microscopy

Song Biotechnologies, LLC provides the research community direct access to the highest level of in vivo microcirculatory analysis through trans- and epi-illumination techniques. With cutting edge phosphorescence and fluorescence microscopy, our in vivo microcirculatory capabilities include measuring interstitial and intravascular oxygenation, blood flow, endothelial dysfunction, tracking of nano molecules, and much more. Additionally, our setup is configurable to accommodate different experimental animal models and parameters of interest.

Microcirculatory preparations are visualized by trans-illuminated bright field microscopy, which allows for real-time viewing and recording of vascular beds in stunning detail. This striking imagery is produced from thin tissue preparations, such as the rat spinotrapezius muscle, and coupled with top-of-the-line optics from Carl Zeiss and ultra-high-definition (4K) cameras capable of recording at over 90 frames per second.

Investigating the microcirculation - which includes arterioles, capillaries, and venules - is powerful for several reasons, but mainly because it lies as a focal point between whole animal/organ physiology and the sub-cellular and biochemical arena. It is where peripheral blood flow resistance is determined, the majority of gas exchange occurs, and leukocytes infiltrate the peri-vasculature during an acute-phase immunological response. When combined with standard investigational parameters such as cardiovascular workups, blood chemistry and/or molecular bioassays, microcirculatory dynamics provide a complete experimental investigation.

Arterioles are the major resistance vessels contributing to systemic blood pressure. Monitoring changes in their diameters, i.e., dilation or constriction, provides vital information for studies of vasoactivity. In some instances, vasoactive agents are either topically applied or infused intravascularly to determine physiological intactness, while in other studies i.e., hemorrhagic shock and resuscitation, diameter changes are correlated to changes in cardiac function. Furthermore, arterioles are also well suited to measuring rheology (blood flow), intra-vascular oxygen tension (oxygen delivery or oxygen-carrying capacity), shear stress, and much more.
Venules are the primary vessels from which activated leukocytes leave the bloodstream to invade tissues as part of the inflammatory process. Investigating the venules during this activation permits for the visualization of leukocyte rolling and adhesion, which sometimes results in an occlusion that redirects blood flow. Furthermore, venules also provide a post-capillary luminal oxygen tension sample for a good approximation of tissue oxygen extraction (VO2).
Capillaries are the primary point of gas exchange, and their functional density is a significant indicator of tissue oxygen delivery. Being the smallest and leakiest vessels, they are also excellent sites for measuring occlusive or extravasative dysfunction.
Compared to brightfield microscopy, where objects are subjected to external illumination and visualization is a function of the heterogeneous transmissive and absorptive properties of samples, fluorescence microscopy allows select targets to stand out against a dark background. Fluorescent probes bind to and concentrate on specific cells, structural elements, or infusates as designed. A brief or continuous excitation pulse causes them to emit a specific wavelength of light that is captured through an optical filter allowing for visual localization and quantification of experimental interests, colocalization of intervention and targets, and tissue and cell type discrimination in real-time.

Our Carl Zeiss microscope is equipped with the cutting-edge Zeiss Axiocam 702 fluorescence high-definition camera that records at speeds up to 128 frames per second. Under the control of the Zeiss Zen II software, rapid changes to biological or experimental target concentrations and distributions can be captured and quantified. Beyond the science, the coupling of high sensitivity hardware, optics, and software produces stunning videos of microvascular dynamics.

Fluorescence microscopy is also useful for tracking nano- and microparticle transit within and between biological compartments. Quantifying changes in perivascular fluorescence allows for the determination of nano-therapeutic circulatory retention rates and extravasative properties. Fluorescent particles can also measure blood flow, endothelial dysfunction, glycocalyx degradation, and more.
Phosphorescence quenching is an established technique for the non-invasive measurement of oxygen in biological systems. Thin fiber optic leads, tissue-diffusible probes, and barrier films allow for robust sampling of in vitro, ex vivo, and in vivo preparations with single or multiple channels. When combined with our advanced Carl Zeiss microscopy platform, real-time monitoring of oxygen delivery (DO2) dynamics in physiologically intact and living preparations becomes possible with extreme precision.

The measurement of oxygen, which is reported in mmHg as partial pressure (PO2), is facilitated by a diffusible palladium porphyrin phosphorescence probe. Bound to albumin, it does not transit tissue, organ, or vascular barriers permitting compartmental specificity. In vivo, exposed preparations are treated topically to report the PO2 outside the blood vessels, or an intravascular infusion can report blood PO2 from any perfused location. Measurement sites are covered with a transparent, oxygen-impermeable membrane to prevent desiccation and atmospheric oxygen contamination.
Our Carl Zeiss microscope is outfitted with high numerical aperture objectives, a monochromatic excitation laser, an ultra-sensitive Hamamatsu photomultiplier tube, and tailored signal conditioning to provide optimal signal quality and clarity. Site localization and sampling occur with single micrometer resolution allowing for the determination of oxygen gradients across capillaries and individual muscle fibers. Data are collected and digitized with a National Instruments data acquisition (DAQ) system with a temporal resolution of two million samples per second. When the probe is diffused into the interstitium and combined with topical pneumatic compression to arrest blood flow, PQM allows for discrete measurements of oxygen consumption (VO2) with excellent temporal and spatial resolution.
The phosphorescence probe is diffusible into the interstitial fluid (fluid space between cells) and injectable into the vasculature rendering all accessible or perfused tissues and organs suitable for measurement. Fiber optic leads are placed proximal to the target tissue and organ, which, if surgically exposed, is isolated from atmospheric contamination with an oxygen impermeable barrier film. Measurement resolution is one millimeter with a temporal resolution of 10Hz. Multiple tissues and organs can be measured simultaneously to provide an overview of localized systemic responses to experimental intervention.

additional capabilities

In vivo experimentation provides a customizable platform for robust data collection. Continuous measurements of animal physiology paired with discrete biomarker sampling work to create an elegant description of how experimental intervention affects systems biology from the macro to the micro-scale.

Physiology is the study of how biological systems function and their communication with other systems. It is more holistic than reductionist and applies to the organ, tissue, and cellular levels. It reveals the synergistic impact of experimental intervention on the normal operation of these systems and provides a top-down guide to the specific mechanisms involved.

The BIOPAC MP150 physiological data acquisition (DAQ) system provides real-time monitoring and cataloging of physiological parameters. The system is capable of collecting 500,000 samples per second, which is essential in ECG (electrocardiogram) and EEG (electroencephalography) studies. Cardiovascular monitoring, spirometry, temperature, electrical conduction, and more are some of the fundamental measurements that gauge the overall physiological response to experimentation.
Long-term studies often require animals to operate in their healthy habitat without external intervention. Implantable sensors allow for wireless cardiovascular, metabolic, biopotential, and thermal monitoring during these observation periods. By eliminating or reducing the use of anesthesia, handling and invasive instrumentation, stress levels that might affect readings and normal circadian cycles are significantly reduced.

Blood, fluid, and cellular micro-sampling during experimentation help resolve the development of transcriptional, translational, and cell-to-cell signaling processes. Endpoint harvest and post-processing of tissues, organs, and fluids allow for focused and global assessments of experimental outcomes.

The ABL90 Flex analyzes small samples (65 µL) to produce a comprehensive electrolyte, metabolic, oximetric, and hemoglobin profile. Measurement time is approximately one minute, allowing for tight temporal resolution of rapid changes to animal physiology. Each sample equates to 0.3 % of a rat’s total blood volume, meaning multiple samples can be assessed even in situations (trauma) where blood volume is a critical variable.
The ROTEM Delta® is used in hospitals to provide rapid assessments and characterization of various coagulopathies, which means the data provided are eminently translatable. In the research setting, the ROTEM tracks the development of abnormal coagulation statuses and the efficacy of resolution efforts. The contributions of both extrinsic and intrinsic coagulation pathways can be teased apart for greater mechanistic understanding.
The STA Compact Max® is a clinical-grade hemostatic analyzer for coagulation status and pathological characterization. Clotting, chromogenic, and immunologic measurements provide a detailed breakdown of clotting factors and constituents in terms of concentrations and activities. It provides a critical perspective in tailoring treatments to situations of trauma-induced coagulopathy.

longitudinal studies

Song Biotechnologies, LLC provides the research community direct access to the highest level of in vivo microcirculatory analysis through trans- and epi-illumination techniques. With cutting edge phosphorescence and fluorescence microscopy, our in vivo microcirculatory capabilities include measuring interstitial and intravascular oxygenation, blood flow, endothelial dysfunction, tracking nanomolecules, and much more. In addition, our setup is configurable to accommodate different experimental animal models and parameters of interest.

Sterile surgical interventions include, but are not limited to: dermal wounding, ischemia/reperfusion injury, organ infarct, and biomedical device implantation. Animals are then monitored and sampled according to the experimental schedule.
Basic toxicology studies where a test agent is introduced into a specific animal/transgenic model coupled with end-point analysis of target tissues and organs.
These studies extend toxicology studies to focus on pertinent biometrics to the test agent and help establish dose-response relationships to animal physiology. Assessment techniques can be found throughout our core capabilities and will be packaged with the intent of the research having translational potential.
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