Intravital Microfluidic Windows for Delivery of Chemicals, Drugs and Probes

Abstract

Intravital imaging, the microscopic visualization of cellular events in living animals, is becoming a powerful tool for the study of many disease processes. The development of the mammary imaging window has been shown to be advantageous for high resolution imaging for the study of cell movements within an organism and can be used to allow the precise observation of cells and tumor angiogenesis for many days [1,2] [3]. A major disadvantage of intravital imaging, however, is that the imaging windows are typically sewn into the skin of the animal, and therefore, the imaging area is not amendable to chemical or drug treatments. The addition of local agents by syringe needle is problematic as injection can elicit unwanted wound responses that complicate elucidation of cellular-response pathways to both secreted factors and pharmacological agents. Systemic treatments can be harmful to the animal and reagents may not make their way to the area under observation. We have designed microfluidic glass windows that will alleviate these complications and provide real time spatial and temporal addition of chemical treatments to cells and tissues.Our system allows the careful titration of reagents to various tissues during imaging. While we can make micron-sized ports for setting up gradients, we can also make larger openings for the addition of various labeled cell types to watch their infiltration into a various organ or tissue. Such a system, for instance, would also allow the addition of controlled amounts of pathogens to visualize a site of infection and the immune response.With glass, it is possible to etch channels and ports down to a few hundred nanometers [4]. They are also durable, optically superior, easy to unclog and fill, and are reusable. Channels were fabricated using a laser ablation system which consists of a diode-pumped frequency doubled Neodymium-doped Yttrium Orthovanadate (Nd:YVO4) laser pump (Verdi), a Ti:Sapphire laser oscillator (Tsunami) and a regenerative amplifier (RegA9000). The substrate position was controlled by an Aerotech ANT95 nanopositioning system and is capable of carefully coordinating the alignment and positioning of the design features through software-controlled stages with nanometer precision.We have previously shown that we can make several chemotaxis devices in glass coverslips [5]. Here, we demonstrate the usefulness of intravital microfluidic windows by staining subcellular organelles in tissues, obtaining cellular responses by adding ligands, and have also set up chemical gradients, all in a living animal. We have imaged tissues and cells with epifluorescence and 2 photon microscopy. In addition to adding reagents, we can also potentially collect cells and effluent for histological and proteomic analysis during and after chemical treatments. 1352

Development and validation of a novel microfluidic device for the manipulation of skeletal muscle microvascular blood flow in vivo

“…There are few examples in the literature where microfluidic devices have been applied to study physiology in vivo 12 . At the time of writing, conceptualized microfluidic devices for studying microvascular blood flow have been proposed, 13 but to date, there have been no studies that apply this technology to deliver drugs to skeletal muscle in vivo.…”