resolved by that objective. Using a zoom setting to create lateral resolution in beyond Nyquist
will not improve the resolution of a confocal image. However, the exception is collecting images
at 2X Nyquist, or 4.6 times the objective resolution, can be useful for deconvolution.
6.11 Obtaining a Z-Series.
The axial resolution for an objective lens is generally 2 to 5 times poorer than the lateral resolution due
to spherical aberrations in the optical path and sample. The sample must be oversampled in the z-axis in
order to achieve maximal resolution, just is is required in the lateral axes. The Fluoview software
calculates the optimal z-step for each combination of objective and wavelength.
The focus steps for objectives in the table below provide sampling to match the Nyquist theorem for
resolution. Smaller values will result in oversampling that may increase detectible resolution at the
expense of inflated file sizes and additional photobleaching.
Table 4. Optimal Z-Step Values
Objective Mag.
Resolution (um)
Lateral Axial
Resolution (um)
Lateral Axial
Optimal Sampling (um)
Pixel Size Z-Step
Optimal Sampling (um)
Pixel Size Z-Step
4X
1.504
54.258
0.654
23.6
10X/.40
0.601
8.56
0.261
3.72
20X/.75
0.319
2.12
0.139
0.921
40X/1.30 Oil
0.181
0.948
0.079
0.412
60X/1.35 Oil
100X/1.4 Oil
0.168
0.778
0.073
0.338
60X/1.20 Water
The resolutions calculated by the Fluoview software tend to be somewhat pessimistic. Axial resolution
is improved by reducing the z-step to 1/2 of these values.
6.12 Resolution.
The lateral and axial resolution limits of the objective lens is set by its NA, not by its magnification.
The resolution limits for any lens are constrained by wavelength, specimen preparation, signal intensity,
noise and sampling frequency. Sampling and resolution on the confocal are properties of zoom and box
size. Most biological specimens do not possess optical properties
Resolution can be defined in 2 ways: 1) How much can you resolve with any given objective lens, 2)
How much resolution do you need to answer your questions? If you are looking for cells that can be
identified by labeling in nuclei that are 5
µ
m in diameter, then you do not need the same resolution as if
you are looking for synapses.
Olympus Fluoview-1000 User’s Guide
V.M. Bloedel Hearing Research Center, Core for Communication Research
Center on Human Development and Disability, Digital Microscopy Center
May 11, 2011
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