Microscopy
Song Biotechnologies provides detailed, in vivo microcirculatory analysis through trans- and epi-illumination modalities. With cutting edge phosphorescence and fluorescence microscopy, we can monitor interstitial and intravascular oxygenation, blood flow, endothelial barrier function, nano molecules distribution, and more. Additionally, our setups can be adapted to various experimental animal models and instruments.
Intravital
Microcirculatory preparations are visualized by trans-illuminated bright field microscopy, which allows for real-time viewing and recording of vascular beds in stunning detail. Images and videos are 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 cameras capable of recording at over 90 frames per second.
The microcirculation, which includes arterioles, capillaries, and venules, connects organ systems to cellular biochemical processes. It determines blood flow resistance, facilitates gas exchange, and allows leukocyte infiltration during immune responses. Combined with cardiovascular assessments and molecular bioassays, studying microcirculatory dynamics offers a comprehensive analysis.
Arterioles are key resistance vessels affecting systemic blood pressure. Monitoring their diameter changes, such as dilation or constriction, is crucial for studying vasoactivity. Vasoactive agents may be applied topically or infused to check physiological responses, while in studies like hemorrhagic shock and resuscitation, diameter changes correlate with cardiac function. Arterioles are also ideal for measuring blood flow, intra-vascular oxygen tension, shear stress, and more.
Venules are the main vessels where activated leukocytes exit the bloodstream to enter tissues during inflammation. Studying post-capillary venules allows observation of leukocyte rolling and adhesion. 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 (perfusion) is a significant indicator of tissue oxygen delivery and tissue/organ function. Being the smallest and leakiest vessels, they are also excellent sites for measuring occlusive dysfunction and large particle extravasation.
Fluorescence
Fluorescence microscopy allows for selective and specific illumination of biological structures and molecules using molecular probes and optical filtration. Unlike brightfield microscopy, where tissues are illuminated broadly and structures are visualized based on non-specific absorptive and transmissive properties, fluorescence emission and capture are fine-tuned to highlight specific elements against a dark background. Measurements of localization, morphology, quantification, and tissue/cell/macromolecule discrimination are achieved with high temporal resolution throughout experiments.
Our Carl Zeiss microscope is equipped with the Zeiss Axiocam 702 fluorescence high-definition camera, capable of recording at speeds up to 128 frames per second. Controlled by the Zeiss Zen II software, it captures and quantifies rapid changes in biological or experimental target concentrations and distributions. Additionally, the combination of sensitive hardware, optics, and software generates detailed videos of microvascular dynamics.
Fluorescence microscopy is also beneficial for tracking nano- and microparticle movement within and between biological compartments. By quantifying changes in perivascular fluorescence, it is possible to determine nano-therapeutic circulatory retention rates and extravasative properties. Fluorescent particles can also be used to measure blood flow, endothelial function, glycocalyx integrity, and more.
Phosphorescence Quenching
Phosphorescence quenching is an established technique for the non-invasive measurement of oxygen in biological systems. Precision optics, 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 high resolution.
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 remains confined to tissue, vascular, and organ compartments. 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 equipped with high numerical aperture objectives, a monochromatic excitation laser, an ultra-sensitive Hamamatsu photomultiplier tube, and customized signal conditioning to provide superior signal quality and clarity. Site localization and sampling are conducted with micrometer precision, enabling the determination of oxygen gradients across capillaries and individual muscle fibers. Data collection and digitization are handled by a National Instruments data acquisition (DAQ) system capable of processing two million data points per second. When the probe diffuses into the interstitium and is combined with topical pneumatic compression to halt blood flow, PQM facilitates accurate measurements of oxygen consumption (VO2) with exceptional temporal and spatial resolution.
This technology uses high-sensitivity signal processing from PQM with fiber optic leads for multi-site measurements in larger animals. The phosphorescence probe can be diffused into interstitial fluid and injected intravascularly, making all accessible tissues and organs measurable. The fiber optic leads are then placed near the target tissue or organ, which, if exposed surgically, is protected from atmospheric contamination by an oxygen-impermeable film. Measurements have a one-millimeter resolution at 10Hz. Multiple tissues and organs can be measured simultaneously to observe localized systemic responses to experimental interventions.