Functional imaging of enzyme activity in living systems provides valuable insights into biological processes and disease states [1]. While in vitro experiments can be used to make fundamental observations using simplified models, in vivo imaging provides information regarding the regulation of enzyme activity in more complex and therapeutically relevant models of human health and disease [2]. In vivo imaging experiments can provide detailed biological information about enzyme function at various levels, including tissues, organs, systems, and whole organisms. Additionally, in vivo imaging methods developed for clinical use can provide activity-based measurements of disease state and progression that can be used for diagnostics, therapy monitoring and surgical guidance [3].

Molecular imaging tools for a variety of imaging modalities have enabled non-invasive analysis of enzyme activity in vivo. In particular, enzyme probes suitable for use in magnetic resonance imaging (MRI) [4,5] have high spatial resolution and unrestricted depth penetration in tissues [6]. However, due to the low sensitivity of MRI imaging, enzymes of interest must have fast kinetics and/or high local concentrations to be detected by probes [7]. Enzyme-responsive positron emission tomography and single-photon emission computed tomography probes are also highly sensitive and provide imaging through any tissue depth [8]. Yet, the time and resources required for isotope production and/or on demand probe synthesis limits their use outside of specialized facilities. Further, radiation exposure can limit applications in longitudinal imaging. Recent advances in ultrasound probes that respond to enzyme activity show promise for diverse imaging applications in deep tissues within the vascular and gastrointestinal systems [9].

Optical imaging agents can provide sensitive, molecular-level information about enzyme activity at low cost, using safe and non-ionizing wavelengths of light [10]. There have been a wide range of fluorescent tools, including “always-on” targeted probes, and “turn-on” probes, developed for monitoring enzyme activity [11]. However, in vivo imaging requires measurements of signals within deep tissues, posing challenges for optical imaging agents which have limited penetration depth [12]. These challenges include variable probe distribution among organs and tissues, differences in cellular uptake and permeability, inconsistent incident illumination, and impact of biological environments on overall signal intensity. Optical tomography methods rely on estimations of scattering and absorption to provide quantitative volumetric measurements but cannot exclude signal from probes taken up in healthy tissues. Thus, obtaining quantitative data for both biological and clinical applications is more challenging in vivo compared to live cell or ex vivo imaging methods [13].

Ratiometric imaging is a benchmarking strategy that involves recording and analyzing two linked signals as a ratio. Ratiometric imaging can overcome challenges associated with quantifying optical probe signals in vivo, as ratiometric signals can be normalized to eliminate effects from dosing, illumination variability, and pharmacokinetics. In this review, we explore recent strategies and applications of in vivo ratiometric imaging probes. These strategies include using caging groups which change the photophysical properties of a single chromophore upon enzymatic cleavage (Figure 1a), using two fluorophores that interact photophysically, such as through Förster resonance energy transfer (FRET)), that respond to enzymatic processing (Figure 1b) and using a single fluorophore that can be benchmarked to a separate static fluorescent signal (Figure 1c). Here, we describe probes with a change in either excitation or emission wavelengths upon enzyme processing that enable ratiometric readout. However, fluorescence lifetime is another parameter that can be modulated by enzyme activity [14]. Recently, fluorescent lifetime-based ratiometric imaging methods such as, macroscopic fluorescence lifetime imaging have been developed for applications including drug-target engagement studies [15].

The studies highlighted here provide insights for the development of robust and widely applicable probes to enable longitudinal tracking of enzyme activity in vivo. We focus on recent examples of fluorescent, ratiometric optical imaging probes developed and applied in preclinical in vivo models. Readers are encouraged to refer to existing work, particularly reviews on chemical probes for ratiometric fluorescence imaging [16,17] and optical imaging of enzyme activity [18, 19, 20] for more comprehensive overviews of the surrounding fields.