Folie 1

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Using Multi-Element Detectors to Create Optimal
Apertures in Confocal Microscopy
This work was supported in part by CenSSIS, the
Center for Subsurface Sensing and Imaging
Systems, under the Engineering Research Centers
Program of the National Science Foundation (Award
Number EEC-9986821) and also by the NSF and NIH
NIBIB under grants DBI-0138425 and 532100P304129
respectively.
Brynmor J. Davis (BU), M. Selim Ünlü (BU),
William C. Karl (BU), Anna K. Swan (BU), Bennett
B. Goldberg (BU)
Reconstructions
Abstract A scheme is proposed for utilizing the
out-of-focus light usually rejected at the
pinhole of a confocal microscope. It is shown
that the ideal detection aperture varies as a
function of the spatial frequency being imaged. A
method for calculating such optimal detection
apertures is given, an example calculation is
shown and a detector array is suggested as a
means to approximate these varying detection
apertures. The performance is evaluated for a
system using a small array of detectors rather
than a standard pinhole. Simulated data sets from
the detector array elements are combined in an
optimal linear manner to give a single set of
data with superior noise characteristics.
Reconstructions from these data are compared to
those from conventional confocal systems with a
variety of pinhole sizes. The reconstruction
error is examined for each instrument as a
function of signal strength. At all signal levels
and all pinhole sizes, the multi-detector system
is seen to outperform the standard single-pinhole
instruments.

• State of the Art
• Confocal microscopy is a highly useful and
  widely used technique in biology due to its
  ability to achieve good axial sectioning (see
  Fig. 1).
• A confocal detection pinhole is used to reject
  the out-of-focus light, however this does mean
  wasted photons.
• Photodetector arrays have now reached speeds and
  sensitivities such that their application to
  confocal microscopy is possible1,2.

Fig. 6. Reconstructions from a single-pinhole
system and the Partitioned Detection Aperture
(PDA) instrument.

• Accomplishments up through Current Year
• Two provisional patents prepared see
  Technology Transfer.
• Two conference papers and one journal paper (on
  a related subject) published see Publications
  Acknowledging NSF Support.
• Journal paper in preparation.
• Encouraging theoretical and simulation results
  demonstrated.
• Initial steps taken toward a physical
  demonstration .

Fig. 7. Optimal apertures at illustrative spatial
frequencies for the example system considered.
Magnitudes are displayed on the top row and
phases on the bottom. All detector areas are 2µm
by 2µm in demagnified space. Notice how aperture
size and shape correspond to the spatial
frequency imaged.

• A Partitioned Detection Aperture (PDA) system
  can be constructed using an array of finite-sized
  apertures and the same mathematical framework.

• Challenges and Significance
• The need for biological imaging systems that are
  both high speed and high resolution has been
  recognized.3
• This work uses a small photodetector array to
  collect the otherwise rejected light2, which is
  then processed usefully into the image.
• The improved efficiency results in higher image
  quality for the same imaging speed or higher
  imaging speed for the same image quality.

• Technology Transfer
• Two provisional patents covering this work are
  being processed through the Boston Univeristy
  Office of Technology Development
• Case Number BU05-05 Partitioned
  Detection-Aperture Confocal Microscope, Brynmor
  J. Davis, Bennett B. Goldberg, William C. Karl,
  Anna K. Swan, M. Selim Ünlü. (Filed with US
  Patent Office)
• Case Number BU05-10 Multi-element
  Photodetector as a Smart Pinhole in Confocal
  Microscopy, M. Selim Ünlü, Brynmor J. Davis.

• Technical Approach
• Each position in the focal plane can be thought
  of as an individual detector producing its own
  data set.
• If these data sets are filtered and summed in
  such a way that the SNR is maximized at each
  spatial frequency, the resulting weightings
  spatial distribution describes the optimal
  aperture as a function of spatial frequency.

Fig. 8. PDA systems approximate the ideal case.

• Publications Acknowledging NSF Support
• Using out-of-focus light to improve image
  acquisition time in confocal microscopy, Brynmor
  J. Davis, William C. Karl, Bennett B. Goldberg,
  Anna K. Swan, M. Selim Ünlü, Proceedings of the
  SPIE, Vol. 5701, 2005
• Sampling below the Nyquist rate in
  interferometric fluorescence microscopy with
  multi-wavelength measurements to remove aliasing,
  Brynmor J. Davis, William C. Karl, Bennett B.
  Goldberg, Anna K. Swan, M. Selim Ünlü,
  Proceedings of the 11th IEEE Digital Signal
  Processing Workshop, 2004
• Capabilities and limitations of pupil-plane
  filters for superresolution and image
  enhancement, Brynmor J. Davis, William C. Karl,
  Anna K. Swan, M. Selim Ünlü, Bennett B. Goldberg,
  Optics Express, Vol. 12, 2004
• Reconstruction of objects with a limited number
  of non-zero components in fluorescence
  microscopy, Brynmor J. Davis, William C. Karl,
  Anna K. Swan, Bennett B. Goldberg, M. Selim Ünlü,
  Marcia B. Goldberg, Proceedings of the SPIE, Vol.
  5324, 2004
• Towards nanoscale optical resolution in
  fluorescence microscopy, Anna K. Swan, Lev
  Moiseev, Charles, R. Cantor, Brynmor J. Davis,
  Stephen B. Ippolito, William C. Karl, Bennett B.
  Goldberg, M. Selim Ünlü, IEEE Journal of Selected
  Topics in Quantum Electronics, Vol.9, 2003

• Plans
• Derive optimal patterns for the case where the
  noise is not Poisson distributed.
• Provide a physical demonstration.
• Pursue patenting and licensing options.

References 1 Ultrasensitivity, speed, and
resolution optimizing low-light microscopy with
the back-illuminated electron-multiplying CCD, C.
G. Coates, D. J. Denvir, N. G. McHale, K. D.
Thornbury, and M. A. Hollywood, Confocal,
Multiphoton, and Nonlinear Microscopic Imaging,
Proc. SPIE Vol. 5139 (2003). 2 CCDiode an
optimal detector for laser confocal microscopes,
J. B. Pawley, M. M. Blouke, and J. R. Janesick,
Three-Dimensional Microscopy Image Acquisition
and Processing III, Proc. SPIE Vol. 2655
(1996). 3 - High speed microscopy in biomedical
research, H. R. Petty, Optics and Photonics News,
Vol. 15 (2004).

• A confocal system was modeled with an excitation
  wavelength of 488nm, a detection wavelength of
  530nm and a numerical aperture of 1.35.

Contact Info Brynmor Davis, PhD
Candidate Department of Electrical and Computer
Engineering Boston University 8. Saint. Marys
Street, Boston MA 022215 bryn_at_bu.edu