Alternatives to Point-Scan Confocal Microscopy
Two different methods to accomplish
Three Different Microscopic System
• Point Scanning Confocal Systems– Conventional confocal microscope setup
• Area Scanning Confocal Systems– Multi pinhole spinning disk provides improved
scan speed
• Fluorescence Grating Imager Systems– Optional sectioning in conventially illuminated
reflecting microscope
Point Scanning Confocal Systems Review
• Pinhole aperture used to remove out of focus image
• Point scan moved through image
Physical movement to scan image:
Images from Paddock et al. Olympus Resource Center
Area Scanning Confocal Systems
• Significant qualities of point-scanning confocal systems
– depth discrimination– high resolution
• Multiple pinholes increase scanning area
• Light microlens array pinhole array objective specimen
• Emitted light pinhole array dichromatic mirror lens CCD
Images from Paddock et al. Olympus Resource Center
More Advantages
• CCD camera located on image plane • Improved image acquisition speed
– Currently scans as fast as 1000 frames/sec• Dependant on CCD speed (recent EMCCDs can do 500
frames/sec)
• Reduced photobleaching and phototoxicity– CCD on image plane collects emitted light at higher
efficiency than from photomultiplier tubes
and Disadvantages
• Pinhole size – Diameter of source and detector pinholes
cannot be varied independently– Cannot change based on objective
• Low illumination– Recent inclusion of microlenses in second
disk improves efficiency
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.
Fluorescence Grating Imager Systems
Movable grating in the field iris plane of incident-light illuminator
Multiple exposures used to remove out of focus light
Grating oscillation only apparent in plane of focus
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
Signal Computation
)sincos(2/)( 110 baaACtermtermDCf period) grating 1/4 90 (where shift phasea For o
Fluorescence detected at a pixel consists of:•Steady or DC component due to out-of-focus background•Oscillating or AC component due to moving of stripes across in-focus structures•Noise due to both DC and AC components
ooo
2/1218090
2900
1/2-
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2/1227090
21800
ooo
2/120240
2240120
21200
1/2
180-90-0
])())[((2 images 3 period, 1/4
270-180-90-0
])())[(2/(1 images 4 period, 1/4
240-120-0
])()())[(3/(2 images 3 period, 1/3
iiii
iiii
iiiiii
Commonly used shift sequences, and optical section formula: Note that first two
provide uniform exposure
Example
“Optical section computation from image data showing the actin cytoskeleton in a 3T3 fibroblast.” A: i0 B: i90 C: i180
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
Spatial HarmonicsThe projected grating includes spatial harmonics. Only odd harmonics occur for square wave gratings:
...)5sin5cos()3sin3cos()sincos()( 553311 bababaDCf
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 5th harmonic ≥ Abbe’s resolution limit for the objective
Alternatively, if the 5th harmonic period = Abbe’s resolution limit (λ/NA) fundamental period = 5x(λ/2NA)3rd 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
Optical Sectioning
}][/{)2/( 2/122 NAnn
/2NA L: period grating projection
: thicknesssection optical
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:
}][))/(/{[)2/( 2/1222/122 NAnLNAn
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
SNR and Sampling
• Signal (S) and Background (B) photocounts are Poissen distributed variables
• Subtraction removes background mean (NB) but noise remains equal to √(NB)
• SNR ≈ S/√(S +2B) – since SNR is S:RMS sum of noise in the signal and the
background
• When noise is not a limiting factor, the finest period resolvable grating is Abbe’s resolution limit– Camera pixel spacing / total magnification ≤ λ/4NA
Advantages and Disadvantages
• Minimal modification to existing microscope• Optical sectioning similar in accuracy to point confocal• Light exposure 1.5x normal• Optical sectioning accomplished without deconvolution
– Immediately processed
• Standard filter sets usable– Wavelength only limited by aberration in UV
• Slower than spinning disk confocal• Higher background levels than point confocal• Possible striping artifact
ReferencesImaging 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
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.
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.
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.
Spinning-Disk Confocal Microscopy – A Cutting Edge Tool for Imaging Membrane Traffic, Akihiko Nakano, Cell Structure and Function 27: 349-355 (2002)
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,