Detecting immune cells with label-free two-photon autofluorescence and deep learning

Label-free imaging has gained broad interest because of its potential to omit elaborate staining procedures which is especially relevant for in vivo use. Label-free multiphoton microscopy (MPM), for instance, exploits two-photon excitation of natural autofluorescence (AF) from native, metabolic proteins, making it ideal for in vivo endomicroscopy. Deep learning (DL) models have been widely used in other optical imaging technologies to predict specific target annotations and thereby digitally augment the specificity of these label-free images. However, this computational specificity has only rarely been implemented for MPM. In this work, we used a data set of label-free MPM images from a series of different immune cell types (5,075 individual cells for binary classification in mixed samples and 3,424 cells for a multi-class classification task) and trained a convolutional neural network (CNN) to classify cell types based on this label-free AF as input. A low-complexity squeezeNet architecture was able to achieve reliable immune cell classification results (0.89 ROC-AUC, 0.95 PR-AUC, for binary classification in mixed samples; 0.689 F1 score, 0.697 precision, 0.748 recall, and 0.683 MCC for six-class classification in isolated samples). Perturbation tests confirmed that the model is not confused by extracellular environment and that both input AF channels (NADH and FAD) are about equally important to the classification. In the future, such predictive DL models could directly detect specific immune cells in unstained images and thus, computationally improve the specificity of label-free MPM which would have great potential for in vivo endomicroscopy.