Microscope part 2 BY DR. C. P. ARYA (B.Sc. B.D.S.; M.D.S.; P.M.S.; R.N.T.C.P.)
Final Presentation
1. Improved Surface Imaging using Movable
Microlenses with Localized Plasmon
Structured Illumination Microscopy
Alefia Kothambawala
University of California, Davis
SDNI REU
Anna Bezrydina
Zhaowei Liu
3. • 1878 – Ernst Abbe correlates
resolution to the wavelength of
light (d=0.61λ/NA)
• 1893- August Koehler developed
the Koehler Illumination
• 1903 – Richard Zsigmondy
develops the ultramicroscope
• 1932 – Frits Zernike invents the
phase-contrast microscope
• 1938 – Ernst Ruska develops the
electron microscope.
• 1981 – Gerd Binnig and Heinrich
Rohrer invent the scanning
tunneling microscope
http://www.zeiss.com/corporate/en_de/history/technological-milestones/microscopy.html
http://photonicsforenergy.spiedigitallibrary.org/article.aspx?articleid=1166239
http://www.microscopy-uk.org.uk/mag/artmar06/go-phase.html
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4. Vocabulary
• Magnification- ability to make small objects seem
larger
• Resolution- ability to distinguish two objects from
each other
• Diffraction- breaking up of electromagnetic waves
as they encounter an obstacle
• Diffraction limit- finite limit beyond which it is
impossible to resolve separate points
• Abbe's equation- describes resolution in a
perfect optical system
d = 0.61λ where NA= nsinα
nsinα
www.cartoonstock.com
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5. High-ResolutionMicroscopy
• STORM- Stochastic switching of single-molecule fluorescence signal
• PALM- Developed using photoactivatable fluorescent proteins as switchable probes
• STED- Selective deactivation of fluorophores located away from center of excitation
• Developers of these techniques (Erik Betzig, Stefan W. Hell and W. E. Moerner)
were awarded Nobel Prize in Chemistry in 2014
• Drawbacks- high laser intensity irradiation, short lifetime of fluorescent dyes, limited
imaging speed
http://huanglab.ucsf.edu/STORM.html
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6. STORM PALM
STED
STORM/ PALM:
slow imaging speed
STED: laser
damage and small
field of view
BUT WE CAN RESOLVE THESE ISSUES…
http://www.precoptic.pl/Products/18/18/nSTORM https://www.activemotif.com/catalog/627/sted-microscopy-products
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/palm/introduction
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7. Structured Illumination Microscopy (SIM)
• Illuminates fluorescently labeled samples with patterned intensity (grating)
o Acquire multiple images with superimposed striped patterns
o High-resolution information become visible in form of low-resolution Moiré fringes
o Use computer reconstruct higher resolution image
http://physics.aps.org/articles/v3/40
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8. • Combines surface plasmon interference with SIM to achieve sub-diffraction limited
resolution
o Surface plasmons (SPs)- electromagnetic waves formed by oscillations of electrons
at metal/dielectric interface
• Utilizes grid-like structure to create uniform waves
o Light shined at different angles to collect multiple sub-images
o Wavelength shorter than visible light (enables superior resolution)
• Shows an 3x resolution improvement (2-fold improvement in SIM)
Z. Liu, Plasmonic Structured Illumination Microscopy, 2010
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9. Localized Plasmonic SIM (LPSIM)
• 2D hexagonal array of silver nanodiscs
illuminated at different angles
o Fluorescent samples placed directly on
plasmonic substrate
• Total of nine diffraction-limited sub-images
collected for each LPSIM image frame
• Straightforward reconstruction algorithm used
once sub-images collected
• 3x or more resolution improvement
• Local enhancement allows for strong excitation
of targeted fluorescent labels without using
unnecessarily high-powered laser or irradiating
entire sample with strong intensity
J. Ponsetto, Localized Plasmonic Structured Illumination Microscopy, 2016
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10. What can we do next?
Movable Microlenses
(Microspheres)
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11. Dielectric Microspheres
• Microlenses that collect underlying near-field
object information and magnify it
• Transmit information to far field
o Fluorescent beads send rays of light into
microsphere traceable by ray optics.
o Result is a far-field virtual image that is
enlarged beyond the focal plane and collected
by microscope objective
• Alter sizes and materials of microspheres
immersed in varied liquids to achieve different
resolutions and magnifications
Objective
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12. How can we move these
microspheres?
Optical Tweezers
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13. Optical Tweezers
http://www.physics.nyu.edu/~dg86/hot.htm
• Use radiation pressure from highly focused IR laser beam to attract particle to center
of beam (the highest intensity)
o Photons hit particle and transfer momentum to push particle into center of beam
University of Cambridge: Joanne Gornall’s group
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15. • LPSIM substrate fabricated atop
slide
• Fluorescent beads deposited
(100nm 520/560 and 50nm 490/530)
• Microspheres (in water) deposited
• Microsphere and LPSIM substrate
work in tandem to improve resolution
• Viewed under 0.8NA objective
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0.8 NA
16. • Design substrate that
gives controlled,
changeable, ultrafine
illumination patterns
• Collect 9 images from
different angles
• Use matlab to
reconstruct high-res
image
• Polystyrene spheres
(10nm/ 45nm
diameter, n=1.6, 1.8x
magnification)
• TiO2 spheres (20nm,
n=2.3, 3x
magnification)
• Emerged in water
(n=1.3)
• 0.8NA 60x air
objective and 1.2NA
60x water immersion
objective
• Used to trap and move
spheres
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17. Beads size= 50nm
Diffraction limited image = 350nm
LPSIM image = 115nm
• Resolution Definition:
o Minimum distance between two objects to
be imaged as two instead of one
o Full-width at half-maximum (FWHM) of
point-spread function (PSF) of system
150nm
120nm
115nm
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19. Theoretical
diffraction limited
resolution is
435nm.
With spheres
FWHM is 370nm.
With LPSIM under
spheres FWHM
is 102nm.
Overall 4.3x
resolution
improvement.
Inside Sphere
With Sphere Sphere + LPSIM
112nm
102um
330nm
Outside Moved Sphere
Diffraction Limited
102nm
370nm
Outside Moved Sphere
Diffraction Limited
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20. Reconstruction Under TiO2 Sphere
Inside Sphere
240nm
65nm
Theoretical diffraction limited resolution is
440nm.
With spheres FWHM is 240nm.
With LPSIM under sphere FWHM is 65nm.
Overall 6.5x resolution improvement.
Sphere
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21. • Dielectric microspheres provide straightforward and robust tool to be integrated with a
conventional microscope for superresolution optical microscopy
• Achieved 3x resolution improvement of fluorescent sample beyond classical diffraction
limit with LPSIM
• Introduced TiO2 and polystyrene microlenses
• Moved microlenses with optical tweezers
• Achieved more than 6x resolution improvement using LPSIM under TiO2 microspheres
• Achieved more than 4x resolution improvement using LPSIM under polystyrene
microspheres
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22. • Develop microscopy with resolution below 50nm with 10ms speed
o particles in other shapes could be considered (ex. elliptical)
• Apply to high-resolution biomedical imaging
o Real-time observation of biological phenomena, response of cells/ viruses with
medicine, sub-cellular activity under white light without the need to excite
fluorescence.
https://www.pinterest.com/pin/52495151882172408/
http://dkphoto.photoshelter.com/gallery/Microscopic-Animal-
Cells/G0000jkseY.VFBSk/C0000oyPxKwu0APU http://bestofpicture.com/hiv-virus-under-microscope.html
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23. I would like to give my sincerest thanks to Professor Zhaowei Liu for the
use of his lab. In addition, I would like to thank Dr. Anna Bezryadina for
her kind support, instruction, and guidance throughout the experimental
procedure. I am also grateful towards many of the graduate students
working in the lab for their approachability and willingness to answer any
questions. Lastly, I would like to recognize Ms. Dominga Sanchez for her
outstanding job of organizing the REU program.
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