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eSIP: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration.

Butzlaff M, Weigel A, Ponimaskin E, Zeug A - PLoS ONE (2015)

Bottom Line: However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups.Furthermore, due to the fit of the complete profile, our method is less susceptible to noise.Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurophysiology, Center of Physiology, Hannover Medical School, Hannover, Germany.

ABSTRACT
Fluorescence confocal microscopy represents one of the central tools in modern sciences. Correspondingly, a growing amount of research relies on the development of novel microscopic methods. During the last decade numerous microscopic approaches were developed for the investigation of various scientific questions. Thereby, the former qualitative imaging methods became replaced by advanced quantitative methods to gain more and more information from a given sample. However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups. Multispectral beads with sub-resolution size are often used to describe the point spread function and thus the optical properties of the microscope. More recently, a fluorescent layer was utilized to describe the axial profile for each pixel, which allows a spatially resolved characterization. However, fabrication of a thin fluorescent layer with matching refractive index is technically not solved yet. Therefore, we propose a novel type of calibration concept for sectioned image property (SIP) measurements which is based on fluorescent solution and makes the calibration concept available for a broader number of users. Compared to the previous approach, additional information can be obtained by application of this extended SIP chart approach, including penetration depth, detected number of photons, and illumination profile shape. Furthermore, due to the fit of the complete profile, our method is less susceptible to noise. Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy.

No MeSH data available.


Moiré artefacts using structured calibration samples.Example images of maximum projections, taken from a structured non-homogenous layer (Argolight calibration sample). The depicted images show the same field of view of a vertical grid structure (the “homogenous pattern” of the Argolight slide), which were either scanned perpendicular to the grid (A-D) or in a parallel fashion (E-H) with a decreasing pixel size (increasing number of pixels). A Fourier analysis in x direction of the images (I and J) reveals several instances of moiré artefacts (arrow) in addition to the frequency of the grid itself (arrow head). At low resolution (A and E), these artefacts are dominating the images; the grid structure itself becomes invisible.
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pone.0134980.g004: Moiré artefacts using structured calibration samples.Example images of maximum projections, taken from a structured non-homogenous layer (Argolight calibration sample). The depicted images show the same field of view of a vertical grid structure (the “homogenous pattern” of the Argolight slide), which were either scanned perpendicular to the grid (A-D) or in a parallel fashion (E-H) with a decreasing pixel size (increasing number of pixels). A Fourier analysis in x direction of the images (I and J) reveals several instances of moiré artefacts (arrow) in addition to the frequency of the grid itself (arrow head). At low resolution (A and E), these artefacts are dominating the images; the grid structure itself becomes invisible.

Mentions: Although the preparation of a fluorescent solution-based standard reduces technical effort compared to the production of a thin fluorescent layer, the effort to create a defined standard for calibration could still be improved by a highly standardized alternative that is commercially available. Several aspects should be taken into consideration. The calibration sample has to be highly stable over time in order to allow for quantitative comparison of different experiments. Moreover, the manufacturing of calibration samples must be highly reproducible over various product charges to allow for the comparison between different laboratories. The so far only commercially available product which addresses to fulfil the requirements mentioned above (e.g., the production is standardized and the long term stability of fluorescence is guaranteed for five years) is the Argolight calibration slide. We therefore evaluated an ARGO-M slide (standard version of mid 2014) [23] for its calibration properties and its usability for the eSIP layer approach. These slides contain different line-style patterns imprinted into the slide (http://argolight.com/argo-m-standard-slide/). The basic element of all the structures within the calibration sample is a tube section whose diameter is about 600 nm, length about 4 μm (FWHM) and wall thickness about 250 nm (S4 Fig). This resembles a very complex axial profile. Scanning through the object is not analogous to a homogeneous fluorescent layer sample; it is better approximated by the convolution of a rather large structure, which increases intensity with increasing tube length. Since the structures provided are available at a defined length between 4 and 10 μm, the z-profile of the structure used requires additional fit models. All calibration structures are built up by these tubes and its line shape representative, a double line. Thus, even the tightest structures aligned like a fluorescent square were not recognized as homogeneous under standard confocal conditions. While testing this non-homogenously striped structure as a potential alternative for a thin fluorescent layer, we observed Moiré artefacts, derived from lateral frequency differences between the structure width and the 'pixel width' of the point scanning confocal microscope, similar to a beating (Fig 4). More specifically, the Moiré effect is more pronounced when the pixel size is similar to the line period, i.e. about 600 nm, for a scanning parallel to the lines.


eSIP: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration.

Butzlaff M, Weigel A, Ponimaskin E, Zeug A - PLoS ONE (2015)

Moiré artefacts using structured calibration samples.Example images of maximum projections, taken from a structured non-homogenous layer (Argolight calibration sample). The depicted images show the same field of view of a vertical grid structure (the “homogenous pattern” of the Argolight slide), which were either scanned perpendicular to the grid (A-D) or in a parallel fashion (E-H) with a decreasing pixel size (increasing number of pixels). A Fourier analysis in x direction of the images (I and J) reveals several instances of moiré artefacts (arrow) in addition to the frequency of the grid itself (arrow head). At low resolution (A and E), these artefacts are dominating the images; the grid structure itself becomes invisible.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526552&req=5

pone.0134980.g004: Moiré artefacts using structured calibration samples.Example images of maximum projections, taken from a structured non-homogenous layer (Argolight calibration sample). The depicted images show the same field of view of a vertical grid structure (the “homogenous pattern” of the Argolight slide), which were either scanned perpendicular to the grid (A-D) or in a parallel fashion (E-H) with a decreasing pixel size (increasing number of pixels). A Fourier analysis in x direction of the images (I and J) reveals several instances of moiré artefacts (arrow) in addition to the frequency of the grid itself (arrow head). At low resolution (A and E), these artefacts are dominating the images; the grid structure itself becomes invisible.
Mentions: Although the preparation of a fluorescent solution-based standard reduces technical effort compared to the production of a thin fluorescent layer, the effort to create a defined standard for calibration could still be improved by a highly standardized alternative that is commercially available. Several aspects should be taken into consideration. The calibration sample has to be highly stable over time in order to allow for quantitative comparison of different experiments. Moreover, the manufacturing of calibration samples must be highly reproducible over various product charges to allow for the comparison between different laboratories. The so far only commercially available product which addresses to fulfil the requirements mentioned above (e.g., the production is standardized and the long term stability of fluorescence is guaranteed for five years) is the Argolight calibration slide. We therefore evaluated an ARGO-M slide (standard version of mid 2014) [23] for its calibration properties and its usability for the eSIP layer approach. These slides contain different line-style patterns imprinted into the slide (http://argolight.com/argo-m-standard-slide/). The basic element of all the structures within the calibration sample is a tube section whose diameter is about 600 nm, length about 4 μm (FWHM) and wall thickness about 250 nm (S4 Fig). This resembles a very complex axial profile. Scanning through the object is not analogous to a homogeneous fluorescent layer sample; it is better approximated by the convolution of a rather large structure, which increases intensity with increasing tube length. Since the structures provided are available at a defined length between 4 and 10 μm, the z-profile of the structure used requires additional fit models. All calibration structures are built up by these tubes and its line shape representative, a double line. Thus, even the tightest structures aligned like a fluorescent square were not recognized as homogeneous under standard confocal conditions. While testing this non-homogenously striped structure as a potential alternative for a thin fluorescent layer, we observed Moiré artefacts, derived from lateral frequency differences between the structure width and the 'pixel width' of the point scanning confocal microscope, similar to a beating (Fig 4). More specifically, the Moiré effect is more pronounced when the pixel size is similar to the line period, i.e. about 600 nm, for a scanning parallel to the lines.

Bottom Line: However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups.Furthermore, due to the fit of the complete profile, our method is less susceptible to noise.Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurophysiology, Center of Physiology, Hannover Medical School, Hannover, Germany.

ABSTRACT
Fluorescence confocal microscopy represents one of the central tools in modern sciences. Correspondingly, a growing amount of research relies on the development of novel microscopic methods. During the last decade numerous microscopic approaches were developed for the investigation of various scientific questions. Thereby, the former qualitative imaging methods became replaced by advanced quantitative methods to gain more and more information from a given sample. However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups. Multispectral beads with sub-resolution size are often used to describe the point spread function and thus the optical properties of the microscope. More recently, a fluorescent layer was utilized to describe the axial profile for each pixel, which allows a spatially resolved characterization. However, fabrication of a thin fluorescent layer with matching refractive index is technically not solved yet. Therefore, we propose a novel type of calibration concept for sectioned image property (SIP) measurements which is based on fluorescent solution and makes the calibration concept available for a broader number of users. Compared to the previous approach, additional information can be obtained by application of this extended SIP chart approach, including penetration depth, detected number of photons, and illumination profile shape. Furthermore, due to the fit of the complete profile, our method is less susceptible to noise. Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy.

No MeSH data available.