Multimodal Label-Free Imaging to Assess Compositional and Morphological Changes in Cells During Immune Activation
When individual immune cells are triggered by pathogens or signalling molecules, a highly complex set of pathways regulate the response that involves a diverse range of intracellular components. Typically assessed through either fluorescent labelling or by invasive sequencing techniques, an alternative approach is becoming possible: the label-free measurement of the changes that occur on activation. Label-free analysis that can discriminate between different cell types or activation states uses contrast based on the endogenous molecular components of the cell, and can have sensitivity to both the molecular and morphological phenotype of the cells. Using optical scattering spectroscopy, which highlights molecular characteristics of the cell, along with quantitative phase imaging that provides morphology information, the elucidation of activation states can be achieved using only light. These techniques show that label-free optical methods can be an important part of the framework for future directions in single cell analysis.
First I would like to acknowledge the support of the Uehara Foundation, who supported the research discussed here. Aside from financial support, which is crucial, members of the foundation also expressed their interest in developing new ways to visualize and measure cellular processes. Other recipients of Uehara support also were enthusiastic and provided useful feedback and support of this research. It has been an honor to be a part of this program. I would also like to thank the members of my lab, especially Dr. Nicolas Pavillon and Dr. Alison Hobro, without whom this work would not have been what it is. Also my colleagues and collaborators, particularly in Raman imaging at Osaka university, and in immunology at our institute have been very supportive.
Since joining the IFReC immunology institute, our lab research has been aimed at creating new optical tools to understand some of the less clear aspects of the immune response, especially on the single-cell level. Most of the tools used by researchers to study the cell response involve tagging specific target molecules and seeing how they are expressed in different cell types, and determining how that expression changes during dynamic processes such as the stimulation of different immune pathways. There are a large number of pathways, signaling ions, expression levels of different proteins, RNA, and more that can be targeted (Spiller et al. 2010) and this number continually grows as researchers unlock new information on how the cell is built, and how it responds. The growing number of known pathways means that at any given time, we have had, and continue to have, only a partial view of the basic mechanisms of the cell. This is where the developments in this project can play a role: using label-free optical methods to interrogate the cell, we are able to analyze changes in molecular content and morphology which can correlate with how the cell is responding to immune stimulation. This approach does not give the same level of specificity as using fluorescent markers, PCR, or other analytical chemistry-based techniques, but instead allows the generation of quantitative data which corresponds to the entire cellular composition and shape, which in turn, can be correlated with cell type, cell state, or other features of interest. It also allows mining the data to look for emergent patterns, correlations with other measurements, or to look for new features or indicators of cellular reactions which may not have been observed with other measurements.
Raman imaging produces large amounts of highly useful data, and when performed with conservative laser excitation powers, does not harm or modify the cell. However, it is possible to simultaneously multiplex Raman imaging with other label-free modes (Pavillon et al. 2014) and thereby gain complementary data from the sample. The only downside to this approach is complexity of the optical setup, which once solved, allows simultaneous multimodal quantitative label-free imaging and analysis. To this end we also use quantitative phase imaging, which works by measuring the differences in optical path length across the cells, and provides a spatially resolved map of the sample morphology. From a collimated beam of laser light, a partial mirror splits the beam, sending half of the light through the sample, while the other beam acts as a reference and does not pass through the sample. Both beams are then combined and interfered on a detector. This gives a spatially resolved map of the optical path length and attenuation characteristics of the sample. The technique we use, known as digital holographic microscopy (DHM), provides quantitative morphological data that has been shown to be highly useful, for example demonstrating the ability to predict cell death at an early stage by detecting the changes in cellular ionic homeostasis (Pavillon et al. 2012)
Interestingly, more and more reports are now emerging of how individual cells respond differently, and the expression of different markers can be highly variable. The approaches shown here provide a means to evaluate these single cell level responses, without changing or modifying the sample. While not having the specificity of more traditional techniques such as labelling or invasive PCR type methods, these label-free techniques allow the evaluation of different cellular functions before, during, and after they occur, and can then be used over time to evaluate the progression as well as heterogeneity of cell responses. We also use complementary techniques such as ELISA or other wetlab techniques to validate these methods, and can use the resulting data to form a set of independent measurements, covering both population level responses (from ELISA) and single cell variations in response.
I sincerely appreciate the Uehara foundation funding for supporting this fundamental research and we look forward to future progress building on these results.
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