optical artifact. If the protocol has not established a coating, do not infer one simply because a different cell line used it.
Plan the seeding schedule around the acquisition window. Cells should be attached and in the intended physiological state when imaging begins, while avoiding overgrowth during a long time series. Record the passage or preparation information, seeding time, vessel identity and any coating used so that the optical data can be interpreted alongside the culture history.
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The imaging medium has two jobs: it must support cell viability for the planned duration, and it must be compatible with the instrument’s optical and environmental-control requirements. Keep medium composition and handling consistent among samples that will be quantitatively compared.
For quantitative phase measurements, the refractive index of the surrounding medium is part of the measurement context. Changing the medium can change the phase signal even when the cells themselves have not changed. Therefore, use the medium specified by the instrument or validated method, and document any exchange, dilution or supplement change.
A 2024 live-cell holotomography method using Hep3B cells exchanged the cultures into phenol-red-free medium before imaging. That is an example of one experimental setup, not a blanket requirement for all label-free work. Phenol red, serum content, supplements and buffering choices should be changed only when the cell protocol and instrument workflow support the change.
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When a study compares conditions, prepare the media in a way that keeps the relevant optical properties comparable. If distinct preparations require different media, treat the surrounding-medium refractive index as a recorded experimental variable rather than assuming that phase values are directly interchangeable.
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Maintain temperature and gas conditions appropriate for the cells and the selected medium throughout the imaging period. The required settings depend on the cell type, medium and acquisition duration, so use the validated culture or instrument protocol rather than adopting a single universal setting.
Allow the imaging system and environmental enclosure to reach operating conditions before collecting measurements. A published holotomography example used a pre-equilibrated system. Equilibration reduces the chance that focus, drift or cell behavior will change simply because the instrument is still reaching its operating state.
For long acquisitions, control evaporation and limit unnecessary opening of the enclosure. Evaporation changes the medium volume and can alter its composition and optical conditions. Keep the vessel level and securely positioned, and use the facility’s approved method for gas exchange and environmental monitoring.
6. Inspect the sample before acquiring data
Perform a final inspection using the observation method approved for the system. Look for cells with expected morphology and attachment, a stable liquid layer, and an optical path free of bubbles or debris in the imaging region. Avoid vigorous movement, repeated pipetting near the cells or other handling that can disturb the sample immediately before acquisition.
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Define acceptance criteria for the specific cell model and research question. The available protocols do not establish one universal threshold for confluence, morphology, viability or debris. Instead, record the criteria used in your study and apply them consistently to every condition.
How vessel, medium and environment interact
These choices cannot be optimized independently. A vessel’s bottom thickness interacts with objective working distance and optical aberration. The vessel format must also fit the stage and environmental hardware. Medium composition affects viability, while its refractive index affects quantitative phase interpretation. Temperature, gas control and acquisition duration determine whether that medium remains suitable for the whole experiment.
For that reason, “glass-bottom,” “live-cell,” or “label-free compatible” should be treated as starting descriptions rather than complete specifications. Compatibility is established only when the complete combination—vessel, objective, instrument, medium, environmental control and cell model—works under the intended acquisition conditions.
Choosing between common label-free measurement goals
| Research need | Preparation emphasis | Interpretation issue |
|---|---|---|
| Two-dimensional phase contrast | Stable optical bottom, appropriate focus range and a medium kept consistent across samples. | Phase changes reflect the specimen and its surrounding medium; they are not automatically molecular identities. |
| Three-dimensional holotomography | Vessel and objective geometry must support volumetric acquisition; environmental stability is important during the full scan or time series. | The output is a reconstructed refractive-index volume, so medium refractive index and reconstruction settings are part of the method. |
| Label-free imaging plus fluorescence | Use the optical format and environmental conditions required by both channels, without assuming that the label-free channel replaces molecular markers. | Fluorescence can add molecular specificity, while the label-free channel supplies intrinsic structural or phase information. |
This is a decision framework, not a ranking of instruments. The appropriate method is the one whose optical geometry, environmental control and contrast address the biological question without compromising cell health.
Example: adapting a published live-cell holotomography setup
A 2024 method reported Hep3B cells seeded in 96-well glass-bottom plates, followed by exchange into phenol-red-free medium. The instrument was pre-equilibrated and environmental conditions were controlled during live-cell holotomography. To reproduce that work faithfully, treat each detail as part of the stated setup: use the same cell model and vessel specification, follow the reported medium exchange, and match the instrument’s equilibration and environmental procedure.
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Do not transfer that example unchanged to another cell line, plate geometry or imaging system. A different objective may require another optical-bottom thickness; another cell model may require a different attachment procedure; and another medium may have a different refractive index. The example is useful as a complete workflow pattern, not as a universal recipe.
A 2025 holotomography study measured the refractive index of the surrounding medium and used different media for distinct preparations. This illustrates why medium selection belongs in the method description and data interpretation. If your experiment uses more than one medium, measure or otherwise document the relevant medium property according to the instrument protocol, and avoid treating phase or refractive-index values as directly comparable without accounting for that difference.
Quality-control checklist before the first time point
- Instrument: The vessel format, optical-bottom thickness and objective working distance are approved for the system.
- Culture: Cells were handled with the validated sterile procedure, and any coating or seeding schedule is appropriate for that cell model.
- Medium: The composition is suitable for the planned duration, and the medium condition is consistent across quantitative comparisons.
- Environment: Temperature and gas control match the cell and medium requirements, and the system has reached operating conditions.
- Optical path: The imaging region is free of bubbles, debris and liquid-level problems that could interfere with acquisition.
- Sample state: Cells show the expected attachment and morphology under the study’s predefined criteria.
- Documentation: Record vessel identity, bottom specification, objective, medium, exchange time, environmental settings, equilibration status and acquisition duration.
Troubleshooting common preparation failures
| Symptom | Likely preparation question | Corrective approach |
|---|---|---|
| The objective cannot focus on the cells | Is the bottom thickness or vessel geometry outside the instrument’s working range? | Check the approved vessel and objective specifications before reseeding; do not compensate by forcing the focus or stage. |
| Phase or refractive-index values differ between otherwise similar samples | Was the surrounding medium changed or prepared differently? | Verify medium identity and handling, document the refractive-index context, and repeat comparisons with consistent medium conditions. |
| Cells deteriorate during a time series | Are the medium, temperature, gas conditions and duration appropriate for this cell model? | Review the validated live-cell conditions, reduce unnecessary handling and confirm that the environmental system was equilibrated. |
| Images contain unstable regions or sudden contrast changes | Could bubbles, evaporation, movement or environmental drift be affecting the optical path? | Inspect the vessel and enclosure, stabilize the sample, and control evaporation before starting another acquisition. |
| Cells attach poorly | Was a cell-specific coating or seeding procedure required? | Return to the validated culture procedure for that cell type instead of adopting a generic coating or density. |
What to record for reproducible label-free data
Because label-free contrast depends on both the specimen and its optical surroundings, a minimal method description should identify more than the cell line. Record the vessel format and optical-bottom specification, objective and instrument, coating if used, seeding procedure, medium identity and any exchange, environmental conditions, equilibration status, acquisition duration and the criteria used to accept or reject a field.
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Also note whether the result is a two-dimensional phase image or a three-dimensional refractive-index reconstruction. That distinction determines what a change in contrast can legitimately mean. A label-free image can reveal intrinsic structure and dynamics, but it does not by itself establish the molecular identity of a feature.
Bottom line
Prepare label-free live-cell samples as an integrated instrument-and-cell workflow: verify the optical path first, use a compatible glass-bottom or chamber format, culture and seed with cell-specific procedures, select a viability-supporting and optically documented medium, equilibrate environmental control, and inspect the sample before acquisition. Treat published vessel and medium details as method-specific examples, not universal standards.
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