Label-Free Cell Imaging

The Use of Ptychography for Quantitative Label-Free Live Cell Imaging

  • Fig.1: Schematic to illustrate the steps of using ptychographic imaging.Fig.1: Schematic to illustrate the steps of using ptychographic imaging.
  • Fig.1: Schematic to illustrate the steps of using ptychographic imaging.
  • Fig.2: Quantitative phase image of live, unlabelled B104 Neuronal cells obtained using ptychography.
  • Fig. 3:  An individual A549 cell was tracked from mitosis through to the subsequent mitosis of its daughter cell. Calculated nuclear volume is plotted against time, which demonstrates the change in nuclear volume during the stages of the cell cycle (A). Representative images highlight the cell at each point of the cell cycle (B).
  • Fig.4: Characterization of apoptosis. A single B104 neuronal cell undergoing apoptosis was tracked and the calculated cellular thickness was plotted against time (A). Correlative images show the state of the cell at the proposed stage of apoptosis (B).
  • Fig.5: Images of HEK 293 were acquired every 6 min over 40 h. Cells were challenged with different amounts of serum to induce variable rates of cell proliferation. Representative images from time lapse video taken at 6, 20 and 40 h; cultured with either 2.5% or 5.0% serum. Graph (5B) shows the quantification of the cell count over time (B).

The Imaging and Cytometry Laboratory at the University of York [1] provides a facility committed to bringing state of the art technology to researchers with varying levels of expertise. A significant effort is devoted to training scientists in imaging and cytometry techniques to advance their projects. The facility is available to both internal and external users. Staff provides fully assisted technical support for the infrequent user while the facility is an excellent equipment resource for the experienced user.

Among the exciting techniques being used is a novel means of label-free imaging for easy cell tracking and growth analysis. Working together with Phasefocus [2], we report on biological applications of Phasefocus Virtual Lens ptychographic microscopes. These high-contrast artefact free images are ideal for cell cycle and cell mass/volume analysis. This work is also being supported through the Technology Strategy Board's Knowledge Transfer Partnership Programme and the EPSRC.

Ptychography - An Overview of Label-Free Cell Imaging

For label-free cell imaging, the Phasefocus VL20/ VL21 (upright/invert microscopes, respectively) instruments combine ptychography [3] with a light microscope equipped with a variety of lenses to provide quantitative phase images along with conventional brightfield and fluorescence imaging [4].

Collecting images is a simple process. A specimen is sampled by a patch of illumination referred to as the ‘probe.' Unlike point-scanning methods such as confocal microscopy, the probe area is typically much larger than the desired resolution.  Its phase and amplitude distribution are automatically computed, and deleterious effects of any non-uniformities in the illumination can therefore be eliminated [5].

The probe is then either shifted to a predetermined number of overlapping positions on the specimen or, the specimen can be shifted with respect to a stationary probe. At each position, the transmitted or reflected diffraction pattern is recorded on a standard two-dimensional array detector (e.g. a CCD). Data is then treated using a proprietary phase retrieval algorithm which processes the diffraction patterns to create an image pair from the specimen: an amplitude image and a phase image.  The amplitude image is similar to a conventional brightfield microscope image, and is a quantitative map of the specimen's transmittance or reflectance.

The specimen's phase function is a quantitative measure of the phase delay introduced as the wavefront travels through, or is reflected by, the specimen.

Depending upon the specimen and the wavelength, the phase data may be used to measure dry mass, thickness and refractive index.

The Challenges of Label-Free, Live-Cell Imaging

Traditional light microscopy techniques apply a variety of contrast enhancement techniques. The use of fluorescence stains bring several advantages: they add contrast, aid identification of sub-resolution objects (e.g. proteins, DNA, lipids); enable discrimination between similar objects; enable multi-component analysis; and offer quantitative analysis, cell segmentation and tracking. However, there are negatives to this approach: fluorescent labels are not always desirable because they can perturb the natural cell state and normal cell function. For these reasons, it is not always possible/desirable to label live primary cell lines. Given these limitations of conventional fluorescent labels and other stains, we have explored ptychography to give us label/stain-free imaging [6].
Label-free imaging has enabled us to achieve increased contrast quantitative data as shown in figure 2.

Applications Examples

We have gone on to complete a number of important biological applications using ptychography. These include: cell proliferation; following the cell cycle, monitoring apoptosis and cell tracking. In the examples shown here, we recommend that the reader reviews the videos that we captured for each application as these are far more effective at demonstrating the contrast gains, quantitative nature and tracking abilities.

Label-Free Characterization of the Cell Cycle

All cells in a time-lapse video may be analyzed independently and simultaneously. For example, Figure 3A shows the projected nuclear volume for a single A549 cell as it progresses through the cell cycle. This suggests that mitosis can be clearly identified by the distinct peaks, however focusing between these mitotic events the nuclear volume shows little change throughout the proposed G1 stage of the cell cycle. During the S phase of the cycle the cell begins to replicate its DNA, in the graph this is clearly visible as an increase in nuclear volume. Representative images of an individual cell (fig. 3B) correlate with the graph.

Label-Free Characterization of Apoptosis

Figure 4A shows that the projected cellular thickness of a single B104 neuronal cell increases during the healthy stage, but plateaus at the proposed early apoptotic stage. Once the cell is committed to apoptosis, an increase in thickness is clearly seen. As the cell ruptures the thickness drops dramatically, with no further fluctuations. Representative images from the time lapse series (fig. 4B) correlate with the graph.

Label-Free Quantification of Cell Proliferation

Images of HEK 293 (fig. 5A) were acquired every 6 minutes over 40 hours. Cells were challenged with different amounts of serum to induce variable rates of cell proliferation. A, Representative images from time lapse video taken at 6, 20 and 40 hours; cultured with either 2.5% or 5.0% serum. Graph (fig. 5B) shows the quantification of the cell count over time.

Looking Ahead

The future of quantitative phase imaging using ptychography is looking extremely positive. We now have more than six research projects running on our systems. Samples have included the study of cancer and stem cells and applications relating to immunology and neurology. We fully expect this project list to keep on growing as we develop our ability look in even more detail at cell cycles and behavioral studies. There is good evidence to suggest we will also be able to develop significant assays which will assist in early disease identification and diagnosis, and in pharmaceutical drug discovery.

We believe this is one of the most important breakthroughs in imaging. It addresses many of the fundamental problems inherent in current microscopy techniques. Put simply, what you get is more than what you see!

References
[1] The Imaging & Cytometry Laboratory, Department of Biology, University of York, UK: http://www.york.ac.uk/biology/technology-facility/imaging-cytometry/
[2] Phasefocus, UK: www.phasefocus.com
[3] Rodenburg J. M. and Faulkner H. M. L.: Appl. Phys. Lett. 85, 4795 (2004)
[4] A. M Maiden et al.: Proc. SPIE 7729, Scanning Microscopy 2010, 77291I (June 03, 2010)
[5] Maiden A. M. and Rodenburg J. M.: Ultramicroscopy 109, 10 (2009)
[6] Marrison J. et al.: Nature Scientific Reports 3, August (2013)

Authors
Dr. Peter O'Toole
 
Dr. Rakesh Suman, Phasefocus

The Imaging & Cytometry Laboratory
Department of Biology
University of York
York, UK

Contact

University of York
Heslington Road
York, Yorkshire YO10 5DD
United Kingdom

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