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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.


Related in: MedlinePlus

Gaining information on X and Y from a commercially available calibration sample.Neither the layer- nor the solution-based eSIP approach provide information about the lateral imaging properties. Utilizing defined fluorescent structures makes it possible to describe aberrations in these dimensions, too. The grid structure on the commercially available Argolight slide was imaged using two excitation wavelength (440 nm and 488 nm) and maximized field of view (A). The analysis of this grid structure revealed strong distortions which can be quantified using second order polynomial fits. To depict this aberration, the second order term (multiplied with 105) is shown next to the corresponding fit (white lines) for the first excitation (B). The difference in the two excitations is depicted in C as vectors at the position of grid crossings reflecting the direction and the size of the shift (the size of the arrows are multiplied by 100 for visibility).
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pone.0134980.g005: Gaining information on X and Y from a commercially available calibration sample.Neither the layer- nor the solution-based eSIP approach provide information about the lateral imaging properties. Utilizing defined fluorescent structures makes it possible to describe aberrations in these dimensions, too. The grid structure on the commercially available Argolight slide was imaged using two excitation wavelength (440 nm and 488 nm) and maximized field of view (A). The analysis of this grid structure revealed strong distortions which can be quantified using second order polynomial fits. To depict this aberration, the second order term (multiplied with 105) is shown next to the corresponding fit (white lines) for the first excitation (B). The difference in the two excitations is depicted in C as vectors at the position of grid crossings reflecting the direction and the size of the shift (the size of the arrows are multiplied by 100 for visibility).

Mentions: Although the ARGO-M slide we tested is not an optimal sample for the eSIP approach as it is, the ARGO-M slide with its defined structures can be utilized easily to include an evaluation of lateral aberrations (Fig 5): the distortion can be quantified by a polynomial fit (second order) to estimate the linearity of the grid structure. A clear distortion towards the outer areas of the field of view is evident (Fig 5B). Comparing the grid structure upon excitation at different wavelengths clearly demonstrates that the aberrations obtained in this optical system are wavelength-dependent (Fig 5C).


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

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

Gaining information on X and Y from a commercially available calibration sample.Neither the layer- nor the solution-based eSIP approach provide information about the lateral imaging properties. Utilizing defined fluorescent structures makes it possible to describe aberrations in these dimensions, too. The grid structure on the commercially available Argolight slide was imaged using two excitation wavelength (440 nm and 488 nm) and maximized field of view (A). The analysis of this grid structure revealed strong distortions which can be quantified using second order polynomial fits. To depict this aberration, the second order term (multiplied with 105) is shown next to the corresponding fit (white lines) for the first excitation (B). The difference in the two excitations is depicted in C as vectors at the position of grid crossings reflecting the direction and the size of the shift (the size of the arrows are multiplied by 100 for visibility).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134980.g005: Gaining information on X and Y from a commercially available calibration sample.Neither the layer- nor the solution-based eSIP approach provide information about the lateral imaging properties. Utilizing defined fluorescent structures makes it possible to describe aberrations in these dimensions, too. The grid structure on the commercially available Argolight slide was imaged using two excitation wavelength (440 nm and 488 nm) and maximized field of view (A). The analysis of this grid structure revealed strong distortions which can be quantified using second order polynomial fits. To depict this aberration, the second order term (multiplied with 105) is shown next to the corresponding fit (white lines) for the first excitation (B). The difference in the two excitations is depicted in C as vectors at the position of grid crossings reflecting the direction and the size of the shift (the size of the arrows are multiplied by 100 for visibility).
Mentions: Although the ARGO-M slide we tested is not an optimal sample for the eSIP approach as it is, the ARGO-M slide with its defined structures can be utilized easily to include an evaluation of lateral aberrations (Fig 5): the distortion can be quantified by a polynomial fit (second order) to estimate the linearity of the grid structure. A clear distortion towards the outer areas of the field of view is evident (Fig 5B). Comparing the grid structure upon excitation at different wavelengths clearly demonstrates that the aberrations obtained in this optical system are wavelength-dependent (Fig 5C).

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.


Related in: MedlinePlus