There are many diseases and biological processes that involve circulating cells in the bloodstream, such as cancer metastasis, immune reaction/inflammation, reproductive medicine, and stem cell therapies. This has driven significant interest in new technologies for the study of circulating cells in small animal research models and clinically. Most currently used methods require drawing and enriching blood samples from the body, but these suffer from a number of limitations. In contrast, “in vivo flow cytometry” (IVFC) refers to set of technologies that allow study of cells directly in the bloodstream of the organism in vivo. In recent years the IVFC field has grown significantly and new techniques have been developed, including fluorescence microscopy, multi-photon, photo-acoustic, and diffuse fluorescence IVFC. In this paper we review recent technical advances in IVFC, with emphasis on instrumentation, contrast mechanisms, and detection sensitivity. We also describe key applications in biomedical research, including cancer research and immunology. Last, we discuss future directions for IVFC, as well as prospects for broader adoption by the biomedical research community and translation to humans clinically.

Nanomaterials are of enormous value for biomedical applications because of their customizable features. However, the material properties of nanomaterials can be altered substantially by interactions with tissue thus making it important to assess them in the specific biological context to understand and tailor their effects. Here, a genetically controlled system is optimized for cellular uptake of superparamagnetic ferritin and subsequent trafficking to lysosomes. High local concentrations of photoabsorbing magnetoferritin give robust contrast in optoacoustic imaging and allow for selective photoablation of cells overexpressing ferritin receptors. Genetically controlled uptake of the biomagnetic nanoparticles also strongly enhances third-harmonic generation due to the change of refractive index caused by the magnetiteprotein interface of ferritins entrapped in lysosomes. Selective uptake of magnetoferritin furthermore enables sensitive detection of receptor-expressing cells by magnetic resonance imaging, as well as efficient magnetic cell sorting and manipulation. Surprisingly, a substantial increase in the blocking temperature of lysosomally entrapped magnetoferritin is observed, which allows for specific ablation of genetically defined cell populations by local magnetic hyperthermia. The subcellular confinement of superparamagnetic ferritins thus enhances their physical properties to empower genetically controlled interrogation of cellular processes with deep tissue penetration.The interest in overexpressed ferritin has recently been revived in attempts to create biomagnetic actuators aimed at evoking controlled cellular responses to magnetic fields. In this regard, ferritin was used to manipulate cellular processes via magnetic hyperthermia or magnetomechanical transduction, [4] although the biophysical mechanisms underlying these effects are so far not understood well on a theoretical level. [5] Fully genetically encoded sensors and actuators, which do not necessitate supplementation of synthetic components, have become the frequently preferred solutions for, e.g., fluorescent Ca 2+ imaging and optogenetics. However, with regard to noninvasive techniques with deep tissue penetration such as optoacoustic (OA) imaging or MRI, synthetic nanostructures often still possess superior material properties as compared to genetically encodable biomaterials.Semigenetic approaches, which consist of a genetic component that interacts with an exogenous compound, can exploit the superior physical properties of synthetic nanostructures for remote cell actuation and deep tissue imaging and at the same time benefit from genetic targetability. [6] A recent study showcased the use of ferritin in a semigenetic approach to obtain cellular MR-contrast. [7] It was hypothesized that the enhanced contrast resulted from ferritin agglomeration inside lysosomes after cellular uptake through murine T cell immunoglobulin domain and mucin domain 2 (Tim-2) receptor. This contrastenhancing effect could be exploited even more effectively if the...

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