Histological Methods to Determine Blood Flow Distribution with Fluorescent Microspheres

Abstract

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lee's stain or Richardson's stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Visualizing Regional Myocardial Blood Flow in the Mouse

“…Although our study was performed in an ex vivo heart preparation, the microsphere methods can be easily applied to living mice, other organs, and physiological conditions. We used an Imaging Cryomicrotome, 11 but a number of different approaches, [34][35][36] including paraffin-embedded histological methods, 37 can be used to count and determine the locations of fluorescent microspheres within organs. Investigators willing and able to dissect mice organs into multiple regions and then use established methods to count the microspheres 38,39 can now be confident in using samples with <400 microspheres per piece.…”

Gelatin‐embedded cell–polymer constructs for histological cryosectioning

“…Unlike water-soluble embedding media, gelatin is meant to remain in the section after histological processing, acting as an exogenous biological component of the sample. Gelatin serves this role well, and has been used in other histological applications requiring greater specimen continuity, such as brain tissue, 11,12 tissues perfused with fluorescent microspheres, 13 ultrathin sections of cells for immunocytochemistry, 14 -16 and whole mounts of developing embryos. 17,18 As shown in Table I, gelatin-embedded samples can also be sectioned reproducibly, with the critical advantage of remaining adherent to the slides afterwards.…”

Characterization of steady-state fluorescence properties of polystyrene latex spheres using off- and online spectroscopic methods

“…Commercially available microspheres are widely used as tools in numerous scientific research disciplines (e.g., DNA hybridization probes, as tracers for blood flow and neuronal pathways), diagnostics (e.g., immunoassays), and size calibrations (e.g., flow cytometry and microscope calibration) (e.g., Härmä et al, 2000;Hiesinger et al, 2001;Katz and Iarovici, 1990;Luchtel et al, 1998;Schwartz et al, 1998;Spiro et al, 2000). Fluorescent polystyrene latex spheres (PSLs) play a particularly important role in the characterization and calibration of instruments that rely on particle autofluorescence (also called intrinsic fluorescence) detection.…”

“…The main principle common to these techniques is the detection of intrinsic fluorescence from fluorophores such as amino acids, coenzymes, vitamins, and pigments that ubiquitously occur in aerosols of biological origin (e.g., Hill et al, 2009;Pan et al, 2010;Pöhlker et al, 2012Pöhlker et al, , 2013. These PBAP represent a diverse and dynamic subset of airborne particles, consisting of whole organisms like bacteria, viruses, archaea, algae, fungi, and related reproductive units (e.g., pollen, bacterial and fungal spores), as well as decaying biomass and fragments from plants or insects (e.g., Deepak and Vali, 1991;Després et al, 2012;Jaenicke, 2005;Madelin, 1994;Pöschl, 2005). They are ubiquitous in the Earth's atmosphere, where they affect many environmental mechanisms and, therefore, represent an important link between ecosystem activities and atmospheric processes (e.g., Andreae and Crutzen, 1997;Després et al, 2012;Fröhlich-Nowoisky et al, 2016;Fuzzi et al, 2006;Huffman et al, 2013;Möhler et al, 2007;Morris et al, 2014).…”