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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.


Using section imaging property parameters to optimize microscope system settings.To estimate the correction collar setting at a Zeiss LSM 510 confocal microscope, a series of eSIP measurements can be used to define the influence on single eSIP parameters. (A) 2d plot of the intensity parameter for correction collar settings of a 40x/1.2 W C-Apochromat (Zeiss) ranging from 0.14 to 0.19 obtained at a wavelength range from 500 nm to 740 nm with pinhole setting 1.0 AU is shown. The data is derived from a 115 x 115 μm centre region of each plane. Although using an Apochromat the emission wavelength dependency is evident in this measurement. However the optimal settings can easily be found. In analogue fashion the influence of the correction collar was analysed based on the ωFWHM parameter (B). Interestingly, there is no wavelength dependency for this parameter. Analysing the collimator setting in a lux-FRET paradigm with two different excitations at a Zeiss LSM 780 utilizing a 40x/1.2 W C-Apochromat objective revealed different optimal settings in respect to the observed parameter (C and D). In contrast to an optimization according to the maximal intensity (black), we found a different optimal setting, when the axial position difference between first (440 nm) and the second excitation (488 nm) is measured (red). For the collimator setting series we tested the solution-based approach (C) as well as the Argolight calibration slide (D).
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pone.0134980.g003: Using section imaging property parameters to optimize microscope system settings.To estimate the correction collar setting at a Zeiss LSM 510 confocal microscope, a series of eSIP measurements can be used to define the influence on single eSIP parameters. (A) 2d plot of the intensity parameter for correction collar settings of a 40x/1.2 W C-Apochromat (Zeiss) ranging from 0.14 to 0.19 obtained at a wavelength range from 500 nm to 740 nm with pinhole setting 1.0 AU is shown. The data is derived from a 115 x 115 μm centre region of each plane. Although using an Apochromat the emission wavelength dependency is evident in this measurement. However the optimal settings can easily be found. In analogue fashion the influence of the correction collar was analysed based on the ωFWHM parameter (B). Interestingly, there is no wavelength dependency for this parameter. Analysing the collimator setting in a lux-FRET paradigm with two different excitations at a Zeiss LSM 780 utilizing a 40x/1.2 W C-Apochromat objective revealed different optimal settings in respect to the observed parameter (C and D). In contrast to an optimization according to the maximal intensity (black), we found a different optimal setting, when the axial position difference between first (440 nm) and the second excitation (488 nm) is measured (red). For the collimator setting series we tested the solution-based approach (C) as well as the Argolight calibration slide (D).

Mentions: Unless otherwise noted, experiments were carried out on an inverted and motorized microscope (Axio Observer Z.1) equipped with a 40x/1.20 W C−Apochromat objective. The attached laser scanning unit (LSM 780, Zeiss, Jena, Germany) enabled confocal imaging. For excitation, 440 nm and 488 nm lasers were used. Unless otherwise noted pinhole was set to 0.5 AU. Detailed scanning parameters are listed in the corresponding figures / figure captions. Images were recorded at 16-bit pixel depth. For analysis, they were processed in MATLAB scripts utilizing the eSIP formulas described above. As start parameters for the fitting we used the parameters obtained from the lookup approach [11]. The fluorescein/perylenediimid-layer reference layer, thickness ≈ 110 nm, nD ≈ 1.59 was a generous gift by J. M. Zwier. The fluorescent solution used in Fig 2 is a dilution of fluorescein in distilled water (Uranine AP, λem = 516 nm, OD = 1.37 for 1 cm at 487 nm). To account for a wider emission wavelength range needed for Fig 3 Rhodamine 6G (λem = 555 nm) was added and adjusted to an equal fluorescence intensity (both fluorophores from Applichem, Darmstadt, Germany). The solution was imaged in an ibidi μ-Dish with 170±5 μm glass bottom (ibidi GmbH, Martinsried, Germany).


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

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

Using section imaging property parameters to optimize microscope system settings.To estimate the correction collar setting at a Zeiss LSM 510 confocal microscope, a series of eSIP measurements can be used to define the influence on single eSIP parameters. (A) 2d plot of the intensity parameter for correction collar settings of a 40x/1.2 W C-Apochromat (Zeiss) ranging from 0.14 to 0.19 obtained at a wavelength range from 500 nm to 740 nm with pinhole setting 1.0 AU is shown. The data is derived from a 115 x 115 μm centre region of each plane. Although using an Apochromat the emission wavelength dependency is evident in this measurement. However the optimal settings can easily be found. In analogue fashion the influence of the correction collar was analysed based on the ωFWHM parameter (B). Interestingly, there is no wavelength dependency for this parameter. Analysing the collimator setting in a lux-FRET paradigm with two different excitations at a Zeiss LSM 780 utilizing a 40x/1.2 W C-Apochromat objective revealed different optimal settings in respect to the observed parameter (C and D). In contrast to an optimization according to the maximal intensity (black), we found a different optimal setting, when the axial position difference between first (440 nm) and the second excitation (488 nm) is measured (red). For the collimator setting series we tested the solution-based approach (C) as well as the Argolight calibration slide (D).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134980.g003: Using section imaging property parameters to optimize microscope system settings.To estimate the correction collar setting at a Zeiss LSM 510 confocal microscope, a series of eSIP measurements can be used to define the influence on single eSIP parameters. (A) 2d plot of the intensity parameter for correction collar settings of a 40x/1.2 W C-Apochromat (Zeiss) ranging from 0.14 to 0.19 obtained at a wavelength range from 500 nm to 740 nm with pinhole setting 1.0 AU is shown. The data is derived from a 115 x 115 μm centre region of each plane. Although using an Apochromat the emission wavelength dependency is evident in this measurement. However the optimal settings can easily be found. In analogue fashion the influence of the correction collar was analysed based on the ωFWHM parameter (B). Interestingly, there is no wavelength dependency for this parameter. Analysing the collimator setting in a lux-FRET paradigm with two different excitations at a Zeiss LSM 780 utilizing a 40x/1.2 W C-Apochromat objective revealed different optimal settings in respect to the observed parameter (C and D). In contrast to an optimization according to the maximal intensity (black), we found a different optimal setting, when the axial position difference between first (440 nm) and the second excitation (488 nm) is measured (red). For the collimator setting series we tested the solution-based approach (C) as well as the Argolight calibration slide (D).
Mentions: Unless otherwise noted, experiments were carried out on an inverted and motorized microscope (Axio Observer Z.1) equipped with a 40x/1.20 W C−Apochromat objective. The attached laser scanning unit (LSM 780, Zeiss, Jena, Germany) enabled confocal imaging. For excitation, 440 nm and 488 nm lasers were used. Unless otherwise noted pinhole was set to 0.5 AU. Detailed scanning parameters are listed in the corresponding figures / figure captions. Images were recorded at 16-bit pixel depth. For analysis, they were processed in MATLAB scripts utilizing the eSIP formulas described above. As start parameters for the fitting we used the parameters obtained from the lookup approach [11]. The fluorescein/perylenediimid-layer reference layer, thickness ≈ 110 nm, nD ≈ 1.59 was a generous gift by J. M. Zwier. The fluorescent solution used in Fig 2 is a dilution of fluorescein in distilled water (Uranine AP, λem = 516 nm, OD = 1.37 for 1 cm at 487 nm). To account for a wider emission wavelength range needed for Fig 3 Rhodamine 6G (λem = 555 nm) was added and adjusted to an equal fluorescence intensity (both fluorophores from Applichem, Darmstadt, Germany). The solution was imaged in an ibidi μ-Dish with 170±5 μm glass bottom (ibidi GmbH, Martinsried, Germany).

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.