Depth of sharpness of optical microscope

What is the definition ?
Depth of Sharpness of the Optical Microscope mainly concerns the Depth of field (DOF), which studies how an objective can be focused without any appreciable change in image sharpness. It is also the ability to produce a sharp image from a non-flat surface and the distance along the optical axis either side of the focus point that remains in acceptable focus.

DOF can be improved by closing the aperture on the objective, however with the expense of optical resolution and with a significant loss of light.

Depth of Field or Depth of Focus?
Depth of Field is sometimes referred to as Depth of Focus, but although the exact definition of both is slightly different and there has been some discussion as to if we should instead be talking about Depth of Focus instead of Depth of Field.

We will use the term Depth of Field on this piece of paper since the term specifically defines the distance in front of the objective, which remains sharp above and below the object being imaged.

The variable depth of field makes you to see uneven surfaces in sharp contrast. Or alternatively to see individual layers of living tissues without the higher or lower layers interfering with the image quality.

How to increase the depth field ?
DOF can be improved by closing the aperture on the objective, however with the expense of optical resolution and with a significant loss of light.

The discovery of how to increase Depth of Field up to levels significantly beyond normal limits is of profound importance in microscopy. Extended depth of field is a vital component in the ability to observe nanometer size objects, live and in natural color, and therefore increase its sharpness- something that cannot be done with conventional light microscopes mainly due to their inability to solve the depth of field and resolution issues.

There is a part of a computer chip seen under the microscope. Adding depth of field provides significantly more details allowing a better sharpness of structures and understanding of the structure with a 3 dimensional appearance as you can see in the image above. The total depth of field is given by the sum of the wave and geometrical optical depths of field as:

dtot = λn/NA2 + (n/M•NA)e

where λ is the wavelength of illumination, n is the refractive index of the imaging medium, NA is the objective numerical aperture, M is the objective lateral magnification, and e is the smallest distance that can be resolved by a detector that is placed in the image plane of the objective.

We have to notice that the diffraction-limited depth of field (the first term on the right-hand side of the equation) decreases inversely with the square of the numerical aperture, while the lateral limit of resolution is reduced with the first power of the numerical aperture. The table below make the comparison between the magnification, numerical aperture, depth of field and image depth ( notice that a high level of image depth will lead to a higher sharpness of the image).

Practical values for visual depth of field
In DIN/ISO standards, the depth of field on the side of the object is defined as the "axial depth of the space on both sides of the object plane within which the object can be moved without detectable loss of sharpness in the image, while the positions of the image plane and the objective are maintained". Particularly at low magnifications, the depth of field can be significantly increased by reducing the numerical aperture. Yet, the smaller the numerical aperture, the lower the lateral resolution. It is therefore a matter of finding the optimum balance of resolution and depth of field depending on the structure of the object.

Depth of field in digital image processing
The Multifocus module of the Leica Application Suite (LAS) was developed with the objective of extending field depth of the automated microscope many times over. The illumination, image brightness and all other camera parameters can be set individually to optimize the quality of the resulting image. The LAS Multifocus module provides an easy solution to capturing the extended field depth of live images by integrated control of the microscope with the motorfocus. The automatic capture of z stacks together with the intelligent image combination algorithms guarantees easy photography and storage of sharply focused images.