Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation

Bailey, Brent; Dl, Farkas; Taylor, D. Lansing; Lanni, Frederick

doi:10.1038/366044a0

Cited by 313 publications

(244 citation statements)

References 21 publications

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“…The latest published configuration of an SWFM generates the standing-wave field by splitting a laser beam into two and focusing each beam into the respective back focal plane of two objective lenses which face the specimen from opposite sides (Bailey et al, 1993. From the objective lenses the light emerges as collimated, counterpropagating beams which interfere in the specimen to form the desired standing-wave field.…”

Section: Introductionmentioning

confidence: 99%

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Development of a standing‐wave fluorescence microscope with high nodal plane flatness

Freimann

Pentz

Hörler

1997

Journal of Microscopy

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SummaryThis article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness.As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible.The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique.The instrument developed uses a green-emitting heliumneon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces.The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods.To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655. To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen.A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.

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Section: Introductionmentioning

confidence: 99%

“…Bailey et al (1993) report on a maximization of fringe flatness by adjusting the optics, indicating that nodal plane flatness was a problem.…”

Section: Introductionmentioning

confidence: 99%

Development of a standing‐wave fluorescence microscope with high nodal plane flatness

Freimann

Pentz

Hörler

1997

Journal of Microscopy

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“…This condition is not fulfilled in a standing-wave microscope that uses a flat standing-wave pattern in a conventional epifluorescent microscope (Bailey et al, 1993). In a standing-wave microscope, the PSF is characterised by three to five side lobes on either side which gain relative strength with lateral defocus.…”

Section: Discussionmentioning

confidence: 96%

4Pi-Confocal Microscopy Provides Three-Dimensional Images of the Microtubule Network with 100- to 150-nm Resolution

Nagorni

Hell

1998

Journal of Structural Biology

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We show the applicability of 4Pi-confocal microscopy to three-dimensional imaging of the microtubule network in a fixed mouse fibroblast cell. Comparison with two-photon confocal resolution reveals a fourfold better axial resolution in the 4Pi-confocal case. By combining 4Pi-confocal microscopy with Richardson-Lucy image restoration a further resolution increase is achieved. Featuring a threedimensional resolution in the range 100-150 nm, the 4Pi-confocal (restored) images are intrinsically more detailed than their confocal counterparts. Our images constitute what to our knowledge are the best-resolved three-dimensional images of entangled cellular microtubules obtained with light to date. Academic Press

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“…SRM techniques are therefore an attractive option to study molecular processes at synapses but imaging an axially aligned synapse is challenging as this requires maintaining the super resolution along the zaxis away from the coverslip. Improvement in axial resolution has been addressed with standing-wave excitation fluorescence microscopy [18], I5M [19] and 4Pi methods [3,20] since the 1990s but these methods require special sample preparation along with complex optical alignment of illumination and/ or imaging paths on both sides of the sample and have not been widely taken up in biological research. To date, SRM of the IS has been limited to imaging "artificial synapses" formed by a lymphocyte and an activating ligand on a horizontal coverslip, e.g.…”

Section: Biophotonicsmentioning

confidence: 99%

3‐D stimulated emission depletion microscopy with programmable aberration correction

Lenz

Sinclair

Savell

et al. 2013

Journal of Biophotonics

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We present a stimulated emission depletion (STED) microscope that provides 3‐D super resolution by simultaneous depletion using beams with both a helical phase profile for enhanced lateral resolution and an annular phase profile to enhance axial resolution. The 3‐D depletion point spread function is realised using a single spatial light modulator that can also be programmed to compensate for aberrations in the microscope and the sample. We apply it to demonstrate the first 3‐D super‐resolved imaging of an immunological synapse between a Natural Killer cell and its target cell. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation

Cited by 313 publications

References 21 publications

Development of a standing‐wave fluorescence microscope with high nodal plane flatness

Development of a standing‐wave fluorescence microscope with high nodal plane flatness

4Pi-Confocal Microscopy Provides Three-Dimensional Images of the Microtubule Network with 100- to 150-nm Resolution

3‐D stimulated emission depletion microscopy with programmable aberration correction

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