Emerging technologies for diagnostic pathology.

Many technological advances have been made in the recent years, several of them with a great potential of significantly improving the diagnostic pathology field. This article discusses three of the most promising technologies, which emerged in the last one year. Fluorescent in situ sequencing can lead to the simultaneous identification of the transcriptome-wide RNA in individual cells across a tissue sections. 3D microscopy together with advanced image analysis can be used in diagnostic pathology and will especially be useful in hard to diagnose cases where the spatial relationship of the tissue components is important. Expansion microscopy physically expands the biological specimen, and is of great interest for diagnostic pathology since the cheap conventional microscopes can be used to image a symmetrically expanded tissue. In addition, digital analysis and computational pathology are an integral part of each of these three emerging technologies, which underline their importance for the future developments in diagnostic pathology.


Introduction
Several technologies with great potential in significantly advancing the diagnostic pathologies have emerged in the recent years. This article focuses on three revolutionary technologies that emerged in the last one year. These methods promise to advance the diagnostic in pathology by adding another dimension (3D diagnosis), sequencing in situ (fluorescent in situ sequencing) and tissue expansion (expansion microscopy). These technologies combined with automated pathology have the potential of being widely employed by the pathologists in the near future.
Researchers and pathologists understand now more than ever the importance of personalized medicine. The potential impact of next-generation sequencing and whole-genome analysis in medicine and, specifically, in clinical laboratory practice is well recognized 3,4 . However, the current sequencing techniques, although high-throughput, do not provide much information on the exact location of its target. Moreover, DNA/RNA FISH identifies the exact location of the target, however, it is not a high-throughput technique 5 , since it is limited in the number of probes that can be simultaneously or consecutively employed. Identifying the exact location of the sequenced or identified RNA/DNA of interest on the tissue section would bring an enormous benefit not only to the research community, but also to the diagnostic pathology field. Fluorescence in situ sequencing (FISSEQ) is a highly multiplexed subcellular RNA sequencing in situ, which permits massively parallel detection of genetic elements, including gene transcripts, important in analyzing cellular phenotypes, gene regulation, and cellular environment in situ 6,7 . This transcriptome-wide RNA sequencing in situ was validated on multiple specimen types and spatial scales. Fisseq is compatible with fresh frozen brain tissue sections and whole-mount embryos, although the authors only present the complete sequencing of cell lines 6 .
Briefly, the cells/tissue are fixed and the RNA is revers transcribed into cDNA using random hexamer primers. cDNA is then in situ amplified and cross-linked, forming the single-stranded DNA nanoballs (also named rolonies or cDNA amplicons). Fluorescent sequencing, which consists in using a fluorescent probe hybridized to the adaptor sequence and imaged by confocal microscopy, is next performed on the DNA nanoballs 6,7 .
If successful on tissue sections, this method would enable the simultaneous identification of the transcriptome-wide RNA in individual cells across a tissue sections. This will not only help scientists to study in situ cellular interactions (such as the ones from epithelium and stroma in several types of malignancies (breast, gastro-intestinal tumors etc)), immune cells and cancer associated fibrobalsts/tumor cells etc) but will also help in diagnosis and post-diagnosis follow up of many pathologies, including cancers.

3D diagnosis in pathology
Since the early 20 th century, pathological classification of many types of malignancies has been primarily based on the visual analysis of hematoxylin and eosin stained image slides using conventional two dimensional (2D) microscopy 8 . With the recent development of new state-of-the-art microscopy platforms, such as the fluorescent lightsheet microscopy (the Nature Methods' method of the year 2014) 9-11 and multiphoton microscopy (2015 Brain Prize given for the development of 2 photon microscopy) 12 , the rapid acquisition of three dimensional (3D) images directly from tissue samples up to several millimeters in thickness is now possible 11 .
Recent results presented a successful pipeline of formalin fixed paraffin embedded tissue sample preparation, imaging with a 3D advanced microscopy and extraction of quantitative image measurements from the 3D images to build classification models in breast tissue. This classification models help in differentiating normal tissue from non invasive breast cancer and different grades of invasive breast cancer 13,14 .
Thus, 3D microscopy together with advanced image analysis can be used in diagnostic pathology and will be especially useful in hard to diagnose cases where the spatial relationship of the tissue components is important.

Expansion microscopy
Researchers have successfully created state of the art microscopes, which are under continuous development, in order to provide high resolution images and to see in details the structures of the different types of cells and tissue 9-12 . However, a revolutionary idea recently came to life when a 4-5 fold expansion of the human brain tissue was reported with minimal distortion 15 . The authors named this method, of physical magnification of the specimen itself, expansion microscopy (ExpM) 15 . ExpM would be a great asset in diagnostic pathology, since one could use a cheap conventional microscope to image a symmetrically expanded tissue.
ExpM uses a "swellable" polymer network in a biological sample that can expand the sample by 4-5 fold 15,16 . The method uses the well known properties of the polyelectrolyte gels, which after water addition they expand. The tissue is placed in a polyelectrolyte gel, followed by its treatment with a protease to homogenize its mechanical characteristics and the placement of the specimen in water for expansion 15 .
Expanded tissues (such as the brain) have the great advantage that the process makes them completely or partially transparent. Thus, these tissues are well fit for lightsheet microscopy, which requires transparent specimens, so there is no need for a preclearing step 17 .
In conclusion, these are only few of the several important technological advances that could help the diagnostic pathology field in the near future. Although these methods are still under development, they have an extraordinary potential in transforming diagnosis of different pathologies. Moreover, these three techniques can be combined together and employed for analysis of the same samples, leading to valuable results for both research and diagnosis.