Pinhole Controls Optical Slicing

To measure intensity profiles in axial direction, the microscope must be equipped with a motorized focus drive. Usually, the software will provide a tool for acquisition of a 3D image stack or – more elegant – a quick profile section, called xz scan. Profile sectioning is performed by scanning the illumination point on a single straight line while simultaneously incrementing the focus position. The displayed image is an xz cut through the sample. If the sample contains structures that are sufficiently smaller than the diffraction limited resolution, the image of the structure is the actual PSF. Software packages for measuring intensity profiles through such an xz cut are widely available.

To monitor the performance of a system, a planar mirror is usually used as sub-diffraction structure (which is true in z dircetion only, of course). Reflected light confocal microscopy yields thinner optical sections as compared to fluorescence, but sample preparation and execution of the measurement are somewhat simpler. These measurements allow system performance to be monitored over long time periods or for comparison with other systems. It is vital, though, to ensure proper sample preparation. For instance, oil-immersion lenses corrected for covered samples need mirrors covered with a coverslip and mounted with immersion oil on both sides of the coverslip. The z positioning must be better than the z resolution and must be calibrated. The pinhole diameter must be calibrated and should be varied between 0.5 and 2 Airy-disc diameter (Airy Unit, AU).

As most applications imply fluorescence, the measurement of actual section sizes should use fluorescent beads. For classical true confocal systems, beads of diameter 50 … 100 nm are appropriate. Preparations of these samples are a bit more cumbersome and fluorescence markers tend to bleach with time.

The measurement is very sensitive to any kind of sample imperfection, refractive index mismatch, temperature and other parameters. In order to avoid unnecessary frustration, only experienced microscope operators should do these tests.

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