Scanning microscope: Please find technical details in the published article.
As can be seen from the picture, the motorized stage holds 8 slides. The
workstation controlling the microscope and acquisition is the right one, while the
left computer processes the image data. The digital camera is connected with
fiber-optics to the workstation. The scanning and the processing workstations
communicate through a standard 100 Mbit ethernet connection.

Workstations: The computer that controls the motorized microscope is a
standard 2 GHz Microsoft Windows workstation. The processing computer is
a 2.6 GHz Microsoft Windows workstation.

Pixel size and resolution limit: A pixel size of 0.26 um corresponds to more
than 90 000 dpi. Even this is however not the highest possible digitization
resolution. With our microscope settings the laws of optics postulate that the
maximum resolution with a NA (Numerical Aperture) of 1.3 is < 0.10 um. This
would provide even more details in the virtual slides, but would also result in
a more than four-fold increase in scanned image data amount. As described
in the published article, the 0.26 um resolution was considered satisfactory
by most pathologists.

Objectives and autofocusing: As discussed in the article, slide scanning with
a 10x or 20x objective would have resulted in much fewer image tiles and
autofocusing  operations during image capture, but the resulting image quality
was disappointing. The main reason for this is that with a 10x or 20x objective,
the area of the view field is much larger, resulting in bad autofocusing due to
always present height variations in the slides. Also, the subjective visual image
quality when scanning with the oil-40x objective was clearly superior. We intend
to publish test series of the same slide scanned with different objectives and
camera settings.

The depth of field: For most histological specimens the depth of field of the
light microscope is much smaller than the thickness of the specimen. In a
microscope this limitation can be overcome by continually adjusting the focus,
by moving the zone of sharpness up and down the optical axis and thus
getting a mental image of a structure’s three-dimensional shape. The depth of
field depends on the objective lens’s numerical aperture, in that a high quality
objective such as the one used to scan the virtual slides on this website, yields
a narrow depth of field. Capturing image data with an objective with a lower
numerical aperture would increase the depth of field. The numerical aperture
can also be lowered by simply closing down the condenser iris diaphragm.
But as the depth of field increases, there is a corresponding loss of resolution
in the captured image. The theoretical resolving power as a function of a lens’
numerical aperture can be calulated as

                     R =  λ / ( 2 N.A.)

where R is the resolving power in µm, λ is in µm, and N.A. is the numerical
aperture.

There are, however other ways to extend the depth of field in a virtual slide,
except for using a smaller numerical aperture. A functional solution is to aquire
and use multiple focal planes in the same virtual slide, or digitally merge them.

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