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VayTek offers Advanced Image Processing Systems, including both hardware and software for:
- Microscopy
- Industrial Inspection
- Medical Imaging
- Quality Control
- Non-Destructive Testing
- Deconvolution of Confocal Images
- 3D Volume Visualization and Measurement
VayTek, Inc.
305 West Lowe Ave.
Fairfield, IA
Tel 641-472-2227
Fax 641-472-8131
Email vaytek@vaytek.com
VayTek's Homepage
VayTek Website Links
Application Notes
Cameras
Contact Us
Customers
Distributors
FAQ
Free Demos
Imaging Mall
Microscopes
Product Guide
Sending Files
Site Map
Stable Table
VoxBlast 3-D Software
VoxBlast 3-D Movies
Who We Are
XY Stages
VayTek offers Advanced Image Processing Systems, including both hardware and software for:
- Microscopy
- Industrial Inspection
- Medical Imaging
- Quality Control
- Non-Destructive Testing
- Deconvolution of Confocal Images
- 3D Volume Visualization and Measurement
VayTek, Inc.
305 West Lowe Ave.
Fairfield, IA
Tel 641-472-2227
Fax 641-472-8131
Email vaytek@vaytek.com
Deconvolution
Formula for computing optimal step size for deconvolution
Table for lenses for air objectives
Table for lenses for water objectives
Table for lenses for oil objectives
Visual Basic Code to compute the above tables
The formula used by VolumeScan for calculating the depth of field is:
Where:
D = depth of field
W = wavelength of light (emission)
n = refractive index of medium between the lens and the specimen
M = magnification of the lens plus optical relay to the camera
e = resolution limit of the microscope/camera system
The formula can be found on page 48 of Video Microscopy by Inoue' and Spring, 1997, Plenum Press. Other formulae have been proposed by other authors.
The refractive index for air is 1.0 and 1.3 for water. The refractive index for oil is printed on the side of the bottle of immersion oil, usually about 1.5.
The magnification is the multiple of the lens magnification and the magnification of the relay optics between the lens and the camera, usually 10x.
The NA appears on the side of the objective. The wavelength of light, in fluorescent applications, is given by the emission wavelength of the filter. For brightfield applications, use the average of the spectrum - about .540.
e, the resolution limit of the microscope/camera combination, is a little more difficult to calculate. For most high-end digital cameras, it is the resolution limit of the microscope. To derive this term:
1) Calculate the resolution limit of the microscope using
Where:
d = the limit of resolution
W = the wavelength of light
NA = the numerical aperture of the lens
2) Calculate the smallest object that can be resolved on the
CCD array of the camera:
Where:
c = the resolution limit of the camera
P = the size of a single pixel in the ccd camera array
M = the magnification of the relay optics between the lens and
the camera
3) Compare the results from steps 1 and 2. Select the largest number.
Notice from the formula that:
1) the axial resolution will always be worse than the lateral resolution. The axial resolution, relative to the lateral resolution, is significantly worse as the NA decreases.
2) the magnification of the image contributes only a small portion to the depth of field.
3) blue light produces a depth of field that is half as thick as that produced by red light.
4) the depth of field is thinner with an air lens than with an oil lens.
Other Factors Affecting the Depth of Field
Calculating the theoretical depth of field using the above formula is straightforward. However, in practice there are other factors that can affect the depth of field.
If you plan to use deconvolution on the images after they
have been captured, especially with a constrained iterative and
measured PSF, the depth of field can be further reduced. The
exact decrease in the depth of field is variable and depends
on the algorithm used, the quality of the images, the PSF used,
the camera used to capture the images, etc. The literature on
the subject suggests a 30 to 50% reduction is possible. There
is an option in VolumeScan (Deconvolution candidate checkbox)
that will reduce the depth of
field as calculated in the above formula by another 30% if it
is selected.
The refractive index can have a significant impact on the depth of field. In experiments performed with VolumeScan, the accuracy of the predicted depth of field from the formula above was confirmed as long as the correct medium was used with the chosen lens. However, if the medium did not match the lens, the measured depth of field was significantly different from the predicted depth of field.
Matching the refractive index of the medium and the specimen is important also. If a glass cover slip is used with a specimen immersed in water, and an oil immersion lens is used to image the specimen, there can be significant, non-linear distortions in the depth of field. In this instance, the further the lens is focused down through the specimen, the greater the depth of field will be. A water immersion lens, in this instance, will give the same depth of field through the entire depth of the specimen.
