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Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index.

Besseling TH, Jose J, Van Blaaderen A - J Microsc (2014)

Bottom Line: We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account.There was however a strong deviation with the theoretical predictions using (high-angle) geometrical optics, which predict much lower scaling factors.As an illustration, we measured the PSF of a correctly calibrated point-scanning confocal microscope and showed that a nearly index-matched, micron-sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.

View Article: PubMed Central - PubMed

Affiliation: Soft Condensed Matter, Debye Institute for NanoMaterials Science, Utrecht University, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

A fluorescent PMMA sphere dispersed in an index matching mixture of 24 wt% cis-decalin in CHB, recorded with a confocal microscope (Leica SP8). (A) 3D view constructed from a XYZ image stack. (B) A single XY image shows that x and y distances are equal. (C) The reconstructed XZ view of the image shows that there is a small (5.8%) elongation in the z-direction. Due to the refractive index mismatch between the suspension ( = 1.49) and the oil immersion ( = 1.52) an elongation in the z-direction of 2% was expected. (D) Intensity profiles along different lines trough the sphere, as indicated in the figure. The profiles were normalized and shifted for better visualization.
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fig04: A fluorescent PMMA sphere dispersed in an index matching mixture of 24 wt% cis-decalin in CHB, recorded with a confocal microscope (Leica SP8). (A) 3D view constructed from a XYZ image stack. (B) A single XY image shows that x and y distances are equal. (C) The reconstructed XZ view of the image shows that there is a small (5.8%) elongation in the z-direction. Due to the refractive index mismatch between the suspension ( = 1.49) and the oil immersion ( = 1.52) an elongation in the z-direction of 2% was expected. (D) Intensity profiles along different lines trough the sphere, as indicated in the figure. The profiles were normalized and shifted for better visualization.

Mentions: As a second method to calibrate the axial distance in a confocal microscope, we exploited the well-defined 3D geometry of large spherical PMMA particles (average diameter σ = 50 μm and polydispersity larger than 10%), dyed with a thin fluorescent shell (∼ 500 nm). We used these particles to determine the z-calibration of a point-scanning confocal microscope (Leica SP8). We first confirmed correct calibration of the xy-distances of the microscope by imaging a calibration grid (Ted Pella, grid spacing 0.01 mm) in reflection mode using a 100x/1.4 oil immersion objective (Leica). Then we imaged a single particle in 3D using the same objective. Figure4(A) shows a 3D image-stack of a particle dispersed in an RI-matching mixture of 24 wt% cis-decalin/CHB. In Figure4(B), a single xy-image shows that the diameter of the particle in the x- and y-direction is equal. However, a reconstructed xz-view of the particle (Fig.4C) shows that there is an elongation in the z-direction. From the intensity profiles, shown in Figure4(D), we determined the diameter of the particle in the x-, y- and z-direction, and found an elongation of 5.8% in the z-direction. We also deconvolved the 3D image stack with a theoretical depth-dependent PSF. The resulting intensity profile in the z-direction is indicated with the (blue) dashed line in Figure4(D). The deconvolution resulted in a decrease of the width of both peaks, however, there was no significant change in the distance between them. Additionally, we acquired images for different scan-speeds and different image-sizes and found similar results. Due to the (small) RI mismatch between the suspension ( = 1.490) and the immersion oil ( = 1.516) we expected, based on Eq. (4), an axial scaling factor in the z-direction of only f(1.49) = 0.98. We therefore conclude that there is a small but significant elongation in the z-direction of 3.7%, which is most likely due to an incorrect calibration of the microscope. To confirm this statement, we measured the height of our calibration cell when it was filled with immersion-oil (Fig.2B) with the same microscope and objective as used for the image-stack in Figure4, and found a distance of μm. This indicated a similar deviation of 3.0% in the axial direction.


Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index.

Besseling TH, Jose J, Van Blaaderen A - J Microsc (2014)

A fluorescent PMMA sphere dispersed in an index matching mixture of 24 wt% cis-decalin in CHB, recorded with a confocal microscope (Leica SP8). (A) 3D view constructed from a XYZ image stack. (B) A single XY image shows that x and y distances are equal. (C) The reconstructed XZ view of the image shows that there is a small (5.8%) elongation in the z-direction. Due to the refractive index mismatch between the suspension ( = 1.49) and the oil immersion ( = 1.52) an elongation in the z-direction of 2% was expected. (D) Intensity profiles along different lines trough the sphere, as indicated in the figure. The profiles were normalized and shifted for better visualization.
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Related In: Results  -  Collection

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fig04: A fluorescent PMMA sphere dispersed in an index matching mixture of 24 wt% cis-decalin in CHB, recorded with a confocal microscope (Leica SP8). (A) 3D view constructed from a XYZ image stack. (B) A single XY image shows that x and y distances are equal. (C) The reconstructed XZ view of the image shows that there is a small (5.8%) elongation in the z-direction. Due to the refractive index mismatch between the suspension ( = 1.49) and the oil immersion ( = 1.52) an elongation in the z-direction of 2% was expected. (D) Intensity profiles along different lines trough the sphere, as indicated in the figure. The profiles were normalized and shifted for better visualization.
Mentions: As a second method to calibrate the axial distance in a confocal microscope, we exploited the well-defined 3D geometry of large spherical PMMA particles (average diameter σ = 50 μm and polydispersity larger than 10%), dyed with a thin fluorescent shell (∼ 500 nm). We used these particles to determine the z-calibration of a point-scanning confocal microscope (Leica SP8). We first confirmed correct calibration of the xy-distances of the microscope by imaging a calibration grid (Ted Pella, grid spacing 0.01 mm) in reflection mode using a 100x/1.4 oil immersion objective (Leica). Then we imaged a single particle in 3D using the same objective. Figure4(A) shows a 3D image-stack of a particle dispersed in an RI-matching mixture of 24 wt% cis-decalin/CHB. In Figure4(B), a single xy-image shows that the diameter of the particle in the x- and y-direction is equal. However, a reconstructed xz-view of the particle (Fig.4C) shows that there is an elongation in the z-direction. From the intensity profiles, shown in Figure4(D), we determined the diameter of the particle in the x-, y- and z-direction, and found an elongation of 5.8% in the z-direction. We also deconvolved the 3D image stack with a theoretical depth-dependent PSF. The resulting intensity profile in the z-direction is indicated with the (blue) dashed line in Figure4(D). The deconvolution resulted in a decrease of the width of both peaks, however, there was no significant change in the distance between them. Additionally, we acquired images for different scan-speeds and different image-sizes and found similar results. Due to the (small) RI mismatch between the suspension ( = 1.490) and the immersion oil ( = 1.516) we expected, based on Eq. (4), an axial scaling factor in the z-direction of only f(1.49) = 0.98. We therefore conclude that there is a small but significant elongation in the z-direction of 3.7%, which is most likely due to an incorrect calibration of the microscope. To confirm this statement, we measured the height of our calibration cell when it was filled with immersion-oil (Fig.2B) with the same microscope and objective as used for the image-stack in Figure4, and found a distance of μm. This indicated a similar deviation of 3.0% in the axial direction.

Bottom Line: We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account.There was however a strong deviation with the theoretical predictions using (high-angle) geometrical optics, which predict much lower scaling factors.As an illustration, we measured the PSF of a correctly calibrated point-scanning confocal microscope and showed that a nearly index-matched, micron-sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.

View Article: PubMed Central - PubMed

Affiliation: Soft Condensed Matter, Debye Institute for NanoMaterials Science, Utrecht University, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus