Application of Laser Feedback Interference Microscopy to Localize the Ciliary Vector

Ben Ovryn, Ph.D.,1 Tabrez Alam2 and Peter Satir,Ph.D.1 

Introduction

References

Acknowledgements

Abstract

Discussion

Fluorescence ImagingCells from culture dishes were plated onto glass coverslips. Upon reaching 90-95% confluency, cells were starved for 24-48 hrs in serum-free DMEM media with Pen-Strep. They were then prepared for immunofluorescence with mouse anti-acetylated alpha-tubulin (1:1000 in 0.1% BSA) as the primary antibody and either anti-mouse Cy5 or anti-mouse Alexa 488 (1:500 in 0.1% BSA) as the secondary antibody. The coverslips were mounted onto a glass specimen slide and imaged with a Nikon TE2000U microscope with 60x, NA=1.3 oil-immersion objective. 405nm (DAPI), 488nm (Alexa 488), and 637nm (Cy5) wavelength beams were used for excitation.

Primary Cilia grew in both cell types under 24-hr and 48-hr starvation. At 48-hr serum starvation, the K-wt cells had elongated cilia, averaging at 4.02 m while MEF-wt cilia ranged at 2.70 m. In the IFT-88 knockdown, little or stunted cilia grew in both cell types. In the following select fluorescent images, the blue indicates the nuclear stain, DAPI, whereas the green indicates regions where the secondary antibody has attached to the primary antibody.

We acknowledge the support of: Dennis Guo (Departments of Chemical and Biomedical Engineering, Carnegie Mellon University) who grew all of the primary cilia used in our experiments while he was supported by the Summer Undergraduate Research Program at Einstein (2013). We thank Olatz Pampliega and the lab of Ana Maria Cuervo, M.D., Ph.D. for all cell culture. This work was supported by the Einstein Summer High School Program and grant R01GM076293 to B.O.

[1] Christensen, S., Pedersen, S. F., Satir, P., Schneider, L., Veland, I. R. (2008). “The Primary Cilium Coordinates Signaling Pathways in Cell Cycle Control and Migration During Development and Tissue Repair.” Curr. Top. Dev. Biol. 85 (2008): 261-301[2] Atılgan, Erdinç, and Ben Ovryn. "Reflectivity and topography of cells grown on glass-coverslips measured with phase-shifted laser feedback interference microscopy." Biomedical optics express 2.8 (2011): 2417-2437.[3] Feng, S., Winful, H. G. “Physical Origin of the Gouy Phase Shift.” Optics Letters 26.8 (2001): 485[4] Jiang, H., English, B. P., Hazan, R. B., Wu, P. and Ovryn, B. (2015), “Tracking Surface Glycans on Live Cancer Cells with Single-Molecule Sensitivity,” Angew. Chem., 127: 1785–1789.

We have demonstrated the attributes associated with interference imaging of primary cilium. As compared with fluorescence imaging, the label-free interference method is not impacted by bleaching and may be used to localize the axial position of the tip of the primary cilium with sub-micron precision. Interference imaging of the primary cilium was on fixed cells, but we have also shown its utility of live cell imaging. Using laser feedback interferometry combined with a high numerical aperture microscope objective, we have observed that protein density within the primary cilium is large enough so as to cause a measurable change in reflectivity. Correlated fluorescence imaging of the primary cilium demonstrates that the change in signal is associated with the primary cilium. The next steps in this project are: (1) grow the cells in a microfluidic chamber and demonstrate controlled displacements of the primary cilium and (2) implement our single molecule tracking methods and measure the dynamics of selected molecular signal components as they traffic from the PDGFRreceptor.4

1Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, NY; 2Bronx High School of Science, Bronx, NY

We have developed a form of interference microscopy, called phase-shifted, laser-feedback interference microscopy, that can measure the reflectivity and topography of a reflective surface.2 These reflections can arise from dense protein aggregations or organelles or the surface of glass. We have completely calibrated the instrument and demonstrated that it can produce “images” that appear to look like those obtained from a CCD and that the technique is extremely sensitive to small changes in index of refraction. The interferometer is based upon a continuous-wave helium-neon laser that illuminates the sample. Reflected light from the sample travels back to the laser cavity. Images are produced pixel-by-pixel by scanning the object.

10 m

A B C

Select images of cells with primary cilia. (A) shows a MEF-WT grown under 24 Hr Serum Starvation. (B) shows a Kidney Epithelial Cell Wild-Type under 48 Hr Serum Starvation. (C) shows a Mouse Embryo Fibroblast IFT Knockdown under 24 Hr Starvation with deformed cilia.

In migrating fibroblasts with primary cilia, the primary cilium points in the direction of migration, but why this is so is unknown. The objective of our work is to investigate whether directional signaling from the primary cilium (the ciliary vector) affects the direction of cell migration or vice versa and to determine what molecules and mechanisms are involved. Using a well defined cilium-based RTK signaling pathway whose receptor (PDGFR specifically localizes and signals from the ciliary membrane, we use micropipettes to apply a variable concentration of directional stimulus to the cilium and phase shifted laser feedback interference microscopy to determine the transverse and axial position of the cilium in living fibroblasts.

We have obtained data which demonstrate that we can correlate fluorescence imaging of GFP labeled cilia with our interference imaging. Although analogous to confocal imaging, our interferometer is a form of label-free imaging that has high sensitivity to dense protein aggregations and can determine their axial position with an accuracy and precision of better than 10nm in live cells. We have obtained data which indicates that we obtain a strong reflective signal from the primary cilium that we can use for localization. We plan to use this signal in a feedback loop so that we can introduce control perturbations to the direction of the cilium, with an AFM like probe, while simultaneously tracking signaling molecules.

As long known, the primary cilium points in the direction of migration. We have shown, for example, that in wound healing experiments with mouse embryo fibroblasts (MEFs), all the primary cilia along the edge of the wound point in the direction of migration.1 In Tg737 MEFs, with abortive primary cilia, migration is random, suggesting that there is a deterministic relationship between the ciliary direction and the direction of migration. In MEFs, the receptor tyrosine kinase PDGFR is upregulated at ciliogenesis and tracks to the ciliary membrane.. When the PDGFR ligand, PDGF-, is present, the receptor in the cilium dimerizes and signals via a phosphorylation cascade to molecules controlling cell division and migration. However, it is not known how signaling through PDGFR affects ciliary direction, and whether the direction of migrations determines the ciliary direction or conversely, if the cilium determines the migration direction.

Measuring Phase Change Through Focus

Primary Cilia Cell CultureMouse kidney epithelial wild-type (K-wt) and mouse embryo fibroblast wild-type (MEF-wt) cells were compared to their respective IFT-88 knockouts which served as experimental controls. Because Intraflagellar Transport (IFT) is responsible for the transport of materials across axonemal microtubules and the assembly of the primary cilium, IFT cell types demonstrate a lack of cilia and/or stunted cilia. All cell types were cultured in DMEM with 10% FBS and Penicillin-Streptomycin (100 IU and 100 g/mL respectively). The MEF-IFT88 cell-type was a knockdown variant accomplished through shRNA, and thus required selection through puromycin.

Laser Feedback Interference Microscopy

Calibration using a microscopy reference standard.

Correlated Fluorescence and Interference

K-wt Primary cilium immunofluorescence image. m

In a confocal microscope, defocus of the sample rapidly reduces the intensity. In an interference microscope, the imaging is much more complex. As a result of Heisenberg’s uncertainty principle, an observe can measure either the position or momentum of a wave at focus with arbitrary precision, but not both at the same time. This uncertainty introduces a non-linearity into the phase and a shift of as an object is scanned through focus.3

Effect of defocus on the interference signal. (a) Change in the measured visibility or reflectivity (dotted lines) as the coverslip-buffer interface was translated by away from the objective lens along the z-axis. (b) Determined position of the interface, measured z, as a function of known defocus (dotted. (c) Difference between measured and predicted phase, within 2 m near focus (dotted). Plot of predicted phase change (solid black line).

kd

Cover Slip

Objective Lens

“Poker”

Results

Atilgan and Ovryn measured and modeled this effect by determining the phase and reflectivity (fringe visibility) as a coverslip was translated linearly through focus.2 As defocus increases, (a) the reflectivity decreases and (b) the linear phase change is recovered. Near focus (c), however, the non-linear phase variation is apparent and pronounced.

Live Cell Interference ImagingWe have demonstrated that we can measure the reflectivity and topography at focal and nascent adhesions in live, motile cells.2 Each of the scans contains 60 x 40 pixels and the reflectivity (top, measured in parts per thousand) and topography (bottom, measured in nm) was determined at each pixel in approximately 25 seconds. The temporal separation between scans was about 6 minutes. It may be observed that as the cell moves, it retracts its filopodia.

A B CThe primary cilium points towards the leading edge the direction of migration. Differential Interference Contrast (DIC) and immunofluorescence microscopy of a migrating cell in a scratch assay. The red arrowhead and the dotted-line represent the direction of migration of the cell into the wound as visualized by video DIC microscopy. For immunofluorescence, nuclei were stained with DAPI (blue) and the primary cilium was visualized with anti-Acetylated -tubulin (Ac-tub, green arrow).

CCD image “reflectivity” image

Determining the Distance Between the Primary Cilium’s Tip and a Glass Coverslip

Ultimately, we plan to culture cells in a custom microfluidic chamber produced with parallel top and bottom glass slides such that an actuated cantilever can be introduced from the side to displace the primary cilium. This approach will enable simultaneous interference imaging and tip displacement. For proof-of-principle experiments, however, we have grown cells on MatTek glass-bottom dishes and used an additional glass coverslip. We have demonstrated that we can localize the position of the primary cilium in three-dimensions, but we have not yet introduced controlled displacements.

As shown above, incident laser light reflects from both the glass coverslip and from the primary cilium. These two reflected fields combine and this interference changes the intensity of the laser. We introduce controlled phase-shifts using an electro-optic modulator and we can separately determine the “reflectivity” of the primary cilium and the position of the primary cilium above the glass coverslip. The latter is determined from the measured phase, , which is the scalar or dot product between the displacement vector, d, of the cilium and the wavevector, k, that is formed by the imaging geometry.

In interference microscopy, if a reflective object is translated through the fixed focus of a high-NA objective, the phase changes smoothly near the focus, but non-linearly at the focus. This non-linear phase change is called the “Gouy” phase shift. We verified the measurement of this phase change.

m

Laser feedback interference reflectivity.

We produced correlated images of fluorescent primary cilium and interference images so to localize the primary cilium in three-dimensions. As demonstrated below, the reflectivity (visibility) of the primary cilium changes abruptly as the primary cilium is translated through the fixed focus of a high-NA objective. This reflectivity change is considerably more pronounced than the intensity change that would be measured in a confocal microscope. Furthermore, as expected from theory, the phase changes non-linearly near focus. This is demonstrated in the figure below where we measure the change in position as the primary cilium is linearly translated through focus; from 0 to 10 m, we input a linear translation and we measure a linear displacement. Near focus, at about 10 m defocus, the displacement changes non-linearly. This enables the measurement of the distance of the primary cilium above the glass coverslip

z (m)

Mea

sure

d z

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Non-linear phase change at focus.

Mea

sure

d z

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z (m)

Vis

ibil

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z (m)

Axial scan of cilium through focus

Laser spot and ciliumDAPI GFP (cilium)

Fluorescent primary cilium and laser spot.

Transverse (xy) scan of cilium at fixed focus