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Nanometric depth resolution from multi-focal images in microscopy.

Dalgarno HI, Dalgarno PA, Dada AC, Towers CE, Gibson GJ, Parton RM, Davis I, Warburton RJ, Greenaway AH - J R Soc Interface (2011)

Bottom Line: To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound.The approximations used in the analytical treatment are tested using numerical simulations.Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

View Article: PubMed Central - PubMed

Affiliation: Physics, SUPA/IIS, School of Engineering and Physical Sciences, Heriot-Watt University, , Edinburgh EH14 4AS, UK.

ABSTRACT
We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

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A fly (Drosophila melanogaster) egg chamber expressing a green fluorescence-tagged protein and imaged using epifluorescence, a 0.7NA dry 20× objective and the DOE-based attachment with 7.3 µm separation between the in-focus planes. Left to right the three images show: the surface of the egg chamber within the egg chamber; the far side of the oocyte and nurse cells deeper within the egg chamber. Scale bar, 20 µm.
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RSIF20100508F1: A fly (Drosophila melanogaster) egg chamber expressing a green fluorescence-tagged protein and imaged using epifluorescence, a 0.7NA dry 20× objective and the DOE-based attachment with 7.3 µm separation between the in-focus planes. Left to right the three images show: the surface of the egg chamber within the egg chamber; the far side of the oocyte and nurse cells deeper within the egg chamber. Scale bar, 20 µm.

Mentions: Briefly, a DOE in the form of an off-axis Fresnel lens has a different focal length in each diffraction order. If this DOE is combined carefully with a lens of high optical power, the system focal length in each diffraction order can be arranged to deliver in-focus and spatially separated images in which the image in each diffraction order is equivalent to an image that could have been recorded in a z-stack sequence. The etch depth (thus phase modulation) of the DOE determines the relative brightness of the images in the various diffraction orders. The lateral resolution, depth of focus and depth of field of the image in each diffraction order is equal to the performance that would have been achieved from the equivalent image in a sequential z-stack. Used as a simple three-dimensional snapshot system, the technique delivers images of the sort shown in figure 1; see also [28]. The DOE combines the function of the beamsplitter and defocus lens in other multi-focus techniques (e.g. [16]).Figure 1.


Nanometric depth resolution from multi-focal images in microscopy.

Dalgarno HI, Dalgarno PA, Dada AC, Towers CE, Gibson GJ, Parton RM, Davis I, Warburton RJ, Greenaway AH - J R Soc Interface (2011)

A fly (Drosophila melanogaster) egg chamber expressing a green fluorescence-tagged protein and imaged using epifluorescence, a 0.7NA dry 20× objective and the DOE-based attachment with 7.3 µm separation between the in-focus planes. Left to right the three images show: the surface of the egg chamber within the egg chamber; the far side of the oocyte and nurse cells deeper within the egg chamber. Scale bar, 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104330&req=5

RSIF20100508F1: A fly (Drosophila melanogaster) egg chamber expressing a green fluorescence-tagged protein and imaged using epifluorescence, a 0.7NA dry 20× objective and the DOE-based attachment with 7.3 µm separation between the in-focus planes. Left to right the three images show: the surface of the egg chamber within the egg chamber; the far side of the oocyte and nurse cells deeper within the egg chamber. Scale bar, 20 µm.
Mentions: Briefly, a DOE in the form of an off-axis Fresnel lens has a different focal length in each diffraction order. If this DOE is combined carefully with a lens of high optical power, the system focal length in each diffraction order can be arranged to deliver in-focus and spatially separated images in which the image in each diffraction order is equivalent to an image that could have been recorded in a z-stack sequence. The etch depth (thus phase modulation) of the DOE determines the relative brightness of the images in the various diffraction orders. The lateral resolution, depth of focus and depth of field of the image in each diffraction order is equal to the performance that would have been achieved from the equivalent image in a sequential z-stack. Used as a simple three-dimensional snapshot system, the technique delivers images of the sort shown in figure 1; see also [28]. The DOE combines the function of the beamsplitter and defocus lens in other multi-focus techniques (e.g. [16]).Figure 1.

Bottom Line: To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound.The approximations used in the analytical treatment are tested using numerical simulations.Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

View Article: PubMed Central - PubMed

Affiliation: Physics, SUPA/IIS, School of Engineering and Physical Sciences, Heriot-Watt University, , Edinburgh EH14 4AS, UK.

ABSTRACT
We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

Show MeSH
Related in: MedlinePlus