Optical probes that can be used to measure certain quantities with subcellular resolution give us access to a new level of information at which physics, chemistry, life sciences, and medicine become strongly intertwined. The emergence of these new technologies is owed to great advances in the physical sciences. However, evaluating and improving these methods to new standards requires a joint effort with life sciences and clinical practice. In this Account, we give an overview of the probes that have been developed for measuring a few highly relevant parameters at the subcellular scale: temperature, pH, oxygen, free radicals, inorganic ions, genetic material, and biomarkers. Luminescent probes are available in many varieties, which can be used for measuring temperature, pH, and oxygen. Since they are influenced by virtually any metabolic process in the healthy or diseased cell, these quantities are extremely useful to understand intracellular processes. Probes for them can roughly be divided into molecular dyes with a parameter dependent fluorescence or phosphorescence and nanoparticle platforms. Nanoparticle probes can provide enhanced photostability, measurement quality, and potential for multiple functionalities. Embedding into coatings can improve biocompatibility or prevent nonspecific interactions between the probe and the cellular environment. These qualities need to be matched however with good uptake properties, colloidal properties and eventually intracellular targeting to optimize their practical applicability. Inorganic ions constitute a broad class of compounds or elements, some of which play specific roles in signaling, while others are toxic. Their detection is often difficult due to the cross-talk with similar ions, as well as other parameters. The detection of free radicals, DNA, and biomarkers at extremely low levels has significant potential for biomedical applications. Their presence is linked more directly to physiological and clinical manifestations. Since existing methods for free radical detection are generally poor in sensitivity and spatiotemporal resolution, new reliable methods that are generally applicable can contribute greatly to advancing this topic in biology. Optical methods that detect DNA or RNA and protein biomarkers exist for intracellular applications, but are mostly relevant for the development of rapid point-of-care sample testing. To elucidate the inner workings of cells, focused multidisciplinary research is required to define the validity and limitations of a nanoparticle probe, in both physical and biological terms. Multifunctional platforms and those that are easily made compatible with conventional research equipment have an edge over other techniques in growing the body of research evidencing their versatility.