Alternatives to Point-Scan Confocal Microscopy

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Alternatives to Point-Scan Confocal Microscopy

  1. 1. Alternatives to Point-Scan Confocal Microscopy Two different methods to accomplish
  2. 2. Three Different Microscopic System <ul><li>Point Scanning Confocal Systems </li></ul><ul><ul><li>Conventional confocal microscope setup </li></ul></ul><ul><li>Area Scanning Confocal Systems </li></ul><ul><ul><li>Multi pinhole spinning disk provides improved scan speed </li></ul></ul><ul><li>Fluorescence Grating Imager Systems </li></ul><ul><ul><li>Optional sectioning in conventially illuminated reflecting microscope </li></ul></ul>
  3. 3. Point Scanning Confocal Systems Review <ul><li>Pinhole aperture used to remove out of focus image </li></ul><ul><li>Point scan moved through image </li></ul><ul><li>Physical movement to scan image: </li></ul>Images from Paddock et al. Olympus Resource Center
  4. 4. Area Scanning Confocal Systems <ul><li>Significant qualities of point-scanning confocal systems </li></ul><ul><ul><li>depth discrimination </li></ul></ul><ul><ul><li>high resolution </li></ul></ul><ul><li>Multiple pinholes increase scanning area </li></ul><ul><li>Light  microlens array  pinhole array  objective  specimen </li></ul><ul><li>Emitted light  pinhole array  dichromatic mirror  lens  CCD </li></ul>Images from Paddock et al. Olympus Resource Center
  5. 5. More Advantages <ul><li>CCD camera located on image plane </li></ul><ul><li>Improved image acquisition speed </li></ul><ul><ul><li>Currently scans as fast as 1000 frames/sec </li></ul></ul><ul><ul><ul><li>Dependant on CCD speed (recent EMCCDs can do 500 frames/sec) </li></ul></ul></ul><ul><li>Reduced photobleaching and phototoxicity </li></ul><ul><ul><li>CCD on image plane collects emitted light at higher efficiency than from photomultiplier tubes </li></ul></ul>
  6. 6. and Disadvantages <ul><li>Pinhole size </li></ul><ul><ul><li>Diameter of source and detector pinholes cannot be varied independently </li></ul></ul><ul><ul><li>Cannot change based on objective </li></ul></ul><ul><li>Low illumination </li></ul><ul><ul><li>Recent inclusion of microlenses in second disk improves efficiency </li></ul></ul>
  7. 7. Artifacting “ Selected frames from a 500 frame kinetic series showing effect on synchronisation banding as CSU22 disk speed is changed from 5000 to 1800 rev/min. A 19.44 ms EMCCD exposure time was employed, corresponding to synchronisation with the 1800 rev/min final disk speed.” From Chong et al.
  8. 8. Fluorescence Grating Imager Systems <ul><li>Movable grating in the field iris plane of incident-light illuminator </li></ul><ul><li>Multiple exposures used to remove out of focus light </li></ul><ul><li>Grating oscillation only apparent in plane of focus </li></ul>Diagram: Imaging Neurons and Development, RM Yuste and A Konnerth, eds. Cold Spring Harbor Laboratory Press, 2004; Flourescence Grating Imager Systems for Optical-Sectioning Microscopy, F. Lanni
  9. 9. Signal Computation <ul><li>Fluorescence detected at a pixel consists of: </li></ul><ul><li>Steady or DC component due to out-of-focus background </li></ul><ul><li>Oscillating or AC component due to moving of stripes across in-focus structures </li></ul><ul><li>Noise due to both DC and AC components </li></ul>Commonly used shift sequences, and optical section formula: Note that first two provide uniform exposure
  10. 10. Example “ Optical section computation from image data showing the actin cytoskeleton in a 3T3 fibroblast.” A: i 0 B: i 90 C: i 180 D: (A-B) E: (B – C) F: optical section, Pythagorean summation of D and E Diagram: Imaging Neurons and Development, RM Yuste and A Konnerth, eds. Cold Spring Harbor Laboratory Press, 2004; Flourescence Grating Imager Systems for Optical-Sectioning Microscopy, F. Lanni
  11. 11. Spatial Harmonics The projected grating includes spatial harmonics. Only odd harmonics occur for square wave gratings: The harmonics are considered error terms and minimized by different methods: Adjusting sequence period – a 1/3 period compensates for 3rd harmonic (first error term then from 5th harmonic) Selecting grating such that the 5 th harmonic ≥ Abbe’s resolution limit for the objective Alternatively, if the 5 th harmonic period = Abbe’s resolution limit (λ/NA)  fundamental period = 5x(λ/2NA) 3 rd harmonic period = λ/NA  fundamental period = 3x(λ/2NA) Finally, the amplitude of the error drops off as 1/n, so for increasingly large harmonic the error is more insignificant
  12. 12. Optical Sectioning With a projection grating period of L and know NA, n, and λ , the opical section can be computed as: The minimal section thickness is measured when the selected grating period (L) is equal to λ /NA (twice Abbe’s resolution limit) and is equal to: Typical axial response of grating imager based on Zeizz Axiovert 200M with 1.30 NA objective and 1.33 um projected grating period Optical section thickness = graphical full-width at half-maximum = 0.65um; compares well with equation value of 0.623um Diagram: Imaging Neurons and Development, RM Yuste and A Konnerth, eds. Cold Spring Harbor Laboratory Press, 2004; Flourescence Grating Imager Systems for Optical-Sectioning Microscopy, F. Lanni
  13. 13. SNR and Sampling <ul><li>Signal (S) and Background (B) photocounts are Poissen distributed variables </li></ul><ul><li>Subtraction removes background mean (N B ) but noise remains equal to √(N B ) </li></ul><ul><li>SNR ≈ S/√(S +2B) </li></ul><ul><ul><li>since SNR is S:RMS sum of noise in the signal and the background </li></ul></ul><ul><li>When noise is not a limiting factor, the finest period resolvable grating is Abbe’s resolution limit </li></ul><ul><ul><li>Camera pixel spacing / total magnification ≤ λ /4NA </li></ul></ul>
  14. 14. Advantages and Disadvantages <ul><li>Minimal modification to existing microscope </li></ul><ul><li>Optical sectioning similar in accuracy to point confocal </li></ul><ul><li>Light exposure 1.5x normal </li></ul><ul><li>Optical sectioning accomplished without deconvolution </li></ul><ul><ul><li>Immediately processed </li></ul></ul><ul><li>Standard filter sets usable </li></ul><ul><ul><li>Wavelength only limited by aberration in UV </li></ul></ul><ul><li>Slower than spinning disk confocal </li></ul><ul><li>Higher background levels than point confocal </li></ul><ul><li>Possible striping artifact </li></ul>
  15. 15. References <ul><li>Imaging Neurons and Development, RM Yuste and A Konnerth, eds. Cold Spring Harbor Laboratory Press, 2004; Flourescence Grating Imager Systems for Optical-Sectioning Microscopy, F. Lanni </li></ul><ul><li>Optimization of Spinning Disk Confocal Microscopy: Synchronization with the Ultra-Sensitive EMCCD, F. K. Chonga, Colin G. Coatesa, Donal J. Denvira, Noel McHaleb, Keith Thornburyb and Mark Hollywoodb. </li></ul><ul><li>Theory of Confocal Microscopy, Olympus Corporation, Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310. </li></ul><ul><li>Introduction to Confocal Microscopy, Olympus Corporation, Stephen W. Paddock - Laboratory of Molecular Biology, Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin 53706. Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310. </li></ul><ul><li>Spinning-Disk Confocal Microscopy – A Cutting Edge Tool for Imaging Membrane Traffic, Akihiko Nakano, Cell Structure and Function 27: 349-355 (2002) </li></ul><ul><li>A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells, Michael C. Adams,a Wendy C. Salmon,a Stephanie L. Gupton, Christopher S. Cohan,Torsten Wittmann, Natalie Prigozhina, and Clare M. Waterman-Storera, </li></ul>

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