Alternatives to Point-Scan Confocal Microscopy

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-

oooo

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,