Using Multi-Element Detectors to Create Optimal Apertures in Confocal Microscopy This work was supported in part by CenSSIS, the Center for Subsurface.

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)

AbstractA 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.

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 .

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

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

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).

Contact InfoBrynmor Davis, PhD CandidateDepartment of Electrical and Computer Engineering Boston University8. Saint. Mary‘s Street, Boston MA [email protected]

Technology TransferTwo 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.

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.

Value Added to CenSSISThis work falls under CenSSIS Research Thrusts R1(as a new measurement methodolgy is employed) and R2 (as physics-based inversion is a key aspect of the system). The physical implementation can be validated using the CenSSIS BioBED platform.

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Fig. 1 – Confocal (left) and standard widefield (right) fluorescence images of a human medulla – www.olympusconfocal.com/theory/confocalintro.html.

Reconstructions

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Fig. 2. The 3D object above is used to test the reconstruction performance through simulation. Fig. 3. Confocal microscope illustration

– www.bioteach.ubc.ca/CellBiology.

Standard Pinhole

Fig. 4. Circular pinhole - 1 Airy width.

Partitioned Aperture

Fig. 5. Detection aperture array – 0.5 Airy element side length.

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

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

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.

Fig. 8. PDA systems approximate the ideal case.

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

Fig. 9. The PDA system gives a higher SNR than the single-pinhole systems at all frequencies

Fig. 10. Simulations show an improved reconstruction from the PDA system at all signal levels.