Resource detail

Live-cell imaging: Is it beneficial to adopt label-free methods?

Live cell imaging is becoming a vital part of the research process, with advances made in the methodology and approach taking researchers forward in terms of accuracy and efficiency.

For instance, due to advancements in super resolution microscopy techniques alongside development of fluorophores, fluorescent probes have enabled us to image cellular and molecular components beyond the limits of optical resolution from ~ 250 nm to about ~10 nm (1).

These microscopy techniques require fluorescent dyes, proteins or photo switchable probes, which all come with their own limitations{Jensen, 2012, Use of fluorescent probes: their effect on cell biology and limitations} (2). We know that immunofluorescence techniques require harsh methods of fixation, antigen retrieval that can modify morphology of membranous organelles.

In addition, the current methods demand cumbersome protocols for antibody optimization and control experiments to avoid non-specific staining and means the process is running at less than optimal efficiency. Furthermore, the use of fluorescent tagged proteins has many pitfalls, including transfection efficiency, abnormal localization and function of fused protein.

Where live cell imaging use fluorescent vital stains, we are then limited by the problems of photo-bleaching and photo-toxicity, rendering this approach unsuitable for time-lapse experiments. This has increased the urgency for developing improved, more viable techniques for label-free live cell imaging.

Label-free imaging has several advantages over fluorescence based imaging, in instances where researchers’ are striving to gain insight in the behavior of living cells. These include:

  1. Cells are not damaged due to fixatives, photo toxicity and protein overexpression.
  2. The information captured from the cells is in its native state.
  3. It’s a cost effective and efficient approach, as there is no need for expensive probes, pre-treatment, fixation and staining procedure.
  4. Time-lapse monitoring is continuous, meaning cellular behavior, processes and even subcellular organelles and macromolecules can be tracked in detail.
  5. Imaging can be done under more controlled conditions in media or buffered saline solutions.
  6. The same cells can further be used for end-point assays like immunohistochemistry, flow-cytometry, reducing experimental setup and complexity.
  7. Remote monitoring of cellular processes, cell quality assessment and monitoring cell confluency is a viable option.
  8. This approach is more suitable for high throughput applications like drug screening, along with automated imaging and analysis. This has huge implication in clinical drug discovery studies where capturing multi-parametric data across time points is vital to achieving accurate results



WHAT ARE THE OPTICS OF LABEL-FREE IMAGING?

Whereas the fluorescent imaging approach to live cell imaging is based on excitation and emission of light by fluorescent probes, label-free imaging utilizes the cells inherent properties of contrast mechanisms such as thickness and refractive index (RI) (Fig 1).

Figure 1 | Basis of how phase delay is used to create contrast image. Simplified from Kasprowicz et al, 2017.

The more prevalent label-free techniques rely on phase contrast (PC) and differential interference contrast (DIC), which employ optical setups in order to convert phase shifts caused by cells/ intracellular features into changes in light wave amplitude. Both PC and DIC optics have their limitations, such as poor contrast and a marked lack of quantitative image segmentation.

Compare this with the modern, quantitative phase imaging (QPI) technique which utilizes modalities to quantify the extent of any phase delay introduced by the sample in the form of pixel level information. Here, the pixel intensity is determined by the thickness and refractive index of the sample, which is a measure of the biomolecular composition and organization (3). Fig 2a explains the difference in the optics between PC, DIC and QPI techniques and a comparison of images obtained by the three above techniques is depicted in Fig 2b. QPI techniques are fast evolving to provide vastly improved optical contrast and spatio-temporal information.

Figure 2a | Simplistic flowchart explaining basic difference in optical measurement in PC, DIC and QPI imaging techniques. Adapted from Kasprowicz et al, 2017.


Figure 2b | Representative images and line profile obtained from DIC, PC and QPI images of A549 cells. Note enhanced contrast and spatial resolution obtained by QPI techniques. Scale bar 50µm. Modified from Kasprowicz et al, 2017.


QUESTIONS TO ASK WHEN DETERMINING THE REQUIREMENTS OF THE EQUIPMENT:

Label-free imaging can be deployed in various ways, in order to obtain required information depending on the question in hand. For instance, questions for consideration could include:

  • How large is the cellular population you would like to image?
  • What is the ideal field of view?
  • How long will the experiment take?

Asking these questions or discussing them with your project team, will help determine the spatial and temporal information that can be extracted from the experimental data.

------------------------

Label-free imaging has not just been deployed for biological assays utilizing compact bright-field microscopes equipped with image analysis algorithms. Label-free imaging can also be used to monitor the growth of cell cultures, allowing teams to receive notifications when the desired level of cell confluence is reached with the option to also automatically maintain cells in the optimal conditions for growth and maximum cell viability.

For researchers looking to get insight into the quality of their cell culture during routine cell expansion procedures. We recommend looking at the CytoSMART Lux2.

------------------------

Once these requirements are defined, the next stage of planning an experiment utilizing label-free live cell imaging, is to consider the following important parameters.

1. Sensitivity:

The details of the images that can be obtained from a microscope depends on the FOV (field-of-view) and magnification of the imaging optics. These two parameters are captured in the field-of-view number: The diameter of the view field (in millimeters) decreases when an increase in magnification of the optical set-up occurs.

While the magnification and field of view are negatively correlated, it is in fact possible to achieve a large field of view while maintaining a relatively large magnification. With the use of modern plan-apochromats and other specialized flat-field objectives, the field of view can be enlarged to capture greater range of specimen features. Such features are desirable for experiments that study a large surface area of a cell culture vessel.

e.g. when studying cell migration using wound healing assays. In these experiments the full length of wells is often used to apply the scratch (wound) into a monolayer of cells. Here the ideal FOV is the whole-well of the cell culture, which has typically not been easily achieved, but using the right equipment and approach this can be done successfully.

On the other hand, the overall magnification becomes crucial when imaging single cells (~10-40 μm) or organelles (~1-2 µm). Advanced optics coupled with improved image processing algorithms can provide high resolution and high quality images from even sub-cellular organelles.

2. Speed:

For time-lapse experiments the rates at which the images are captured depends on the speed of the image acquisition system. Dynamic processes such as cell trafficking need higher acquisition speed, as compared to slower processes like cell division (ranging in hours) and cell differentiation (ranging in days).

3. Duration of experiment:

Where an experiment is spread over several hours or days, the cell culture must be maintained in optimum conditions (temperature, CO2 levels, humidity). In such cases it is important to use microscopes that are fully compatible with incubator use or have the flexibility to utilize a controlled chamber for storing the specimen.

4. The Need for Automation:

When planning experiments that require simultaneous image acquisition from multiple specimens and multiple time-points, one might consider the use of automated imaging set-ups in order to streamline the process and ensure accuracy throughout.

For researchers that want to obtain long term time-lapse videos of their specimens in cell based assays we recommend looking at the CytoSMART Omni



ARE YOU CLEAR ON LEVEL OF DETAIL YOU’LL NEED TO OBSERVE?

Label-free imaging facilitates observation and analysis through a range of specificity and detail, making this a scalable approach for your experiments. Leading researchers are already embracing this approach, as illustrated in the below cross section of applications for label-free imaging. Depending on the biological question, the authors involved in the studies below have exploited the advantages of label-free imaging to extract relevant information, providing metrics ranging from cell population level to intracellular level.

Population level metrics

PC and DIC optics have been successfully used to study cell population behavior like cell growth, viability assays, drug efficacy testing, wound repair and gap closure assays (4, 5). In Blum et al, for instance, PC live cell imaging was used to compare cell proliferation and wound closure rate in wild type and calretinin knockout rodent primary mesothelial cells (Fig 3) (5). The scratch area was imaged every 15 min by live cell imaging and image analysis was done by Image J.

Figure 3 | Wound healing rate was measured as the time taken to repopulate the scratch area in WT and Calretinin knockout rodent mesothelial cells in culture, in 72 hours. Image used for representative purpose from Blum et al, 2015.

QPI based imaging provides additional information, such as the dry mass of a group of cells, utilizing its unique phase metric properties. QPI based live cell imaging combines dry mass and RI based measurements in order to accurately measure cell proliferation and wound closure rates (4, 6).

See an example of how whole-well image analysis can help in setting up reliable wound healing assays.


Individual cell data

Genetically identical cells can show heterogeneity, owing to differences in their proteome and transcriptome. It becomes important to assess cell to cell variability in experiments with any clinical outcome, such as the response of tumor cells to chemotherapeutic agents. Cell motility and migration studies are of significance in understanding immune regulation, embryogeneis, inflammation and cancer and in these instances a live or real time approach to cell time lapse imaging can be deployed to monitor individual cell motility and proliferation (7).

Kasprowicz et al, discusses how single cell trajectories were estimated using time-lapse imaging with QPI optics (3). Figure shows the individual cell trajectories in untreated and staurosporine treated MDA-MB-231 cultures for a 72 hour duration. Images were acquired at 10X magnification at regular 10 minute intervals with trajectory analysis carried out using Cell Analysis Toolbox from Phasefocus. Dynamic phenotype metrics obtained with QPI imaging and analysis exhibits increased unidirectional motion in staurosporine treated MDA-MB-231 cells.

Figure 4 | QPI time sequence images and trajectory tracking of individual MDA-MB-231 cells indicate increase in unidirectional motion in staurosporine treated cells. Scale bar 500 μm. Figure modified from Kasprowicz et al, 2017 for review purposes.

Are you interested in tracking individual cells. Here, we provide cellular metrics that can help you in determining the right strategy for image analysis

Cell cycle and lineage

In vitro cell cycles and cell lineage tracing has huge implications in the studying and analysis of cancer and differentiation. Label-free imaging facilitates the long term imaging of successive divisions or differentiation of individual cells without potentially altering native behavior. Both PC and QPI techniques have successfully been used to study mitosis and cell division (8, 9), with enhanced algorithms.


Zhang et al, characterized stem cell differentiation over a 15 day time period utilizing QPI microscopy and evanescent wave microscopy (10). Briefly, two light emitting diodes (LEDs) of 660 nm were used for illuminating the sample from top (QPI) and from bottom for TIRM (total internal reflection microscopy). As 660 nm is in the long wavelength range, it is less photo-toxic enabling imaging for prolonged hours without much damage to cells. Metrics assessed from the data extrapolated via both of these techniques enabled label-free high resolution imaging of stem cell differentiation status, as depicted in Fig 5. Quantitative assessment of cell size and morphology was done using inbuilt algorithms in Matlab (The Mathworks).

Figure 5 | QPI coupled with TIRM (Total Internal Reflection Microscopy) gives high resolution information (single cell lineage tracking) to track stem cell differentiation in a 15 day culture period. Scale bar 50 µm. Modified from Zhang et al, 2018 for representative purpose.


Organelle level imaging

Modification of traditional DIC and QPI techniques has enabled visualization of subcellular organelles like lipid droplets, mitochondria, ER and golgi from label-free live cells. Nishimura et al, devised a modified DIC based imaging system to visualize sub-cellular organelles in the bright field. For successful imaging at this level of detail, powerful lenses are required so you must ensure that the system you choose is sophisticated and robust enough for the task in-hand and capable of accurately verifying the desired optics.

The authors in this example, utilized a RM-DIC microscope equipped with a modulating retardation unit in the illumination path, introducing a pair of shear direction changers to achieve Phase Distribution (PD). The PD images obtained were then converted to ePD images using Imaris Image Analysis software (BITPLANE) to obtain high resolution organelle level images. Fig 6 shows the corresponding images of ER, golgi and mitochondria obtained using Phase Distribution (modified DIC) microscopy.

Figure 6 | Phase distribution (PD- modified DIC) enables visualization of sub-cellular organelles in NIH3T3 cells in bright field. ePD- pseudo color PD image obtained by image processing. GFP fused with localization signals for Mitochondria, ER and Golgi was expressed in NIH3T3 cells and used to visualize the organelles. Images obtained with PD and fluorescence display comparable resolution and clarity of image. Scale bar 10 μm. Image modified from Nishimura et al, 2019 for representative purposes




WHAT ARE THE OPPORTUNITIES MOVING FORWARD?

Ongoing advancements in optics and AI algorithms provide enormous scope for development of label-free live cell imaging techniques in the near future. Furthermore, the increasing demand for remote cell culture monitoring in controlled experimental setups like hypoxia, BSL3/BSL4 facilities has led to development of compact imaging setups with automated cloud based analysis (Drug Discovery World Fall 2019).

Within the drug discovery area of research, the need for high throughput, automated imaging systems will only increase, and it is imperative that technologies evolve in order to fully unlock the benefits of label-free live cell imaging. CytoSMART, along with researchers and other equipment providers will continue innovating and designing to lead on these developments, supporting innovative, accurate label-free live cell imaging techniques and set ups.

Learn About all CytoSMART imaging solutions here



author:

Sreejata Chatterjee

Sreejata has a PhD in Neuroscience with research expertise in Virology, Genomics and Oncology. In her current role as a Senior Scientist (R&D), she is working on optimizing 3D culture based platforms for clinical cancer research and drug discovery. She is a freelance science writer and consultant, especially enthusiastic about the advancements in the field of precision oncology, imaging and genomics.



REFERENCES:

1. Galbraith CG, Galbraith JA. 2011. Super-resolution microscopy at a glance. J Cell Sci 124:1607-11.

2. Jensen EC. 2012. Use of fluorescent probes: their effect on cell biology and limitations. Anat Rec (Hoboken) 295:2031-6.

3. Kasprowicz R, Suman R, O'Toole P. 2017. Characterising live cell behaviour: Traditional label-free and quantitative phase imaging approaches. Int J Biochem Cell Biol 84:89-95.

4. Jonkman JE, Cathcart JA, Xu F, Bartolini ME, Amon JE, Stevens KM, Colarusso P. 2014. An introduction to the wound healing assay using live-cell microscopy. Cell Adh Migr 8:440-51.

5. Blum W, Pecze L, Felley-Bosco E, Schwaller B. 2015. Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration. Respir Res 16:153.

6. Kastl L, Isbach M, Dirksen D, Schnekenburger J, Kemper B. 2017. Quantitative phase imaging for cell culture quality control. Cytometry A 91:470-481.

7. Dosch J, Hadley E, Wiese C, Soderberg M, Houwman T, Ding K, Kharazova A, Collins JL, van Knippenberg B, Gregory C, Kofman A. 2018. Time-lapse microscopic observation of non-dividing cells in cultured human osteosarcoma MG-63 cell line. Cell Cycle 17:174-181.

8. Marrison J, Räty L, Marriott P, O'Toole P. 2013. Ptychography--a label-free, high-contrast imaging technique for live cells using quantitative phase information. Sci Rep 3:2369.

9. Rapoport DH, Becker T, Madany Mamlouk A, Schicktanz S, Kruse C. 2011. A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters. PLoS One 6:e27315.

10. Zhang J, Moradi E, Somekh MG, Mather ML. 2018. Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status. Sci Rep 8:697.