**3. Virtual microscopy: The general principles**

To reduce the potential inaccuracies in the processing and evaluation of biopsy specimens it is important to look for modern solutions in order to maximize the efficiency of use of biop‐ sy specimens. One of the solutions could be application of virtual microscopy having exten‐ sive mathematical basis with fractal and entropy considerations as well as technological support by appropriate software and hardware. Implementation of innovations into practice could significantly increase the effectiveness of liver biopsy specimens.

The digital image analysis [12-14] and computed morphometry in general is considered an important tool in pathology. It can decrease the workload of voluminous repeated measure‐ ments and increase the accuracy and objectiveness of the results. In several fields, e.g., im‐ munohistochemical and molecular typing of breast cancer, the application of digital image analysis is already highly practical [13].

In virtual microscopy, the demands for mathematical basis are higher than in routine histol‐ ogy. This is illustrated by examples of entropy considerations, Delaunay's triangulation or fractal geometry and general non-Euclidean geometry for irregularly shaped biological ob‐ jects [14-16]. Sophisticated software must be elaborated as well. Additional technical re‐ quirements exist for image resolution and size, fast wide-band data transfer as well as digital data storing [12, 13]. The slide scanners and visualisation software are available and improve continuously [12].

Computed morphometry becomes more practical in association with virtual microscopy and digital image analysis as well. As postulated in reference [16] the natural development of science occurs from the ability to recognize, name and classify the object (corresponding to the diagnosis, e.g., chronic viral hepatitis C) to semiquantitative, ordering measurements (e.g., the activity assessment by Knodell or any analogous scale), finally reaching quantita‐ tive characteristics. Descriptive diagnoses and semiquantitative estimates are widely used in the "classic" liver pathology. In order to gain sufficient reliability and fastness, scalar meas‐ urements would require digital assessment [16]. Computed morphometry on the basis of virtual microscopy is a way towards scalar measurements.

The virtual microscopy can be performed in two different ways. Interactive virtual micro‐ scopy by whole slide imaging leaves the conclusion in the hands of pathologist. It changes significantly the working tools from optical microscopy and subjective decisions to comput‐ er screen and objective measurements. The automated virtual microscopy is even more ex‐ citing as computer system should evaluate the diagnoses [14].

In liver pathology, the software develops regarding assessment of steatosis [17-19] and fib‐ rosis [20-24]. Necroinflammatory changes can be quantified as well [16].

Regarding liver ultrastructure, morphometric evaluation of hepatocyte volume can have prognostic significance predicting survival as shown in liver cirrhosis associated with portal hypertension [25]. Morphometric analysis of liver parenchyma in different alcohol-related pathologic conditions has been tested with good results [26]. Thus, changes in the volume fraction of parenchymal interstitial space and in the surface density of hepatocyte plasma membrane, rough endoplasmic reticulum and outer mitochondrial membrane can be of im‐ portance for distinguishing between cirrhosis and non-cirrhotic states. Hepatocyte nuclear volume fraction measurement can predict the survival in case of cirrhosis. Interestingly, few images are necessary to perform these measurements thus helping to characterise even scarce tissue material [26].

Combination of multiplex quantum dot immunostaining with high resolution whole-slide digital imaging and automated image analysis has been described [27].

At present, the two most frequently studied targets for computer-assisted and/or digital im‐ age analysis in liver biopsies include steatosis and fibrosis.
