Download Waveguide excitation fluorescence microscopy: A new tool for

Waveguide excitation fluorescence microscopy:
A new tool for sensing and imaging the biointerface
H. M. Grandin, B. Stadler, M. Textor, J. Voros
Biointerface Group, Lab. For Surface Science and Tech.,
Dept. of Materials, ETH-Z
Presented by: Ayca Yalcin
Outline
• Introduction to WExFM
• OWLS, TIRFM, Zeptoreader
• WExFM setup
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• Surface Patterning
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• Bio results
• Discussion on system performance
• Conclusion
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Introduction to WExFM
• Makes use of an optical grating to incouple laser light into a
waveguide.
• Incident laser beam is diffracted from the grating and
propagates via internal reflections inside the waveguide.
Neff=nsin+l/
• Multiple reflections within the waveguide generate an
evanescent field of intensity I(z), z being the distance
perpendicular to the surface.
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Optical Waveguide Lightmode
Spectroscopy
-
-
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In situ measurement of the surface
immobilization of biomolecules in
aqueous environment.
OWLS is based on incoupling of a laser
into a waveguide by an optical grating.
The incoupling angles are sensitive to
refractive index changes within the
evanescent field generated above the
surface.
The
angles
are
monitored
by
continuously changing the incident angle
of the laser and measuring the
incoupled light intensity with a
photodetector.
From refractive index changes, the
adsorbed mass can be calculated using
de Feijter‘s formula. (dn/dc=0.18cm3/g),
(~2ng/cm2 sensitivity)
Total Internal Reflection
Fluorescence Microscopy
• Total internal reflection obtained by
laser illumination from the periphery of
the back focal plane of an objective
lens.
• Evanescent wave enters the medium
of lower refractive index to ~0.1μm and
excites fluorophores in this thin
interfacial slice leading to high SNR.
• TIRFM is often used for imaging focal
adhesions of cells or monitoring in-situ
adsorption-desorption
kinetics
of
fluorescently labeled proteins.
• Single molecule detection possible,
>1ng/cm2 sensitivity.
Zeptoreader
•
•
•
•
ZeptoREADERTM (Zeptosens AG,
Switzerland) uses the evanescent
wave from a light beam coupled into
OWLS-like waveguides to excite
fluorophores.
The emitted light is imaged in a CCDcamera to gain additional information
about the lateral distribution, although
with poor lateral resolution.
Therefore it is impossible to
investigate cell adhesion or small
patterns with this set-up.
13um resolution, pM sensitivity
(zeptomoles)
SETUP
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Molecular-Assembly
Patterning by Lift-Off
1. Pattern photoresist
2. Dip 30min in solution of poly(l-lysine)-graftpoly(ethylene glycol)/PEG-Biotin
3. Lift-off photoresist in organic solvent
4. Backfill with PLL-g-PEG
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Biotin surface densities: a)26.6, b)13.3, c)5.3, d)2.7, e)0 pmol/cm2
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Cell attachment to peptide modified PLL-g-PEG
Results
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Mass adsorption curve obtained by OWLS (the
symbols), fluorescence intensity obtained with
WExFM (the solid line) after injection of 2ug/ml
streptavidin-488.
- A linear slope is observed at these low
concentrations, indicating a diffusion limited
adsorption process.
- Similar experiments indicated that 1ng/ml
(<20pM) could easily be detected under
these conditions.
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Fluorescence intensity vs. time. As the
concentration increases from 2ng/ml(diamonds) to
20ng/ml(triangles) a 10-fold increase in the slope is
observed, as determined by a linear fit. The flow
rate was 0.2ml/min.
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Epi-fluorescence (a) compared with WExFM images obtained with (b) and (c) without the use of an emission
filter, for a 2ug/ml streptavidin-633 solution in contact with the waveguide. The signal to noise of a:b:c is 1:13:7.
'Focal Adhesions’(:specific types of large
macromolecular assemblies) serve as a
biochemical signalling hub to concentrate
and direct numerous signaling proteins at
sites of integrin binding and clustering.
A comparison of (a) Epi-fluorescence to (b) WExFM imaging of a
fibroblast cell fixed and stained (Alexa Fluor 488) for vinculin, a protein
associated with focal adhesions.
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Discussion
-Lines in the images due to inhomogeneities in or on
the grating.
-If the defect is on the grating this results in less
efficient incoupling of the incident light. If the defect
is at some distance from the grating it will outcouple
the light and cause a complex interference/diffraction
pattern (Paulus and Martin, 2003).
-By changing the position of the laser along the
grating changes in line patterns could be observed.
However, not possible to remove the lines entirely
through a weighted sum of the images obtained in
this way.
- Imperative to good WExFM images is a clean and
defect free grating and waveguide surface.
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Comparison with other
techniques
• The WExFM combines OWLS with the substrate and objective
flexibility of an Epi-FM and with the strong signal and high
surface sensitivity of TIRFM.
• WExFM vs OWLS:
– fluorescence sensitivity along the waveguide vs. mass sensitivity
only on grating area
– laser angle scanned vs. sample rotated
– WExFM: higher sensitivity
• WExFM vs. Zeptoreader:
– Comparable sensitivity
– WExFM: able to monitor dynamic bio-interactions more closely
• WExFM vs. TIRFM:
– Comparable sensitivity
– WExFM: compatible with any magnification
– WExFM: light incoupling away from sensing area
Conclusion
• WExFM has potential for dynamic and quantitative investigation
of bio-interfacial events in situ.
• TIRFM-like surface sensitivity with the additional advantage of
flexibility in objectives used, flow cell compatibility and potential
for quantification of the fluorescence signal.
• High target sensitivity for fluorescence detection, high surface
specificity, large area analysis with sub-um resolution, ‘built-in’
calibration of fluorescent light gain, and capability to perform
multi-color imaging in situ and in real time.
• Sensitivity of the system demonstrated through dynamic
measurements of the streptavidin–biotin binding event.
• SNR compared to conventional FM gives >10-fold improvement.
• Surface specificity illustrated in a comparison of fibroblast focal
adhesion images.
References
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J. Voros, “The Density and Refractive Index of Adsorbing Protein Layers,”
Biophysical Journal Volume 87 July 2004 553-561.
L. J. Bonderer, “Waveguide Excitation Fluorescence Microscopy and its ability to
investigate cell-surface interactions,” Diploma Thesis, ETH, Zurich, March 2005.
D. Falconnet et al., “A combined Photolithographic and Molecular-Assembly
Approach to produce functional micropatterns for applications in the
biosciences,” Advanced Functional Materials, 2004, 14, No.8.