Deep Learning-Based Virtual H& E Staining from Label-Free Autofluorescence Lifetime Images

Label-free autofluorescence lifetime is a unique feature of the inherent fluorescence signals emitted by natural fluorophores in biological samples. Fluorescence lifetime imaging microscopy (FLIM) can capture these signals enabling comprehensive analyses of biological samples. Despite the fundamental importance and wide application of FLIM in biomedical and clinical sciences, existing methods for analysing FLIM images often struggle to provide rapid and precise interpretations without reliable references, such as histology images, which are usually unavailable alongside FLIM images. To address this issue, we propose a deep learning (DL)-based approach for generating virtual Hematoxylin and Eosin (H&E) staining. By combining an advanced DL model with a contemporary image quality metric, we can generate clinical-grade virtual H&E-stained images from label-free FLIM images acquired on unstained tissue samples. Our experiments also show that the inclusion of lifetime information, an extra dimension beyond intensity, results in more accurate reconstructions of virtual staining when compared to using intensity-only images. This advancement allows for the instant and accurate interpretation of FLIM images at the cellular level without the complexities associated with co-registering FLIM and histology images. Consequently, we are able to identify distinct lifetime signatures of seven different cell types commonly found in the tumour microenvironment, opening up new opportunities towards biomarker-free tissue histology using FLIM across multiple cancer types.