Chédiak-Higashi syndrome approached by several different microscopy imaging technologies.

Abstract

Routinely air-dried BM films stained with a Leishman stain were examined by conventional light microscopy (upper left ×100 objective) and with a confocal laser scanning microscope with Airyscan super-resolution (upper right ×63 objective and ×5.5 electronic magnification). An unstained film was examined by fluorescence lifetime imaging (FLIM) (lower left ×63 objective and ×6 electronic magnifications). In this technique, a pulsating laser stimulates the emission of autofluorescence in unstained cells according to their metabolic state and oxygen consumption. It does not quantify fluorophores, but rather their interaction with the environment.1 Our equipment counts fluorescence lifetimes individually for each emitted photon. The mean lifetimes per image pixel are calculated and transformed into pseudocolours, thus permitting the creation of a virtual FLIM image. In parallel, small BM fragments were fixed with Karnovsky solution and OsO4, and embedded in araldite. Ultrathin sections were stained with uranyl acetate and lead citrate for electron microscopy (lower right ×10 000 magnification). In the Leishman-stained film the large azurophilic granules characteristic of Chédiak–Higashi syndrome were observed. It was not possible to detect any internal heterogeneity either by conventional light microscopy or by Airy-scanned super-resolution microscopy. The FLIM technique, however, disclosed the internal heterogeneity of the lysosomes, demonstrated by different pseudocolours. The electron microscopy confirmed this heterogeneity, showing the varying electron-density of the giant lysosomes in detail. In summary, the FLIM technique is able to highlight, in label-free preparations at the light microscopic level, cellular elements with different physicochemical characteristics. This technique has been used for non-invasive imaging in dermatology and ophthalmology, but has seldom been used for cytological studies. In our case, FLIM was able to show more details than confocal Airyscan super-resolution microscopy.