Optical Imaging of Biomolecular Tension with Sub-100 nm ResolutionRichard Park1,2, Yang Liu1,3, Khalid Salaita1,3

1. Emory SURE, Atlanta, GA, 2. Brown University, Providence, RI, 3. Department of Chemistry, Emory University, Atlanta, GA

Integrin receptors transmit forces generated by the cytoskele-

ton and apposed by the elasticity of the ECM, and accordingly, mechan-

ical forces play important roles in their function. For example, integ-

rin-ligand affinity is highly force-dependent. With these mechanical

cues highly dependent on substrate rigidity, morphology and ligand

density, FAs interact with the cytoskeleton, influencing cell fate.

Background

Focal adhesions (FA) are multi-micron sized protein complexes

comprised of at least 150 different proteins that function to anchor

cells to their extracellular matrix (ECM). Typically, FAs dynamically as-

semble at the sites of activated integrin receptors (labeled red in the

figure below), which are transmembrane proteins that bind specific li-

gands within the ECM.

Nanoscale architecture of focal adhesions

Kanchanawang, P. et al., Nature 2010 468, 580-584

Current methods that measure these forces have tension sensi-

tivity limitations and are unable to directly measure the tension be-

tween a single surface ligand and its integrin receptor counterpart. We

seek to overcome this obstacle and image integrin receptor forces with

sub-100 nm resolution.

So how are these biomolecular

forces accurately reported?[ ]

Approach

The MTFM system consists of three components:

• each gold nanoparticle (AuNP) acts as a fluorescence quencher

• each polymer / fluorophore pair is in a mushroom conformation and quenched

while in the relaxed state

• tension forces extend the polymer and de-quench the fluorophore

The fluorescence intensity optically reports mechanical forces that provide new insight

into the spatial and temporal distribution of single integrin receptors within FAs. However,

the spatial resolution of MTFM is restrainted by the diffraction limit of light. We resolve

this by combining MTFM with the superresolution microscopy technique below.

Mertz, J., Nature Methods 2011 8.10, 811-819

Molecular Tension Fluorescence Microscopy (MTFM)

Cell/sensor interaction

Structured Illumination Microscopy (SIM)

SIM resembles conventional fluorescence microscopy

but with the integration of a translating grid pattern in

the path of illumination, which serves to:

• progressively collect contrast information through

several (nine) image collections

• compare the contrast of a single bright pixel across

each image

• post-process to reduce background fluorescence in

the reconstructed image

Visualizing integrin receptor tension of 3T3 fibroblasts with AuNP-based MTFM probes. Representative RICM, fluorescence tension response, and overlay images are shown for three incubation periods. We observed a strong fluorescence signal in the cell periphery correlating with the cell-binding pattern in the RICM. However, the low spatial resolution of these images diminishes the MTFM advantage – unable to accurately report fluorphore density per pixel. The scale bars are 20 μm.

SIM reveals the visually non-uniform nanostructure of focal adhe-

sions with higher resolution than possible with conventional fluores-

cence microscopy. Moving forward, fluorescence intensity and force

analysis, the use of different cell lines, inhibitors and activators,

FA-marker transfected cells, and further optimization of the com-

bined MTFM/SIM approach can shed more light on the mechanosen-

sitivity of individual integrin receptors within focal adhesions.

Richard Park is currently a rising fourth-year undergraduate student at Brown University concentrating in Biomedical Engineering and French Studies.

Richard_Park@Brown.edu

Structured Illumination Microscopy

Conventional SIM Visualizing integrin receptor tension of rat embryonic fibro-blasts with AuNP-based MTFM probes with conventional and structured illumination micros-copies. We observe an increased resolving capability and a clear, non-uniform distribution of ten-sion. The scale bars are 1 μm.

RICM Fluorescence(Tension)

Overlay

30 min

90 min

16 hr

Results

Conventional Fluorescence Microscopy

Tension Force Microscopy (TFM)

van Hoorn, H. et al., Nano Letters 2014, DOI: 10.1021/nl5008773

10µm10µm

Conclusions

Acknowledgements I thank my mentor, Yang Liu, and my PI, Dr. Khalid Salaita, with both of whom I have had the pleasure to work. I also thank Emory University’s SURE and its director

Dr. Molly Embree. My gratitude also extends to the entire Salaita Group whose efforts to help me in my research exceeded any and all personal expectations.

This work was supported by R25HD079102 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and by the Howard

Hughes Medical Institute Science Education Program award #52006923 to Emory University. Any opinions, findings, and conclusions or recommendations expressed

in this material are those of the authors and do not necessarily reflect the views of the Howard Hughes Medical Institute or Emory University.