DNA Vector Polyethyleneimine Affects Cell pH and Membrane Potential: A Time-Resolved Fluorescence Microscopy Study

Polyethyleneimine (PEI) is one of the very efficient nonviral vectors being developed and tested for artificial gene transfer into target cells. One of its serious limitations is the significant cytotoxicity of the large amounts of free PEI in the mixtures of DNA and PEI used for transfection. To further investigate the cellular effects of free PEI, we have analyzed the PEI-induced alterations of various cell parameters such as membrane heterogeneity and fluidity, cytoplasmic pH, and plasma membrane potential in a variety of cells such as Swiss 3T3 fibroblast, Chinese hamster ovary, insect cells SF9, plant cell line BY2, and Saccharomyces cerevisae. Fluorescence probes such as Nile red, SNARF-1, and cyanine dye DiSC₂(3), coupled with the technique of picosecond time-resolved fluorescence microscopy, were used in estimating the above-mentioned cell parameters. It was found that the cell membranes were largely unperturbed by PEI. However, the cytoplasmic pH showed an increase of 0.1–0.4 units when the cells were treated with PEI. The plasma membrane potential was found to be depolarized in S. cerevisae and Swiss 3T3 cells. These results suggest that the cytotoxic effects of PEI may partly originate from inhibition of regulation of cytoplasmic pH and plasma membrane potential. Further, it is proposed that the resultant cell alterations favors the transfection process.     

In vivo and in vitro study of porcine oviductal epithelial cells, cumulus oocyte complexes and granulosa cells: a scanning electron microscopy and inverted microscopy study

The morphology and structure of porcine oviductal epithelial cells (POEC), cumulus-oocyte complexes (COCs) and granulosa cells (GC) were investigated in vivo and in vitro conditions using scanning electron microscopy (SEM) and inverted microscopy. The POEC contained columnar ciliated cells and spherical shaped non-ciliated cells. Both non- and ciliated cells appeared either in groups or distributing among each other. However, the isolation of cells was observed after culture for 48 h. A total of 921 oocytes from 20 ovaries was isolated resulting in an average of 46 oocytes per ovary. They were round in shape, surrounded by zona pellucida with layers of cumulus cells ranging between 89.16 and 144.68 μm in size. As for COCs, they were classified into 4 types; intact-, multi-, partial-cumulus cell layers and completely denuded oocyte. Interestingly, changes in morphology of COCs with intact and multi-cumulus cell layers were observed in the in vitro study. The GCs in the follicular fluid were also round in shape and found as clusters. After culturing in in vitro for 48 h, no change in morphology was observed. The GC appeared in smaller clusters or were present as single cells and their sizes ranged from 6 to 8 μm. The results obtained from this study allow us to have a better understanding of the morphology and nature of cells under both in vivo and in vitro conditions. This information is also important for the study of their secretions and biochemical compositions, which is of great importance to the use of cells as feeder cells in in vitro fertilization in current studies.     

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca²⁺-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca²⁺ concentration image using anin vitro-determined calibration curve. The Ca²⁺ concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

Fluorescent Probe ABM and Estimation of Immune State in Patients with Different Pathologies (Review Article)

The fluorescent probe ABM (3-aminobenzanthrone derivative) one of the fluorescent probes synthesized in Riga Technical University proved to be an excellent, independent model for studying cell membranes. In our work we have investigated the possibility of using the fluorescent probe ABM for detection of immune state in patients with different pathologies. There is a strong correlation among all studied ABM spectral parameters, immunological characteristics, clinical and laboratory investigations of the all observed patients groups. The obtained results suggest that ABM spectral parameters in cell suspension reflect the alterations of the cellular mechanisms of immunity. Therefore fluorescent method could be used as preliminary screening test in immune diagnostics instead of more expensive, time consuming methods (subset detection, radioisotope method etc.) used as routine in clinics. Spectral parameters of ABM reflect a wide range of interrelated (interdependent) characteristics of cells (physico-chemical state and microviscosity of membrane, proliferating and lipid metabolic activity of cells, distribution of cells among subsets). The observed change of the studied parameters reflects alterations of the cellular mechanisms of immunity which is a main focus for its application as preliminary screening test in immune diagnostics. The fluorescence based method is sensitive, less expensive and time consuming, technically simple and convenient.     

Fluorescence lifetime imaging in biosciences: technologies and applications

The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging techniques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved techniques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.      

Antifading embedding media in confocal immunoflourescence microscopy

Fading or bleaching of fluorescence intensity during continuous illumination of stained objects is a serious problem in fluorescence microscopy. Fluorescence intensity as well as bleaching characteristics of dyes are dependent primarily upon physical parameters such as molecular constants (absorption rate and quantum efficiency), excitation energy and brightness (causes photon saturation), and environmental parameters (pH, ions, binding to proteins, etc.) that can strongly influence the properties of fluorochrome molecules. We have studied the effect of various antifading reagents on the behavior of the common dyes fluorescein isothiocyanate (FITC) and phycoerythrin (PE) using immunofluorescent-stained living cells in suspension or membrane-permeabilized dried cells as test systems. As expected, fading cannot be completely eliminated but may be reduced to varying degrees. In our hands, the most efficient antifading reagent for FITC isn-propyl gallate (NPG) dissolved in glycerol. No additive was found to retard fading, but complete dehydration of the cell suspension reduces this effect.     

Raman spectroscopy and microscopy of individual cells and cellular components

Raman spectroscopy provides the unique opportunity to nondestructively analyze chemical concentrations in individual cells on the submicrometer length scale without the need for optical labels. This enables the rapid assessment of cellular biochemistry inside living cells, and it allows for their continued analysis. Here, we review recent developments in the analysis of single cells, subcellular compartments, and chemical imaging based on Raman spectroscopy. Spontaneous Raman spectroscopy provides for the full spectral assessment of cellular biochemistry, while coherent Raman techniques, such as coherent anti‐Stokes Raman scattering is primarily used as an imaging tool comparable to confocal fluorescence microscopy. These techniques are complemented by surface‐enhanced Raman spectroscopy, which provides higher sensitivity and local specificity, and also extends the techniques to chemical indicators, i.e. pH sensing. We review the strengths and weaknesses of each technique, demonstrate some of their applications and discuss their potential for future research in cell biology and biomedicine.     

Laser-induced autofluorescence for medical diagnosis

The naturally occurring autofluorescence of cells and tissues is based on biomolecules containing intrinsic fluorophores, such as porphyrins, the amino acids tryptophan and tyrosine, and the coenzymes NADH, NADPH, and flavins. Coenzymes fluoresce in the blue/green spectral region (fluorecence lifetimes: 0.5–6 ns) and are highly sensitive indicators of metabolic function. Steadystate and time-resolved blue-green autofluorescence is, therefore, an appropriate measure of the function of the respiratory chain as well as of cellular and tissue damage. Autofluorescence in the yellow/red spectral region is based mainly on endogenous porphyrins and metalloporphyrins, such as coproporphyrin, protoporphyrin (fluorescence lifetime of porphyrin monomers: >10 ns), and Zn-protoporphyrin (2 ns). Various pathological microorganisms such asPropionibacterium acnes, Pseudomonas aeruginosa, Actinomyces odontolyticus, Bacteroides intermedius, andSaccharomyces cerevisiae are able to synthesize large amounts of these fluorophores and can therefore be located. This permits fluorescence-based detection of a variety of diseases, including early-stage dental caries, dental plaque, acne vulgaris, otitis externa, and squamous cell carcinoma. The sensitivity of noninvasive autofluorescence diagnostics can be enhanced by time-gated fluorescence measurements using an appropriate time delay between ultrashort laser excitation and detection. For example, videocameras with ultrafast shutters, in the nanosecond region, can be used to create caries images of the teeth. Alternatively, autofluorescence can be enhanced by stimulating protoporphyrin biosynthesis with the exogenously administered porphyrin precursor 5-aminolevulinic acid (ALA). The fluorophore protoporphyrin IX (PP IX) is photolabile and photodynamically active. Irradiation of PP IX-containing tissue results in cytotoxic reactions which correlate with modifications in fluorescence due to photobleaching and singlet oxygen-dependent photoproduct formation. Therefore, on-line autofluorescence measurements during the phototreatment can yield information on the efficiency of ALA-based photodynamic therapy.     

Synthesis of novel fluorescently labeled water-soluble fullerenes and their application to its cellar uptake and distribution properties

Fullerene is a well-known carbon nanomaterial, which can be potentially used for drug manufacture or delivery. Despite several successful examples of utilizing fullerene derivatives as drug candidate materials, their low water solubility under physiological conditions negatively affects the cell penetration efficiency after treatment. In this work, we successfully synthesized two fullerene derivatives with covalently attached fluorescein and boron dipyrromethene (BODIPY) fluorophore moieties, which exhibited cellular uptake and intracellular localization. While both fluorophores decreased their fluorescence intensity in the vicinity of fullerene, the cellar uptake of the fluorescein-modified fullerene was detected via fluorescence microscopy observations. Moreover, decreases in the fluorescence intensities of the intact fluorescein and BODIPY species were observed when both fluorophores and fullerene coexisted in aqueous media.     

Y2O3:Tm,Yb nanophosphors for correlative upconversion luminescence and cathodoluminescence imaging

We present a phosphor nanoparticle that shows both upconversion luminescence (UCL) and cathodoluminescence (CL). With this particle, low-autofluorescence, deep-tissue and wide-field fluorescence imaging can be achieved with nanometer-order high-spatial-resolution imaging. We synthesized Y₂O₃:Tm,Yb nanophosphors that emit visible and near-infrared UCL under 980 nm irradiation and blue CL via electron beam excitation. The phosphors were applied to fluorescent imaging of HeLa cells. The photostability of the phosphors was superior to that of a conventional organic dye. We show that after uptake by HeLa cells, the particles can be imaged with SEM and CL contrast in a cellular section. This indicates that correlative UCL and CL imaging of biological samples could be realized.     

Measurement of Light and pH Dependence of Single-Cell Photosynthesis by Fluorescence Microscopy

Measurement of algal photosynthetic performance with conventional methods requires thousands of cells obtained by isolation and subsequent cultivation. This is a time-consuming process for many species. We describe a new method to study photosynthetic performance of single algal cells under various environmental conditions by a combination of modulated chlorophyll fluorescence, light microscopy, and sample manipulation techniques. Single cell fluorescence was measured with a modulated microfluorometer integrated in an inverted microscope. The algal cell was sucked onto the tip of a glass microcapillary and positioned in the center of the field of view of the microscope by a micromanipulator. A superfusion device was used to generate a flow of experimental solution of variable composition along the alga. The light dependence of Scenedesmus obtusiusculus single-cell photosystem II (PSII) electron flow was measured at various pH. At a high light intensity PSII electron flow was inhibited at pH 6.5 and higher, while at a low light inhibition occurred at pH 9.5. This is in agreement with inhibition of photosynthesis by substrate (CO₂) limitation at alkaline pH. This approach can easily be extended to study the in vivo effects of other abiotic parameters (temperature, nutrients, toxicants, oxygen) on the photosynthetic performance of algae.     

New insights into plant cell walls by vibrational microspectroscopy

Vibrational spectroscopy provides non-destructively the molecular fingerprint of plant cells in the native state. In combination with microscopy, the chemical composition can be followed in context with the microstructure, and due to the non-destructive application, in-situ studies of changes during, e.g., degradation or mechanical load are possible. The two complementary vibrational microspectroscopic approaches, Fourier-Transform Infrared (FT-IR) Microspectroscopy and Confocal Raman spectroscopy, are based on different physical principles and the resulting different drawbacks and advantages in plant applications are reviewed. Examples for FT-IR and Raman microscopy applications on plant cell walls, including imaging as well as in-situ studies, are shown to have high potential to get a deeper understanding of structure–function relationships as well as biological processes and technical treatments. Both probe numerous different molecular vibrations of all components at once and thus result in spectra with many overlapping bands, a challenge for assignment and interpretation. With the help of multivariate unmixing methods (e.g., vertex components analysis), the most pure components can be revealed and their distribution mapped, even tiny layers and structures (250 nm). Instrumental as well as data analysis progresses make both microspectroscopic methods more and more promising tools in plant cell wall research.     

Electroporative Adjustment of pH in Living Yeast Cells: Ratiometric Fluorescence pH Imaging

A number of vital cell functions including modulation of signaling pathways and regulation of the cellular transport critically depends on the cytoplasmic pH. Many pathological cellular changes are related to the abnormal cytosolic pH as well. Reliable and well-calibrated methods for quantification of the cytosolic pH are therefore of high importance. The pH calibration is particularly difficult in walled cells since standard methods fail. In this report we evaluated the new electroporative calibration method of the cytosolic pH in yeasts by the fluorescence microscopy. The calibration was done on living cells using pyranine as a ratiometric pH-sensitive probe. The probe was electroporatively delivered to the cytosol. We have shown that unlike the measurements in suspension the fluorescence microscopy reveals cell subpopulations with different sensitivity to the pH calibration. While the majority of the cells were well calibrated, there was found subpopulation of uncalibrated cell as well as singular cells exhibiting anomalous pH calibration due to the staining of acidic organelles. Resolution of cell subpopulations helps to achieve better pH calibration compared to the calibration in cuvette on a cell suspension.     

Non‐invasive time‐course imaging of apoptotic cells by confocal Raman micro‐spectroscopy

Confocal Raman micro‐spectroscopy (CRMS) was used to measure time‐course spectral images of live cells undergoing apoptosis without using molecular labels or other invasive procedures. Human breast cancer cells (MDA‐MB‐231) were exposed to 300 µM etoposide to induce apoptosis, and Raman spectral images were acquired from the same cells at 2‐h intervals over a period of 6 h. The purpose‐built inverted confocal Raman micro‐spectrometer integrated an environmental enclosure and wide‐field fluorescence imaging. These key instrumental elements allowed the cells to be maintained under sterile physiological conditions (37 °C, 5% CO₂) and enabled viability and apoptosis assays to be carried out on the cells at the end of CRMS measurements. The time‐course spectral images corresponding to DNA Raman bands indicated an increase in signal intensity in apoptotic cells, which was attributed to DNA condensation. The Raman spectral images of lipids indicated a high accumulation of membrane phospholipids and highly unsaturated non‐membrane lipids in apoptotic cells. This study demonstrates the potential of CRMS for label‐free time‐course imaging of individual live cells. This technique may become a useful tool for in vitro toxicological studies and testing of new pharmaceuticals, as well as other time‐dependent cellular processes, such as cell differentiation, cell cycle and cell–cell interactions. Copyright © 2010 John Wiley & Sons, Ltd.     

Fabrication and characterization of ZnTPP:PCBM bulk heterojunction (BHJ) solar cells

Bulk heterojunction (BHJ) solar cells were fabricated based on blended films of a porphyrin derivative 5,10,15,20-Tetraphenyl-21H,23H-porphine zinc (ZnTPP) and a fullerene derivative [6,6]-phenyl-C₆₁ butyric acid methyl ester (PCBM) as the active layer. The ZnTTP:PCBM BHJ solar cells were fabricated by spin-casting of the blended layer. The weight ratios of ZnTPP and PCBM were varied from 1:1 to 0:10. The electronic and optical properties of each cell were investigated. Optical density (OD) of the blended film for each cell was extracted from its reflection and transmission curves. OD and average absorption coefficients of the active materials were used to determine film thicknesses. Absorption spectra of each component material were compared with the spectra of the blended films. Current density–Voltage (J–V) characteristics were recorded under dark as well as under the illumination of AM 1.5G (1 sun) solar spectrum. The BHJ solar cell with ZnTPP:PCBM ratio of 1:9 showed the best performance . The values of RR, V_OC , J_SC , FF and η for these ratios were 106.3, 0.4 V, 1.316 mA/cm², 0.4 and 0.21%, respectively. The cross-section of this device using SEM was also examined.     

A Correlative Analysis of Gold Nanoparticles Internalized by A549 Cells

Fluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (<100 nm) cannot be resolved by conventional light micro­scopy techniques. Especially after uptake into cells, a common fate of the fluorescence label and the particle core cannot be taken for granted. In this study, a correlative approach is presented to image fluorescently labeled gold nanoparticles inside whole cells by correlative light and electron microscopy (CLEM). This approach allows for detection of the fluorescently labeled particle shell as well as for the gold core in one sample. In this setup, A549 cells are exposed to 8 nm Atto 647N‐labeled gold nanoparticles (3.3 × 10⁹ particles mL^?1, 0.02 μg Au mL^?1) for 5 h and are subsequently imaged by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Eight fluorescence signals located at different intracellular positions are further analyzed by TEM. Five of the eight fluorescence spots are correlated with isolated or agglomerated gold nanoparticles. Three fluorescence signals could not be related to the presence of gold, indicating a loss of the particle shell.     

黄藤细胞壁木质素区域化学分子光谱成像研究

整合共聚焦显微荧光和拉曼光谱成像技术系统研究了黄藤藤茎组织中不同类型细胞以及同一细胞不同形态区域的木质素区域化学特点。共聚焦荧光成像表明黄藤藤茎组织中木质素主要汇聚于初生木质部导管、次生木质部导管、维管束间的薄壁组织细胞以及纤维细胞角隅区。基于荧光光谱差异的光谱成像线性拆分结果显示纤维细胞次生壁由宽、窄层交替的同心层状结构组成,且窄层具有更高的木质化程度。比较黄藤、毛竹、芒草、毛白杨和虎皮松拉曼光谱发现黄藤材细胞壁拉曼光谱与阔叶木毛白杨类似,证实了黄藤材的化学组成更加趋近于阔叶木毛白杨。对拉曼光谱中木质素特征峰成像进一步揭示出纤维细胞中木质素不均一的分布规律: 其中细胞角隅胞间层和复合胞间层的拉曼信号强度最高,表明较高的木质化程度,其次是次生壁中的窄层,而次生壁宽层中拉曼特征峰强度最低,这一分布规律与竹材纤维细胞中木质素分布规律类似。宽、窄层中木质素不仅存在浓度上的差异,而且木质素基本结构单元的比例亦不同。采取光谱去卷积的方法排除了碳水化合物的影响,发现窄层中愈创木基(G型)木质素与紫丁香基木质素(S型)比例为0.19,而在宽层中这一比值为0.14,这一结果亦解释了宽、窄层荧光光谱间的差异。该研究结果对探索黄藤细胞壁生物合成及力学响应机制研究具有重要理论指导意义。     

Measurement of Cell Volume Changes by Fluorescence Self-Quenching

At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (M) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.     

Morphological characterization via light and electron microscopy of Atlantic jackknife clam (Ensis directus) hemocytes

The Atlantic jackknife clam, Ensis directus, is currently being researched as a potential species for aquaculture operations in Maine. The goal of this study was to describe the hemocytes of this species for the first time and provide a morphological classification scheme. We viewed hemocytes under light microscopy (using Hemacolor, neutral red, and Pappenheim’s stains) as well as transmission electron microscopy (TEM). The 2 main types of hemocytes found were granulocytes and hyalinocytes (agranular cells). The granulocytes were subdivided into large and small granulocytes while the hyalinocytes were subdivided into large and small hyalinocytes. The large hemocytes had both a larger diameter and smaller nucleus to cell diameter ratio than their smaller counterparts. A rare cell type, the vesicular cell, was also observed and it possessed many vesicles but few or no granules. Using TEM, granulocytes were found to contain both electron-lucent and electron-dense granules of various sizes. These numerous granules were the only structures that took up the neutral red stain. Hyalinocytes had few of these granules relative to granulocytes. Large hyalinocytes had both various organelles and large vesicles in their abundant cytoplasm while small hyalinocytes had little room for organelles in their scant cytoplasm. Total hemocyte counts averaged 1.96 × 10⁶ cells mL⁻¹ while differential hemocyte counts averaged 11% for small hyalinocytes, 12% for large hyalinocytes, 59% for small granulocytes, and 18% for large granulocytes. The results of this study provide a starting point for future studies on E. directus immune function.