Hematoxylin and Eosin Stained Tissue Using Color Brightfield Imaging with the CytationTM 5 to Image Fixed and Stained Tissue

Examination of chromogenically stained tissues has been the hallmark of clinical diagnostic pathology and cancer research for nearly a century. Fixed and stained tissue on microscope slides has been the routinely used method of data storage. Pathological samples to be viewed were routinely fixed and embedded in paraffin for long term storage. Thin slices were then made from the paraffin samples, immobilized on slides, dewaxed and subsequently stained, usually with hemotoxylin and eosin, and sealed with a cover slip. Examination of the pathological slide required that the examiner utilize a staged multi-objective microscope to make their observations. After examination the slide along with the paraffin embedded tissue was routinely archived for later reexamination. If reexamination was required, the slide had to be retrieved or the paraffin fixed tissue had to be reprocessed and a new slide made prior to reexamination. With advancements in digital imaging and data storage colored imagery is becoming more desirable. The combination of the hematoxylin and eosin (H&E) dyes was first used to stain tissues in 1876 by the chemist Wissowzky (1). Hematoxylin, or more correctly its oxidized form hematin binds with a mordant (typically Al3+) to stain DNA in cellular matter. It is thought to bind with the negatively charged phosphate groups that comprise the DNA backbone and then undergo complex coordination or conjugation to become a permanent stain of the nucleus. Together with its Al3+ mordant, the dye produces a blue color in neutral to basic conditions (Figure 2). The imaging and analysis of labeled fixed and chromogenically stained cells has traditionally been accomplished using manual examination of microscopic slides with a multi-objective staged microscope. With the advent of digital color brightfield imagery, samples can be automatically imaged and the data stored for examination independent of the microscope. Here we describe the use of the Cytation 5, a low cost high-value combination cell imager and microplate reader, to rapidly image Hematoxylin and Eosin stained tissue slides.