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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

Construction and measurement of a calibration cell. (A) A sample cell with height H was built with glass cover slips and a standard microscopy slide, glued together with UV-glue. (B) When the (empty) cell was placed in a Fourier Transform Infrared (FTIR) spectrometer, Fabry Perrot (FP) fringes were visible in the transmission spectrum. (C) The height of the cavity ( μm) was determined from the spacing between the FP fringes (Jiang et al., 1999). The error-bars on individual points are smaller than the symbol size.
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fig01: Construction and measurement of a calibration cell. (A) A sample cell with height H was built with glass cover slips and a standard microscopy slide, glued together with UV-glue. (B) When the (empty) cell was placed in a Fourier Transform Infrared (FTIR) spectrometer, Fabry Perrot (FP) fringes were visible in the transmission spectrum. (C) The height of the cavity ( μm) was determined from the spacing between the FP fringes (Jiang et al., 1999). The error-bars on individual points are smaller than the symbol size.

Mentions: To calibrate the axial distances in a point-scanning confocal microscope, we built a custom sample cell with standard glass cover slips (Menzel Gläzer). The glass cover slips had a RI () close to the RI of the oil-immersion liquid (Type F, Leica, ) used for imaging. We avoided using glass capillaries (Vitrocom), often used in confocal studies on colloidal systems, since they provide lower quality imaging which is partially due to their manufacturing process and also due to the RI ( = 1.47). We used a standard No. 1.0 coverslide, which has a thickness between 130 and 160 μm, as specified by the manufacturer (Menzel Gläzer). Although standard confocal microscopy objectives are optimized for a cover slip thickness of 170 μm (Pawley, 2006) and therefore a No. 1.5 cover slip (thickness 160-190 μm) would have been more accurate, we could not however completely image our cell (with a height ∼ 80 μm), due to the limited working distance of the high numerical aperture objectives that we used. As spacers, we used No. 00 cover slips (thickness 55–80 μm) and the individual components of the cell were permanently fixed onto a standard microscopy slide (Menzel Gläzer) with UV glue (Norland 68 Optical Adhesive), see Figure1(A). The resulting height of the cell H was measured with a Fourier Transform Infrared (FTIR) spectrometer, with a selected diameter aperture of 0.25 mm (Vertex 70, Bruker). To avoid additional interference effects from the top cover slip itself, a drop of immersion oil was carefully placed on top of the cell before the measurement. The thickness and irregularities of the much thicker microscopy slide (∼ 1 mm) made it not necessary to correct for its interference effects.


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)

Construction and measurement of a calibration cell. (A) A sample cell with height H was built with glass cover slips and a standard microscopy slide, glued together with UV-glue. (B) When the (empty) cell was placed in a Fourier Transform Infrared (FTIR) spectrometer, Fabry Perrot (FP) fringes were visible in the transmission spectrum. (C) The height of the cavity ( μm) was determined from the spacing between the FP fringes (Jiang et al., 1999). The error-bars on individual points are smaller than the symbol size.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383648&req=5

fig01: Construction and measurement of a calibration cell. (A) A sample cell with height H was built with glass cover slips and a standard microscopy slide, glued together with UV-glue. (B) When the (empty) cell was placed in a Fourier Transform Infrared (FTIR) spectrometer, Fabry Perrot (FP) fringes were visible in the transmission spectrum. (C) The height of the cavity ( μm) was determined from the spacing between the FP fringes (Jiang et al., 1999). The error-bars on individual points are smaller than the symbol size.
Mentions: To calibrate the axial distances in a point-scanning confocal microscope, we built a custom sample cell with standard glass cover slips (Menzel Gläzer). The glass cover slips had a RI () close to the RI of the oil-immersion liquid (Type F, Leica, ) used for imaging. We avoided using glass capillaries (Vitrocom), often used in confocal studies on colloidal systems, since they provide lower quality imaging which is partially due to their manufacturing process and also due to the RI ( = 1.47). We used a standard No. 1.0 coverslide, which has a thickness between 130 and 160 μm, as specified by the manufacturer (Menzel Gläzer). Although standard confocal microscopy objectives are optimized for a cover slip thickness of 170 μm (Pawley, 2006) and therefore a No. 1.5 cover slip (thickness 160-190 μm) would have been more accurate, we could not however completely image our cell (with a height ∼ 80 μm), due to the limited working distance of the high numerical aperture objectives that we used. As spacers, we used No. 00 cover slips (thickness 55–80 μm) and the individual components of the cell were permanently fixed onto a standard microscopy slide (Menzel Gläzer) with UV glue (Norland 68 Optical Adhesive), see Figure1(A). The resulting height of the cell H was measured with a Fourier Transform Infrared (FTIR) spectrometer, with a selected diameter aperture of 0.25 mm (Vertex 70, Bruker). To avoid additional interference effects from the top cover slip itself, a drop of immersion oil was carefully placed on top of the cell before the measurement. The thickness and irregularities of the much thicker microscopy slide (∼ 1 mm) made it not necessary to correct for its interference effects.

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