Laser scanning confocal fluorescence microscopy: an overview

Innovative and important aspects of laser scanning confocal fluorescence imaging (LSCFI) are presented here as a general overview. We have described and discussed the technology of the procedure in some detail. We also report some of our original work with transmembranous uptake of 5S gamma-globulin on living human leukocytes as an example of one specific application of LSCFI. These original data and results are presented, as well as citing other uses and applications, to show the power of LSCFI technique. The article will hopefully be useful for those not familiar with the methodology and utility of laser scanning confocal fluorescence microscopy. Applications of LSCFI are very diverse, and there are new applications of this technology constantly being developed. Interest is growing in LSCFI, particularly in the pharmacologic and therapeutic areas, as demonstrated in this article.     

Xenobiotic transport across isolated brain microvessels studied by confocal microscopy

To identify specific transporters that drive xenobiotics from central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from rat and pig brain using confocal microscopy and quantitative image analysis. Luminal accumulation of daunomycin and of fluorescent derivatives of cyclosporine A (CSA) and ivermectin was concentrative, specific, and energy-dependent (inhibition by NaCN). Transport was reduced by PSC 833, ivermectin, verapamil, CSA, and vanadate, but not by leukotriene C(4) (LTC(4)), indicating the involvement of P-glycoprotein. Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein methotrexate was also concentrative, specific, and energy-dependent. LTC(4), chlorodinitrobenzene, and vanadate reduced transport of these compounds, but PSC 833 and verapamil did not, indicating the involvement of a multidrug resistance-associated protein (Mrp). Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the capillary endothelium and quantitative polymerase chain reaction showed Mrp1 and Mrp2 expression. Finally, the HIV protease inhibitors saquinavir and ritonavir were potent inhibitors of transport mediated by both P-glycoprotein and Mrp. These results validate a new method for studying drug transport in isolated brain capillaries and implicate both P-glycoprotein and one or more members of the Mrp family in drug transport from central nervous system to blood.     

Visualization of skin penetration using confocal laser scanning microscopy.

The use of skin as an alternative route for administering systemically active drugs has attracted considerable interest in recent years. However, the skin provides an excellent barrier, which limits the number of drug molecules suitable for transdermal delivery. Thus, in order to improve cutaneous delivery, it is necessary to adopt an enhancement method, either (i) passively using novel formulations, e.g. microemulsions, liposomes, and colloidal polymeric suspensions, or more conventional skin permeation enhancers, or (ii) with a physical approach, such as, iontophoresis, sonophoresis or electroporation. Although there has been much progress, the precise modes of action of the different techniques used are far from well-understood. The objective of this review, therefore, is to evaluate how confocal laser scanning microscopy may contribute to the determination of the mechanisms of diverse skin penetration enhancement strategies.     

共聚焦激光扫描显微镜在反向银屑病诊断中的应用研究

目的探讨共聚焦激光扫描显微镜在反向银屑病诊断和鉴别诊断中的应用价值。方法选取临床疑诊反向银屑病的患者20例,比较共聚焦激光扫描显微镜扫描检查和常规组织病理检查的结果。结果20例患者共聚焦激光扫描和组织病理检查结果经统计学分析,无显著性差异。结论共聚焦激光扫描为反向银屑病提供了有价值的、无创的、新的诊断方法。     

Atomic force microscopy analysis and confocal Raman microimaging of coated pellets

Polymer-coated pellets with different coating thicknesses have been studied regarding coating morphology and drug release properties with atomic force microscopy (AFM) and confocal Raman microscopy. The results were compared with those from scanning electron microscopy (SEM) and drug release profiles, which have been measured previously for these systems and found to vary depending on coating thickness. Results from AFM studies indicated that these pellets differ in the amount of crystalline material on the surface of the coating. The amount was found to be highest on the pellet with the thinnest coating. Confocal Raman microscopy studies confirmed that the active component (remoxipride hydrochloride monohydrate) is present at or close to the surface and that the amount is higher for the thinnest coating. AFM studies in aqueous media showed that the crystalline material on the surface was almost instantaneously dissolved and released into the liquid. AFM has proven to be a powerful tool in the study of the surface of dry formulations and in the study of the controlled release mechanism of a pharmaceutical in a liquid cell. The method can be combined with Raman, giving the added possibility to identify the chemical composition in selected small areas of the coating surface.     

Intracellular trafficking of transferrin-conjugated liposome/ DNA complexes by confocal microscopy

Intracellular trafficking of transferrin-conjugated dimethyldioctadecyl-ammonium bromide liposome (T1-liposome)/DNA complexes in HeLa cells was studied using the double-labeled fluorescence technique and confocal microscopy. The size of the T1-liposome/DNA complex was about 367 nm in diameter and the zeta-potential of it at a 5:1 (w/w) ratio was almost neutral. The intracellular pathway of the T1-liposome/DNA complex, noted as green (FITC), red (rhodamine) or yellow (FITC + rhodamine) fluorescence, was elucidated from the plasma membrane to the endosome (or lysosome), and finally to the nucleus. The results of this study indicate that plasmid DNA enters into the nucleus not only as a free form but as an associated form complexed with T1-liposome. More interestingly, the T1-liposome undergoes a nuclear location in the form of ordered structures. This could be a very useful piece of information in designing a safe and advanced gene delivery system.     

Photothermal confocal multicolor microscopy of nanoparticles and nanodrugs in live cells

Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers, and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4?nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold–silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potential to use PTM for super-resolution imaging (down to 50?nm), real-time NP tracking, guidance of PT nanotherapy, and multiplex cancer markers targeting, as well as analysis of non-linear PT phenomena and amplification of nanodrug efficacy through NP clustering and nano-bubble formation are also discussed.     

Nanoscale mapping of the Met receptor on hippocampal neurons by AFM and confocal microscopy

Hepatocyte growth factor (HGF), a neurotrophic protein, acting through its tyrosine kinase receptor, Met, facilitates learning and synaptic plasticity. In concert with the role of the HGF/Met system in synaptic plasticity, we demonstrate that Met is localized to brain regions which undergo extensive synaptic remodeling. We demonstrate that Met activation results in an increase in dendritic spine density and functional synapses. Based on these observations, we hypothesized that Met should be associated with post-synaptic elements found on dendritic spines. Thus, the goal of this study was to determine the sub-cellular localization of Met on hippocampal neurons. Using an atomic force microscopy tip decorated with a specific Met antibody, the location of Met was mapped to different cellular compartments of hippocampal pyramidal neurons. Our results indicated that multimeric activated Met was found to be concentrated in the dendritic compartment while the inactivated monomeric form of Met was prominent on the soma.From the Clinical EditorThe goal of this study was to determine the sub-cellular localization of Met on hippocampal neurons using nanotechnology-based techniques, using an atomic force microscopy tip decorated with a specific Met antibody. The authors demonstrate that multimeric activated Met was found to be concentrated in the dendritic compartment while the inactivated monomeric form of Met was prominent in the soma of hippocampal pyramidal neurons.     

Characterization of particle deformation during compression measured by confocal laser scanning microscopy

Direct compression of riboflavin sodium phosphate tablets was studied by confocal laser scanning microscopy (CLSM). The technique is non-invasive and generates three-dimensional (3D) images. Tablets of 1% riboflavin sodium phosphate with two grades of microcrystalline cellulose (MCC) were individually compressed at compression forces of 1.0 and 26.8 kN. The behaviour and deformation of drug particles on the upper and lower surfaces of the tablets were studied under compression forces. Even at the lower compression force, distinct recrystallized areas in the riboflavin sodium phosphate particles were observed in both Avicel PH-101 and Avicel PH-102 tablets. At the higher compression force, the recrystallization of riboflavin sodium phosphate was more extensive on the upper surface of the Avicel PH-102 tablet than the Avicel PH-101 tablet. The plastic deformation properties of both MCC grades reduced the fragmentation of riboflavin sodium phosphate particles. When compressed with MCC, riboflavin sodium phosphate behaved as a plastic material. The riboflavin sodium phosphate particles were more tightly bound on the upper surface of the tablet than on the lower surface, and this could also be clearly distinguished by CLSM. Drug deformation could not be visualized by other techniques. Confocal laser scanning microscopy provides valuable information on the internal mechanisms of direct compression of tablets.     

Quantification of microparticle coating quality by confocal laser scanning microscopy (CLSM)

In this work, a novel protocol was developed for determining film coating thickness and coating quality of microparticles, based on the use of confocal laser scanning microscopy (CLSM). CLSM was found to be an adequate non-destructive technique for the quantification of the coating thickness and coating quality of individual thin-coated small particles. Combined with image analysis, it was possible to derive with high accuracy the coating thickness distribution of a representative number of microparticles. The performance of the novel methodology was assessed by the quantification of the coating thickness and coating quality of protein-coated microparticles produced by fluidized bed coating. It was found that the CLSM data on coating layer thickness were generally in good agreement with the results from chemical analysis, down to a thickness of 1–1.5 μm. Using CLSM the importance of setting up the appropriate distance between the coating nozzle and the powder bed with respect to microparticle coating quality in fluidized bed processing was illustrated. Coating quality was found to decrease with increasing distance the coating droplets have to travel before impinging onto the core particles as a result of spray-drying of the coating droplets. Also, coating quality decreased with increasing viscosity of the coating droplets, resulting in reduced spreading on the cores.     

Quantitative image analysis of drug-induced lung fibrosis using laser scanning confocal microscopy.

Pulmonary fibrosis is a serious lung disorder that in certain cases may be difficult to quantify. It was our objective to evaluate the use of laser scanning confocal microscopy (LSCM) in quantifying fibrosis after exposure to amiodarone (AD) and bleomycin (BLM), two commonly used therapeutic drugs known to cause debilitating lung fibrosis in humans. Male F344 rats were intratracheally dosed with AD (6.25 mg/kg on days 0 and 2), BLM (0.25 and 1.0 mg/kg on day 0), or their respective vehicle controls. The right lung was assayed for hydroxyproline, a biochemical measure of collagen, at day 21 for the BLM groups and day 28 for the AD groups. The left lung was fixed, sectioned into blocks, dehydrated, stained with Lucifer yellow (LY, 0.1 mg/ml), and embedded in Spurr resin. The area of lung tissue stained by LY was quantified by LSCM. A fibrotic response in the AD and BLM groups was confirmed by histopathological assessment and a significant increase (p < 0.05) in total right lung hydroxyproline above control values. The area of connective tissue stained by LY of the two drug-treated groups appeared as bright linear bands in the alveolar septae and was significantly increased (p < 0.05) as measured by image analysis when compared with their respective controls. LSCM, with its advanced image analysis system, is an alternate method to quantify fibrotic lung disease. LSCM could be particularly useful when tissue quantity is limited, such as when tissue has been archived from previous studies, or when analyzing human lung biopsy samples for disease diagnosis, where biochemical analysis is difficult.     

In vivo real-time confocal microscopy for target-specific delivery of hyaluronic acid-quantum dot conjugates

Hyaluronic acid (HA), which is a biocompatible, biodegradable, and linear polysaccharide in the body, has been widely used for various biomedical applications. In this work, real-time bioimaging for target-specific delivery of HA derivatives was carried out using quantum dots (QDs). In vitro confocal microscopy of HA-QD conjugates confirmed the intracellular delivery of HA derivatives to B16F1 cells with HA receptors by HA-receptor-mediated endocytosis. Furthermore in vivo real-time confocal microscopy of HA-QD conjugates successfully visualized the target specific delivery and accumulation of HA-QD conjugates from the fluorescence-labeled blood vessels to the liver tissues. The authors could confirm the feasibility of HA derivatives as a target-specific intracellular drug-delivery carrier for the treatment of liver diseases and the in vivo real-time confocal microscopy as a new bioimaging tool for various drug-delivery applications.From the Clinical EditorThis study demonstrates the possibility of labeling hyaluronic acid with quantum dots for visualization and for targeted intracellular drug delivery in liver disease models.     

激光共焦显微镜观察真菌性角膜炎治疗效果的研究

目的 研究激光共焦显微镜对观察真菌性角膜炎治疗过程中病情转归的意义.方法 对42例(42眼)经门诊诊断为真菌性角膜炎的患眼,临床使用那他霉素眼用混悬液抗真菌治疗,治疗前及治疗后l周、1月分别行患眼的激光共焦显微镜检查,观察真菌菌丝生长情况.并根据临床反应判断治疗结果.结果 治疗后病灶处菌丝密度明显减少,甚至消失,周边炎性细胞浸润减少,基质透亮度增加,胶原纤维增生,组织呈瘢痕化增生.治疗前,激光共焦显微镜下42例中37例见菌丝分布(88.1％),抗真菌治疗l周后42例菌丝阳性为26例(61.9％),1月后患眼菌丝阳性为9例(21.4％),结果差异具有统计学意义(P＜0.05).最终治愈28例(66.7％),好转9例(21.4％),无效5例(11.9％).最终有效率达88.1％.其中无效5例中均为菌丝阳性,好转中菌丝阳性为4例,但密度较治疗前明显减少.结论 激光共焦显微镜对观察真菌性角膜炎病情的转归具有临床意义.     

Mapping microclimate pH distribution inside protein-encapsulated PLGA microspheres using confocal laser scanning microscopy

The pH in the aqueous pores of poly(lactide-co-glycolide) (PLGA) matrix, also referred to as microclimate pH (μpH), is often uncontrolled, ranging from highly acidic to neutral pH range. The μpH distribution inside protein-encapsulated PLGA microspheres was quantitatively evaluated using confocal laser scanning microscopy. The fluorescent response of Lysosensor yellow/blue dextran used to map μpH in PLGA was influenced by the presence of encapsulated protein. The nonprotonated form of pyridyl group on the fluorescence probe at neutral pH was responsible for the interference, which was dependent on the type and concentration of protein. A method for correction of this interference based on estimating protein concentration inside the microspheres was established and validated. After correction of the influence, the μpH distribution kinetics inside microspheres was evaluated for different PLGA 50/50 microsphere formulations under physiological conditions for 4 weeks. Generally, the μpH acidity increased with the progression of incubation time. The coincorporation of poorly soluble base, magnesium carbonate, in the microspheres prolonged the appearance of detectable acidity for up to 3 weeks. Co-addition of an acetate buffer was able to control the μpH over a slightly acidic range (around pH 4.7) after two week incubation. Microspheres prepared from a lower polymer concentration exhibited a higher μpH, likely owing to reduced diffusional resistance to acidic degradation products. The stability of protein was enhanced by addition of MgCO(3), acetate buffer, or by reduced polymer concentration in the preparation, as evidenced by more soluble protein recovered after incubation. Hence, the μpH imaging technique developed can be employed in the future for optimization of formulation strategies for controlling μpH and stabilizing encapsulated proteins.     

Measurement and mapping of pH in hydrating pharmaceutical pellets using confocal laser scanning microscopy

Purpose. pH modifiers are often used to promote drug solubility/stability in dosage forms, but predicting the extent and duration of internal pH modification is difficult. Here, a noninvasive technique is developed for the spatial and temporal mapping of pH in a hydrated pharmaceutical pellet, within a pH range appropriate for microenvironmental pH control by weak acids. Methods. Confocal dual excitation imaging (Ex 488/Ex 568) of pellets containing a single, soluble, pH-sensitive fluorophore with cross-validation from a pH microelectrode. The technique was used to investigate the changing pH distribution in hydrating pellets containing two weak acids of differing solubility. Results. The algorithm developed provided pH measurements over the range pH 3.5-5.5 with a typical accuracy of 0.1 pH units and with excellent correlation with pH microelectrode measurements. The method showed how pellets containing 25%w/w tartaric acid exhibited a rapid but transient fall in internal pH, in contrast to a slower more prolonged reduction with fumaric acid. Conclusions. Spatial and temporal monitoring of pH in pellets was achieved with good accuracy within a pH range appropriate to pH modification by weak acids. However, the method developed is also generic and with suitable fluorophores will be applicable to other pH ranges and other dosage forms.     

眼表综合分析仪联合角膜激光共聚焦显微镜检查对睑缘炎相关性角结膜病变 45例的价值

目的应用眼表综合分析仪和角膜激光共聚焦显微镜( IVCM)探讨睑缘炎相关性角结膜病变( BKC)病人角膜细胞密度和眼表功能的变化及与蠕形螨感染的关系。方法选取 2020年 9月至,2022年 3月就诊于沧州市中心医院的 45例 BKC病人为观察组,同期的健康志愿者 45例为对照组。根据观察组是否感染蠕形螨,分为蠕形螨感染组和未感染组。对所有研究对象使用眼表综合分析仪和 IVCM进行检查,收集观察组和对照组的非侵入式平均泪膜破裂时间( NITBUTav)、非侵入式首次泪膜破裂时间( NIBUTf)、角膜荧光素染色( FL)评分、泪液分泌试验( SⅠt)、蠕形螨感染情况和角膜细胞密度。 Pearson相关性分析蠕形螨感染数量与病人角膜细胞密度及眼表功能的相关性。结果观察组的 NITBUTav［( 6.31±2.95)s比( 14.85±3.88)s］和 NIBUTf［( 4.65±2.03)s比( 11.88±3.14)s］低于对照组, FL评分高于对照组( P<0.05)。观察组蠕形螨感染阳性率高于对照组,央角膜浅基质层的细胞密度低于对照组( P<0.05)。观察组下方角膜基底细胞层和浅基质层的细胞密度低于对照组( P<0.05)。中蠕形螨感染组的 FL评分高于未感染组,下方角膜基底细胞层、浅基质层和中央角膜浅基质层的细胞密度低于未感染组( P<0.05)。蠕形螨感染数量与 BKC病人下方角膜基底细胞层、浅基质层和中央角膜浅基质层的细胞密度呈负相关,与 FL评分呈正相关(均 P<0.05)。结论眼表综合分析仪联合 IVCM可对 BKC病人的眼表损害进行综合评价; BKC病人下方角膜基底细胞层、浅基质层和中央角膜浅基质层的细胞密度降低, FL评分增加,与蠕形螨感染数量增多有关。     

Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis.

Laser scanning confocal microscopy (LSCM) allows us to simultaneously quantitate the degree of lung fibrosis and distinguish various pathological lesions of intact lung tissue. Lucifer Yellow has been shown an ideal fluorescent stain to examine the connective tissue matrix components of embedded lung tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibrosis and compared this procedure with the more traditional method of assessing fibrosis by measuring hydroxyproline, a biochemical assay of collagen. CD/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/100 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCl and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respectively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Using LSCM and ImageSpace software, the tissue areas of ten random scans from ten blocks of tissue for each of the three groups were measured, and three-dimensional reconstructions of random areas of lung were generated. The silica group showed increases of 57% and 60% in the lung areas stained by Lucifer Yellow over the Fe2O3 and control groups, respectively. Regression analysis of hydroxyproline vs. lung tissue area demonstrated a significant positive correlation (p < 0.05) with a correlation coefficient of 0.91. Histological analysis of right lung tissue revealed a marked degree of granulomatous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyproline content and measured tissue area were observed between the Fe2O3 and control groups. LSCM, and its associated advanced image analysis and three-dimensional capabilities, is an alternative method to both quickly quantitate and examine fibrotic lung disease without physical disruption of the tissue specimen.     

Bi-directional relationship of in vitro mast cell-nerve communication observed by confocal laser scanning microscopy

Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Recently, we used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL) cells to study this interaction. Previously, we concentrated mainly on the activation signal from neurites to mast cells (RBL). However, it is proposed that mast cell-nerve communication is not a one-sided relationship but a bi-directional one. In the present work, we studied the communication from mast cells to neurites. We observed that binding of anti-IgE receptor antibodies to mast cells increases calcium ion concentration [Ca2+]i in SCG neurites. This indicates that mast cell-nerve communication is bi-directional. Confocal fluorescence microscopic images indicated that [Ca2+]i in neurites increased after an increase of [Ca2+]i in mast cells. The lag-time of neurite activation was several times longer than that of mast cell activation. The correlation coefficient between the lag-times for mast cell and nerve activation was calculated to be 0.81. In addition, the fluorescence images showed that calcium signals in SCG neurites were able to extend to a long distance (100-200 microm) from the site where mast cells (RBL) attached to neurites.     

Pharmaceutical applications of confocal laser scanning microscopy: the physical characterisation of pharmaceutical systems

The application of confocal laser scanning microscopy (CLSM) to the physicochemical characterisation of pharmaceutical systems is not as widespread as its application within the field of cell biology. However, methods have been developed to exploit the imaging capabilities of CLSM to study a wide range of pharmaceutical systems, including phase-separated polymers, colloidal systems, microspheres, pellets, tablets, film coatings, hydrophilic matrices, and chromatographic stationary phases. Additionally, methods to measure diffusion in gels, bioadhesives, and for monitoring microenvironmental pH change within dosage forms have been utilised. CLSM has also been used in the study of the physical interaction of dosage forms with biological barriers such as the eye, skin and intestinal epithelia, and in particular, to determine the effectiveness of a plethora of pharmaceutical systems to deliver drugs through these barriers. In the future, there is continuing scope for wider exploitation of existing techniques, and continuing advancements in instrumentation.