The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and 8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

The use of high-frequency short bipolar pulses in cisplatin electrochemotherapy in vitro.

Background In electrochemotherapy (ECT), chemotherapeutics are first administered, followed by short 100 µs monopolar pulses. However, these pulses cause pain and muscle contractions. It is thus necessary to administer muscle relaxants, general anesthesia and synchronize pulses with the heart rhythm of the patient, which makes the treatment more complex. It was suggested in ablation with irreversible electroporation, that bursts of short high-frequency bipolar pulses could alleviate these problems. Therefore, we designed our study to verify if it is possible to use high-frequency bipolar pulses (HF-EP pulses) in electrochemotherapy. Materials and methods We performed in vitro experiments on mouse skin melanoma (B16-F1) cells by adding 1-330 µM cisplatin and delivering either (a) eight 100 µs long monopolar pulses, 0.4-1.2 kV/cm, 1 Hz (ECT pulses) or (b) eight bursts at 1 Hz, consisting of 50 bipolar pulses. One bipolar pulse consisted of a series of 1 µs long positive and 1 µs long negative pulse (0.5-5 kV/cm) with a 1 µs delay in-between. Results With both types of pulses, the combination of electric pulses and cisplatin was more efficient in killing cells than cisplatin or electric pulses only. However, we needed to apply a higher electric field in HF-EP (3 kV/cm) than in ECT (1.2 kV/cm) to obtain comparable cytotoxicity. Conclusions It is possible to use HF-EP in electrochemotherapy; however, at the expense of applying higher electric fields than in classical ECT. The results obtained, nevertheless, offer an evidence that HF-EP could be used in electrochemotherapy with potentially alleviated muscle contractions and pain.

In vivo imaging of neuronal calcium during electrode implantation: Spatial and temporal mapping of damage and recovery.

Implantable electrode devices enable long-term electrophysiological recordings for brain-machine interfaces and basic neuroscience research. Implantation of these devices, however, leads to neuronal damage and progressive neural degeneration that can lead to device failure. The present study uses in vivo two-photon microscopy to study the calcium activity and morphology of neurons before, during, and one month after electrode implantation to determine how implantation trauma injures neurons. We show that implantation leads to prolonged, elevated calcium levels in neurons within 150 µm of the electrode interface. These neurons show signs of mechanical distortion and mechanoporation after implantation, suggesting that calcium influx is related to mechanical trauma. Further, calcium-laden neurites develop signs of axonal injury at 1-3 h post-insert. Over the first month after implantation, physiological neuronal calcium activity increases, suggesting that neurons may be recovering. By defining the mechanisms of neuron damage after electrode implantation, our results suggest new directions for therapies to improve electrode longevity.

The second phase of bipolar, nanosecond-range electric pulses determines the electroporation efficiency.

Bipolar cancellation refers to a phenomenon when applying a second electric pulse reduces ("cancels") cell membrane damage by a preceding electric pulse of the opposite polarity. Bipolar cancellation is a reason why bipolar nanosecond electric pulses (nsEP) cause weaker electroporation than just a single unipolar phase of the same pulse. This study was undertaken to explore the dependence of bipolar cancellation on nsEP parameters, with emphasis on the amplitude ratio of two opposite polarity phases of a bipolar pulse. Individual cells (CHO, U937, or adult mouse ventricular cardiomyocytes (VCM)) were exposed to either uni- or bipolar trapezoidal nsEP, or to nanosecond electric field oscillations (NEFO). The membrane injury was evaluated by time-lapse confocal imaging of the uptake of propidium (Pr) or YO-PRO-1 (YP) dyes and by phosphatidylserine (PS) externalization. Within studied limits, bipolar cancellation showed little or no dependence on the electric field intensity, pulse repetition rate, chosen endpoint, or cell type. However, cancellation could increase for larger pulse numbers and/or for longer pulses. The sole most critical parameter which determines bipolar cancellation was the phase ratio: maximum cancellation was observed with the 2nd phase of about 50% of the first one, whereas a larger 2nd phase could add a damaging effect of its own. "Swapping" the two phases, i.e., delivering the smaller phase before the larger one, reduced or eliminated cancellation. These findings are discussed in the context of hypothetical mechanisms of bipolar cancellation and electroporation by nsEP.

Antibacterial Effects of Toothpastes Evaluated in an In Vitro Biofilm Model.

PURPOSE: To test the antibacterial effects of different toothpastes with the slurry method of toothpaste application in an in vitro oral biofilm model including relevant periodontal pathogens. MATERIALS AND METHODS: Four commercially available toothpastes, two containing sodium fluoride (NaF) at different concentrations (1450 and 2500 ppm), two NaF with either triclosan or stannous fluoride, and a control phosphate-buffered saline (PBS) were used. Multispecies biofilms containing 6 species of oral bacteria were grown on hydroxyapatite disks for 72 h and then exposed for 2 min to the toothpaste slurries or phosphate buffer saline (PBS) by immersion, under continuous agitation at 37°C. Biofilms were then analysed by means of real-time polymerase chain reaction (PCR), combined with propidium monoazide (PMA). Statistical evaluation was performed using ANOVA and Student's t-test, with Bonferroni correction for multiple comparisons. RESULTS: The toothpastes containing NaF and stannous fluoride demonstrated superior antimicrobial activity for A. actinomycetencomitans, P. gingivalis and F. nucleatum when compared to those containing NaF and triclosan, 1450 ppm NaF or 2500 ppm NaF in this multispecies biofilm model. CONCLUSION: The proposed model for the evaluation of toothpastes in the form of slurries detected significant differences in the antimicrobial effects among the tested NaF-containing toothpastes, with the stannous fluoride-based formulation achieving better results than the other formulations. The use of toothpaste as slurries and real-time PCR with PMA is an adequate method for comparing the in vitro antimicrobial effect of different toothpastes.

Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles.

Microbubbles (MBs) have been shown to create transient or lethal pores in cell membranes under the influence of ultrasound, known as ultrasound-mediated sonoporation. Several studies have reported enhanced drug delivery or local cell death induced by MBs that are either targeted to a specific biomarker (targeted microbubbles, tMBs) or that are not targeted (non-targeted microbubbles, ntMBs). However, both the exact mechanism and the optimal acoustic settings for sonoporation are still unknown. In this study we used real-time uptake patterns of propidium iodide, a fluorescent cell impermeable model drug, as a measure for sonoporation. Combined with high-speed optical recordings of MB displacement and ultra-high-speed recordings of MB oscillation, we aimed to identify differences in MB behavior responsible for either viable sonoporation or cell death. We compared ntMBs and tMBs with identical shell compositions exposed to long acoustic pulses (500-50,000cycles) at various pressures (150-500kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120s after opening of the pore, this resulted in cell death. Higher acoustic pressures and longer cycles resulted in more displacing MBs and enhanced sonoporation. Non-displacing MBs were found to be the main contributor to cell death, while displacement of tMBs enhanced reversible sonoporation and preserved cell viability. Consequently, each therapeutic application requires different settings: non-displacing ntMBs or tMBs are advantageous for therapies requiring cell death, especially at 500kPa and 50,000cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.

Corticosterone enhances N-methyl-D-aspartate receptor signaling to promote isolated ventral tegmental area activity in a reconstituted mesolimbic dopamine pathway.

Elevations in circulating corticosteroids during periods of stress may influence activity of the mesolimbic dopamine reward pathway by increasing glutamatergic N-methyl-D-aspartate (NMDA) receptor expression and/or function in a glucocorticoid receptor-dependent manner. The current study employed organotypic co-cultures of the ventral tegmental area (VTA) and nucleus accumbens (NAcc) to examine the effects of corticosterone exposure on NMDA receptor-mediated neuronal viability. Co-cultures were pre-exposed to vehicle or corticosterone (CORT; 1 µM) for 5 days prior to a 24 h co-exposure to NMDA (200 µM). Co-cultures pre-exposed to a non-toxic concentration of corticosterone and subsequently NMDA showed significant neurotoxicity in the VTA only. This was evidenced by increases in propidium iodide uptake as well as decreases in immunoreactivity of the neuronal nuclear protein (NeuN). Co-exposure to the NMDA receptor antagonist 2-amino-7-phosphonovaleric acid (APV; 50 µM) or the glucocorticoid receptor (GR) antagonist mifepristone (10 µM) attenuated neurotoxicity. In contrast, the combination of corticosterone and NMDA did not produce any significant effects on either measure within the NAcc. Cultures of the VTA and NAcc maintained without synaptic contact showed no response to CORT or NMDA. These results demonstrate the ability to functionally reconstitute key regions of the mesolimbic reward pathway ex vivo and to reveal a GR-dependent enhancement of NMDA receptor-dependent signaling in the VTA.

Glutathione Depletion Induces Spermatogonial Cell Autophagy.

The development and survival of male germ cells depend on the antioxidant capacity of the seminiferous tubule. Glutathione (GSH) plays an important role in the antioxidant defenses of the spermatogenic epithelium. Autophagy can act as a pro-survival response during oxidative stress or nutrient deficiency. In this work, we evaluated whether autophagy is involved in spermatogonia-type germ cell survival during severe GSH deficiency. We showed that the disruption of GSH metabolism with l-buthionine-(S,R)-sulfoximine (BSO) decreased reduced (GSH), oxidized (GSSG) glutathione content, and GSH/GSSG ratio in germ cells, without altering reactive oxygen species production and cell viability, evaluated by 2',7'-dichlorodihydrofluorescein (DCF) fluorescence and exclusion of propidium iodide assays, respectively. Autophagy was assessed by processing the endogenous protein LC3I and observing its sub-cellular distribution. Immunoblot and immunofluorescence analysis showed a consistent increase in LC3II and accumulation of autophagic vesicles under GSH-depletion conditions. This condition did not show changes in the level of phosphorylation of AMP-activated protein kinase (AMPK) or the ATP content. A loss in S-glutathionylated protein pattern was also observed. However, inhibition of autophagy resulted in decreased ATP content and increased caspase-3/7 activity in GSH-depleted germ cells. These findings suggest that GSH deficiency triggers an AMPK-independent induction of autophagy in germ cells as an adaptive stress response.

Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide.

Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting.

Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery.

This work is focused on the development of a plant virus-based carrier system for cargo delivery, specifically 30nm-sized cowpea mosaic virus (CPMV). Whereas previous reports described the engineering of CPMV through genetic or chemical modification, we report a non-covalent infusion technique that facilitates efficient cargo loading. Infusion and retention of 130-155 fluorescent dye molecules per CPMV using DAPI (4',6-diamidino-2-phenylindole dihydrochloride), propidium iodide (3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide), and acridine orange (3,6-bis(dimethylamino)acridinium chloride), as well as 140 copies of therapeutic payload proflavine (PF, acridine-3,6-diamine hydrochloride), is reported. Loading is achieved through interaction of the cargo with the CPMV's encapsidated RNA molecules. The loading mechanism is specific; empty RNA-free eCPMV nanoparticles could not be loaded. Cargo-infused CPMV nanoparticles remain chemically active, and surface lysine residues were covalent modified with dyes leading to the development of dual-functional CPMV carrier systems. We demonstrate cargo-delivery to a panel of cancer cells (cervical, breast, and colon): CPMV nanoparticles enter cells via the surface marker vimentin, the nanoparticles target the endolysosome, where the carrier is degraded and the cargo is released allowing imaging and/or cell killing. In conclusion, we demonstrate cargo-infusion and delivery to cells; the methods discussed provide a useful means for functionalization of CPMV toward its application as drug and/or contrast agent delivery vehicle.

Antitumor drug delivery in multicellular spheroids by electropermeabilization.

Electrochemotherapy (ECT) is a physical technique that allows cytotoxic molecules to be efficiently released in tumor cells by inducing transient cell plasma membrane permeabilization. The main antitumoral drugs used in ECT are nonpermeant bleomycin and low permeant cisplatin. The method is nowadays applied in clinics as a palliative treatment. In order to improve it, we took advantage of a human 3D multicellular tumor spheroid as a model of tumor to visually and molecularly assess the effect of ECT. We used bleomycin and cisplatin to confirm its relevance and doxorubicin to show its potential to screen new antitumor drug candidates for ECT. Confocal microscopy was used to visualize the topological distribution of permeabilized cells in 3D spheroids subjected to electric pulses. Our results revealed that all cells were efficiently permeabilized, whatever their localization in the spheroid, even those in the core. The combination of antitumor drugs and electric pulses (ECT) led to changes in spheroid macroscopic morphology and cell cohesion, to tumor spheroid growth arrest and finally to its complete apoptosis-mediated dislocation, mimicking previously observed in vivo situations. Taken together, these results indicate that the spheroid model is relevant for the study and optimization of electromediated drug delivery protocols.

Quantification of propidium iodide delivery using millisecond electric pulses: experiments.

The transport mechanisms in electroporation-mediated molecular delivery are experimentally investigated and quantified. In particular, the uptake of propidium iodide (PI) into single 3T3 fibroblasts is investigated with time- and space-resolved fluorescence microscopy, and as a function of extracellular buffer conductivity. During the pulse, both the peak and the total integrated fluorescence intensity exhibit an inverse correlation with extracellular conductivity. This behavior can be explained by an electrokinetic phenomenon known as Field-Amplified Sample Stacking (FASS). Furthermore, the respective contributions from electrophoresis and diffusion have been quantified; the former is shown to be consistently higher than the latter for the experimental conditions considered. The results are compared with a compact model to predict electrophoresis-mediated transport, and good agreement is found between the two. The combination of the experimental and modeling efforts provides an effective means for the quantitative diagnosis of electroporation.

Sonoporation of endothelial cells by vibrating targeted microbubbles.

Molecular imaging using ultrasound makes use of targeted microbubbles. In this study we investigated whether these microbubbles could also be used to induce sonoporation in endothelial cells. Lipid-coated microbubbles were targeted to CD31 and insonified at 1 MHz at low peak negative acoustic pressures at six sequences of 10 cycle sine-wave bursts. Vibration of the targeted microbubbles was recorded with the Brandaris-128 high-speed camera (~13 million frames per second). In total, 31 cells were studied that all had one microbubble (1.2-4.2 micron in diameter) attached per cell. After insonification at 80 kPa, 30% of the cells (n=6) had taken up propidium iodide, while this was 20% (n=1) at 120 kPa and 83% (n=5) at 200 kPa. Irrespective of the peak negative acoustic pressure, uptake of propidium iodide was observed when the relative vibration amplitude of targeted microbubbles was greater than 0.5. No relationship was found between the position of the microbubble on the cell and induction of sonoporation. This study shows that targeted microbubbles can also be used to induce sonoporation, thus making it possible to combine molecular imaging and drug delivery.

[Afferent nerve connection among "Hegu" (LI4), "Neiguan" (PC6), "Futu" (LI18) and thyroid gland region: fluorescent double labelling method].

OBJECTIVE: To observe the peripheral and central neural connection of acupoint "Hegu" (LI4), "Neiguan" (PC6), "Futu" (LI18) and the thyroid gland (TG) region with fluorescence double-labelling method. METHODS: Thirty male Wistar rats were divided into LI4-LI18 group, PC6-LI18 group, TG-LI 18 group, LI 4-TG group, and PC 6-TG group, with 6 rats in each. Fluorescence dyes Propidium Iodide (PI, 10 microL) and Bisbenzimide (Bb, 10 microL) were, separately, injected into those acupoints mentioned above and the TG region. Sixty hours after PI-injection and 12 hours after Bb-injection, the rats under deep anesthesia were transcardiacally perfused with PBS containing 4% polyoxymethylene, followed by taking the spinal cord and dorsal root ganglia (DRGs) of the cervical segments (C1-C8) and thoracic 1 (T1) segment. Fluorescent single- and dual-labeled cells of the sliced DRGs and cervical spinal cord were observed by fluorescence microscope. RESULTS: (1) All PI- and Bb-labeled cells were found to distribute in DRGs from C1-T1 segments. PI single-labeled cells from LI4 and PC6 mainly distributed in DRGs from C4 to C8. Bb single-labeled cells from LI18 and TG region distributed in DRGs from C1-C6. (2) Dual-labeled cells in the LI 4-LI 188 group, PC6-LI18 group, LI4-TG group, and PC6-TG group distributed in DRGs from C3 to C7, suggesting bifurcate peripheral processes of the cervical DRG neurons to innervate LI8, LI4, PC6 and the TG region. And the dual-labeled cells of the TG-LI 18 group distributed mainly in DRGs from C1 to C4. (3) A small number of single-labeled neurons(about 8% of total labeled cells in DRGs) and only several dual-labeled neurons were found in the anterior horn of the cervical spinal cord. CONCLUSION: LI18, LI4 and PC6 and the thyroid gland have the same peripheral cells in DRGs of C3-C7 segments, suggesting that the bifurcate peripheral innervation may provide an anatomic evidence for the analgesic effect of acupuncture of LI18, LI4 and PC6 on thyroidectomy.

Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation.

Ultrasound contrast microbubbles have the ability to enhance endothelial cell permeability and thus may be used as a new way to deliver drugs. It facilitates the transfer of extracellular molecules into cells activated through ultrasound driven microbubbles. The present study is designed to correlate the relationship between microbubble induced cell deformation and enhanced cell membrane permeability. Propidium iodide (PI) was used as a membrane integrity probe. Using high-speed imaging of vibrating microbubbles against endothelial cells and imaging transport of PI into these cells showed a direct correlation between cell deformation and resulting cell membrane permeability. The membrane permeabilization lasted for a short period without affecting endothelial cells viability. We identified that microbubbles are crucial to enhance transient cell membrane permeability. Thus, permeability of individual cells is increased. The roles of ultrasound contrast microbubbles as the trigger for improved drug efficacy are discussed.

Laser-assisted microinjection into targeted animal cells.

A pulsed (17 nanoseconds) Nd:YAG laser (1064 nm) was used to inject impermeable dyes (propidium iodide andiodide and merocyanine 540) and a plasmid (pEGFP-N1) encoding green fluorescent protein (GFP) into human breast adenocarcinoma cells (MCF-7). The cell membrane integrity and viability were fully preserved in this laser-assisted transfer.

Dual fluorescence analysis of DNA apoptosis in sperm.

OBJECTIVE: The objective was to compare fluorochrome Hoechst 33342 (Ho342) with combined Ho342/propidium iodide (PI) stains for assessment of sperm quality. STUDY DESIGN: Washed donor sperm cells were incubated in either 0, 0.15, or 15 micromol/L camptothecin (CAM) or 0.37 or 3.7 mmol/L genistein (GEN) for 4 hours at 37 degrees C. The sperm cells were analyzed for cycle-independent apoptosis and necrosis by single- compared with dual-stained fluorescence microscopy to contrast the relative effectiveness of these two approaches. RESULTS: The single-stain procedure did not detect viability differences (overall 76.1% +/- 2.2% live). In contrast, the dual-stain procedure identified a dose-dependent decrease in viability and increased necrozoospermia for CAM and GEN treatments. Apoptosis was 2-fold higher with topoisomerase inhibitor treatment. CONCLUSION: The two topoisomerase inhibitors were associated with increased apoptosis and dose-dependent necrosis. The data suggested that the dual-stain combination Ho342/PI was more sensitive than the single Ho342 stain analysis and permitted quantifying the apoptosis and necrosis events in sperm.

By-passing of P-glycoprotein using immunoliposomes.

The over-expression of MDR1 P-glycoprotein has been associated with the development of multidrug-resistance in cancer cells. Methods used to overcome multidrug-resistance often involve the co-administration of inhibitors of P-glycoprotein. Here, we test the hypothesis that an immunoliposome-based drug delivery system may be used as an alternative approach to overcome multidrug-resistance since immunoliposomes penetrate target cells by receptor-mediated endocytosis which allows to by-pass membrane-associated P-glycoprotein. Targeting of immunoliposomes was achieved by the use of an anti-transferrin receptor monoclonal antibody (OX26 mAb). Incorporation of radiolabelled digoxin within OX26-immunoliposomes enhanced cellular uptake of digoxin by a factor of 25 in immortalised RBE4 rat brain capillary endothelial cells. Uptake of liposomal digoxin was insensitive to ritonavir, a P-glycoprotein inhibitor, and was reduced in presence of increasing free concentrations of OX26 mAb or nocodazole, a reversible inhibitor of endocytosis. In contrast, uptake of free digoxin was enhanced by a factor of 1.8 in presence of ritonavir and was insensitive to OX26 mAb or nocodazole. Cellular uptake and intracellular accumulation of liposomal digoxin (55% internalisation within 30 min) was demonstrated by acid wash of the cells and was confirmed by confocal microscopy studies. Endosomal release to the cytosol of propidium iodide loaded immunoliposomes was shown. These in vitro studies suggest that immunoliposome-based drug delivery systems can be used to by-pass P-glycoprotein and thus deliver drugs to the cytosol of a target cell.

Ocular drug targeting by liposomes and their corneal interactions.

Topical drug delivery is preferred in the eye to avoid under or over medication and undesired side effects of systemic administration. In order to maintain adequate drug levels in ocular tissues, frequent drug installation is necessary in vision threatening conditions like glaucoma, corneal ulcers due to microbial infections, etc. Only a part of the installed drug reaches the aqueous humor and the rest of it is drained by the nasolacrimal duct. Positively charged liposomes have been found to enhance the penetration of drugs into the cornea. The present study was conducted to visualize the interaction of liposomes with the cornea. Briefly, positively charged liposomes entrapped with Carboxyfluorescein (CF), Propidium iodide (PI), Horseradish peroxidase (HRP), Biotin, Hydroxy benzimide (Hoechst No: 33258), and Ethoxy benzimide (Hoechst No: 33342) were prepared by sonication. Their size and shape were analysed by laser dynamic light spectra and electron microscopy, respectively. The liposome encapsulated and free materials in buffer were instilled, in a volume of 20 microliters, into eyes of anesthetized albino rats. After 30 min and 1 h, the eyes were enucleated and quickly processed for cryosections of 3-5 microns thickness. The interaction of liposome entrapped propidium iodide and HRP was visualized on the outer epithelium of the cornea after specific processing.