Imaging is a central method in life sciences, and the drive to extract information from microscopy approaches has led to methods to fluorescently label specific cellular constituents. However, the specificity of fluorescent labels varies, labeling can confound biological measurements, and spectral overlap limits the number of labels to a few that can be resolved simultaneously. Here, we developed a deep learning computational approach called “in silico labeling (ISL)” that reliably infers information from unlabeled biological samples that would normally require invasive labeling. ISL predicts different labels in multiple cell types from independent laboratories. It makes cell type predictions by integrating in silico labels, and is not limited by spectral overlap. The network learned generalized features, enabling it to solve new problems with small training datasets. Thus, ISL provides biological insights from images of unlabeled samples for negligible additional cost that would be undesirable or impossible to measure directly.