Since Gomori stained activity of phosphatase in tissue sections in the 1930s, histochemistry and cytochemistry (H&C) developed into true disciplines that cannot be done without in science. Especially, in the second half of last century, tremendous progress was achieved in specificity, sensitivity and precision of localization of H&C methods. H&C can be defined as the disciplines that localize specific compounds, such as a specific sequence of DNA or RNA using in situ hybridization, a specific protein using immunoH&C or a product of activity of a specific enzyme using enzyme H&C. The latter method was developed by Gomori to visualize phosphatase activity in tissue sections or cell preparations, respectively, and this was in fact the initiation of H&C .

Especially immunoH&C has become indispensable for microscopic imaging, flow cytometry and cell sorting on the basis of fluorescent or coloured staining of one or more specific proteins in tissue or cell samples. Besides, in situ hybridization has become a valuable technology for the staining of specific sequences of DNA (e.g. DNA of viruses in a human tissue) or RNA (idem of RNA viruses or mRNAs to visualize where in a tissue or in which types of cells a specific gene is expressed). The third major approach, enzyme H&C or metabolic mapping^1,2, that visualizes the product of a specific enzyme reaction as a fluorescent or coloured end product, became almost obsolete in the last 2 decades of last century. Metabolism was considered old fashioned in that time. How did this opinion change in the beginning of this century by the awareness that metabolism is very much involved in major complex diseases such as cancer and diabetes. This awareness initiated studies that targeted therapeutically metabolic pathways that are crucial for diseased tissue or cells. For example, specific mutations in genes encoding for enzymes such isocitrate dehydrogenase 1 and 2 are important steps in the development of a number of types of cancer such as glioma (primary brain tumors), acute myeloid leukemia, cholangiocarcinoma (tumors of bile ducts) or chondrosarcoma (tumors of cartilage)³. Metabolic mapping plays an important role in the search for therapies of types of cancer that have this mutation⁴. It is only one example of metabolic mapping where it has experienced a revival in the present century showing its new role in H&C.

The other major development is 3D histochemistry. Histochemistry has always been performed on thin tissue sections, but since the group of Deisseroth published their Clarity method to look through brain, 3D histochemistry has become the talk of the scientific world⁵. The principles of 3D histochemistry are clearing of the tissue, fluorescence labelling of specific proteins or structures, and visualization of the entire organs or tissue sample using confocal microscopy or light-sheet microscopy. Basically, two approaches exist for clearing of organs or tissue samples. The first one is removal of lipids of cell membranes from the organ or tissue. The lipids cause the opaqueness of tissues and removal of the lipids renders tissues transparent or clear which is the principle of Clarity. The other clearing method is based on matching the refractive index of the tissue sample and the solution in which the tissue sample is embedded . This method works for tissues that contain significant amounts of extracellular matrix (ECM)⁶. The central nervous system hardly contains ECM but large amounts of cell membranes and this makes Clarity ideal for brain but not for tissues that contain considerable amounts of ECM. These tissues can be cleared in solutions that have a similar refractive index such as in iDISCO and BABB methods⁶. The group of Erturk recently showed that an entire rat can be cleared using iDISCO methodology and fluorescence imaging can be performed in 3D in the intact animal⁷. Moreover, iDISCO and BABB can also be used to clear human tissues⁶, because perfusion of the tissue is not needed as is the case with Clarity. Therefore, Clarity can only be applied to experimental animals. The advantages of 3D histochemistry are enormous as molecular and cellular interactions can be studied directly in 3D instead of indirectly using reconstructions on the basis of images of large amounts of serial sections.

These developments in H&C in combination with the rapid developments in microscopy and nanoscopy make imaging of tissues and cells increasingly important tools in the life sciences.

1. Van Noorden CJF (2010) Imaging enzymes at work: Metabolic mapping by enzyme histochemistry. J Histochem Cytochem 58:481-497
2. Van Noorden CJF (2014) Metabolic mapping by (quantitative) enzyme histochemistry. Pathobiology of Human Disease: A Dynamic Encyclopedia of Disease Mechanisms, pp 3760-3774
3. Molenaar RJ et al (2014) The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation. Biochim Biophys Acta 1846:326-341
4. Molenaar RJ et al (2015) Radioprotection of IDH1-mutated cancer cells by the IDH1-mutant inhibitor AGI-5198. Cancer Res 75:4790-4802
5. Chung K et al. (2013) Structural and molecular interrogation of intact biological systems. Nature 497:332-337
6. Azaripour A et al. (2016) A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue. Progr Histochem Cytochem 51:9-23
7. Pan C et al. (2016) Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods DOI:10.1038/NMETH.3964