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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.


Schematic representations of the two basic calibration concepts.The fit approach to measure eSIP parameters using a homogenous fluorescent layer (A) reveals the parameters amplitude (A), full width at half maximum (ωFWHM), the axial position (z0) and the offset (I0). The skewness parameter and the Lorentz-Gauss fraction are not shown. If the solution-based sample is used (B) the steepness of the profile can also be expressed by ωFWHM (compare Eqs 5–9.), and instead of the skewness parameter we included the length constant (LC). Example data are shown in grey, and an appropriate fit is shown in black.
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pone.0134980.g001: Schematic representations of the two basic calibration concepts.The fit approach to measure eSIP parameters using a homogenous fluorescent layer (A) reveals the parameters amplitude (A), full width at half maximum (ωFWHM), the axial position (z0) and the offset (I0). The skewness parameter and the Lorentz-Gauss fraction are not shown. If the solution-based sample is used (B) the steepness of the profile can also be expressed by ωFWHM (compare Eqs 5–9.), and instead of the skewness parameter we included the length constant (LC). Example data are shown in grey, and an appropriate fit is shown in black.

Mentions: I0 is the intensity offset, A the maximal profile intensity, ωFWHM is the axial resolution reflected by FWHM, z0 the axial position of the intensity maximum (Fig 1). Accounting for the fact that the axial intensity profile will not be perfectly symmetric due to slight refractive index mismatch, we approximated the skewness of the profile by introducing a skewness factor s, which leads to a skewness corrected axial position zs for the skewed profile:zs=z⋅es⋅z(2)


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

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

Schematic representations of the two basic calibration concepts.The fit approach to measure eSIP parameters using a homogenous fluorescent layer (A) reveals the parameters amplitude (A), full width at half maximum (ωFWHM), the axial position (z0) and the offset (I0). The skewness parameter and the Lorentz-Gauss fraction are not shown. If the solution-based sample is used (B) the steepness of the profile can also be expressed by ωFWHM (compare Eqs 5–9.), and instead of the skewness parameter we included the length constant (LC). Example data are shown in grey, and an appropriate fit is shown in black.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134980.g001: Schematic representations of the two basic calibration concepts.The fit approach to measure eSIP parameters using a homogenous fluorescent layer (A) reveals the parameters amplitude (A), full width at half maximum (ωFWHM), the axial position (z0) and the offset (I0). The skewness parameter and the Lorentz-Gauss fraction are not shown. If the solution-based sample is used (B) the steepness of the profile can also be expressed by ωFWHM (compare Eqs 5–9.), and instead of the skewness parameter we included the length constant (LC). Example data are shown in grey, and an appropriate fit is shown in black.
Mentions: I0 is the intensity offset, A the maximal profile intensity, ωFWHM is the axial resolution reflected by FWHM, z0 the axial position of the intensity maximum (Fig 1). Accounting for the fact that the axial intensity profile will not be perfectly symmetric due to slight refractive index mismatch, we approximated the skewness of the profile by introducing a skewness factor s, which leads to a skewness corrected axial position zs for the skewed profile:zs=z⋅es⋅z(2)

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.