DETERMINANTS OF PHOTOSENSITIZATION BY MONO-L-ASPARTYL CHLORIN e6

The mono-N-aspartyl derivative of chlorin e6 (MACE) is a new photosensitizer being examined for use in anti-neoplastic photodynamic therapy. Studies were carried out to identify unique aspects of MACE localization by murine leukemia L1210 cells in vitro. Octanol/water partitioning studies were used to quantitate the hydrophobicity of MACE and two analogs, chlorin e6 and mesochlorin. Sites of cellular localization of these dyes were probed by fluorescence studies, and by examining loci of photodamage. These studies indicate that MACE, a hydrophilic dye, partitions to cytoplasmic loci. Data obtained with chlorin e6, a more hydrophobic dye, are consistent with binding at both membrane and cytoplasmic sites. A substantially more hydrophobic product, meso-chlorin, binds primarily to the cell membrane. While the tumor-localizing porphyrin product HPD binds to plasma LDL less than HDL, MACE and CE are predominantly bound to plasma protein and HDL. Patterns of distribution and localization of MACE differ substantially from those observed with HPD and other hydrophobic sensitizers. Phototoxic effects of MACE could not be specifically attributed to membrane or mitochondrial damage.     

Sites of photodamage induced by photodynamic therapy with a chlorin e6 triacetoxymethyl ester (CAME)

The acetoxymethyl ester of chlorin e6 (CAME) was initially designed to be a hydrophobic photosensitizing agent that would be recognized by an endocytic pathway and initially accumulated in lysosomes. This was expected to lead to hydrolysis of the ester groups, followed by redistribution of the free chlorin to other subcellular sites. In this study, we examined the patterns of localization of CAME and of subsequent photodamage in murine leukemia L1210 cells. The drug was initially localized at intracellular sites, yielding a pattern similar to that obtained with a fluorescent probe for acidic intracellular vesicles and endosomes. A brief (30 min) incubation with 10 microM CAME followed by irradiation led to mitochondrial photodamage and apoptotic cell death. At a higher drug level, or with a longer incubation time, we observed additional photodamage to the plasma membrane and to lysosomes. The higher photodynamic therapy dose led to inhibition of apoptosis, with cell death likely occurring via a necrotic process. Distribution of CAME among the components of human plasma was to albumin > high-density lipoprotein > low-density lipoprotein. These results have implications concerning the likely mechanism of CAME accumulation and subcellular distribution.     

Sites of photosensitization by protoporphyrin and tin protoporphyrin in leukemia L1210 cells

Studies with protoporphyrin (PP) and tin protoporphyrin (SnPP) were carried out to assess the effects of tin insertion on the sites of dye localization. Fluorescence emission spectra and studies on the sites of photodamage were consistent with a concentration of PP at membrane loci. In contrast, SnPP photodamage involved an intracellular site.     

Do Liposome‐binding Constants of Porphyrins Correlate with Their Measured and Predicted Partitioning Between Octanol and Water?¶

We tested correlations between lipophilicity parameters and the partitioning of sensitizers into membranes. For this purpose we investigated 17 porphyrins and two chlorins having various chemical structures. Some of these compounds possess an amphiphilic structure (including hematoporphyrin, deuteroporphyrin, mesoporphyrin, chlorin e6 and more). The others are very symmetrical sensitizers [meso-tetra(N-methyl-4-pyridyl)porphyrin, tetra-benzoporphyrin, coproporphyrin I dihydrochloride (CP), meso-tetra(4-carboxyphenyl)porphyrin (TCP) and meso-tetra(m-hydroxyphenyl)chlorin]. Our investigation also included two series of hematoporphyrins and protoporphyrins with varying lengths of alkylcarboxylate side groups. The partitioning of these compounds between the bulk aqueous phase and liposomes was studied by fluorescence methods, and a liposome-binding constant, Kb, was obtained. It was found that CP and TCP do not incorporate into the lipid phase at pH 7.3. An n-octanol-water partition coefficient (log P) and a distribution coefficient (log D) were predicted with a modeling software. The values of log D were also obtained experimentally. We found that for the studied molecules, the predicted log D correlated well with the measured values. The values of log D as well as log P, in turn, did not correlate nicely, for the whole group of studied compounds, with the binding constants to liposomes. However, in the case of porphyrins that share a similar structure, the Kb showed good linear correlation with both log P and log D. For the series of hematoporphyrins and protoporphyrins with different lengths of alkylcarboxyl groups, it was shown that prolongation of this group caused an increase in the lipophilicity and the liposome-binding constant. This effect is more pronounced for the proto- than for the hematoporphyrin series. The results highlight the possible use, as well as limitations, of lipophilicity parameters for the prediction of membrane binding.     

Acid-base properties of chlorin e6: relation to cellular uptake

Chlorins are attractive compounds for photodynamic therapy because of their high absorption in the red spectral region. In this study, the absorbance, fluorescence excitation and fluorescence emission spectra of chlorin e6 have been recorded as functions of pH in phosphate-buffered saline (PBS) solution with and without fetal calf serum (FCS). For pure PBS solutions, variation of the pH of the solution results in a shift of both the absorption and the fluorescence spectrum as well as in a decrease of the fluorescence intensity. Spectrophotometric and fluorimetric titration curves, based on observed changes, have been plotted. There is an indication of aggregate formation at low pH values (pH < 5). The presence of 5% FCS results in a shift of the titration curve, from an inflection point at about 6.5 to one at about 7.6. Pronounced spectral changes of the fluorescence emission spectra of protein-bound chlorin e6 (change of spectral shape, decrease of peak intensity) are also observed. The partition coefficients in the 1-octanol-water system increase with decreasing pH. Thus, relatively more of the drug is incorporated in the octanol phase at low pH. Cellular uptake of chlorin e6 in the presence of serum is significantly higher at pH 6.7 as compared with that at 7.3 and 7.6. We conclude that a change in the pH value of the surrounding medium leads to a change in the lipophilicity of chlorin e6. Such a change is likely to influence its binding to the serum proteins as well as its interaction with the plasma membrane of cells and may also be related to the selective tumor uptake of the drug.     

Novel metal complexes of boronated chlorin e6 for photodynamic therapy

Various structural modifications of chlorins are aimed at optimization of biomedical characteristics of these plant-derived tetrapyrrolic compounds. In particular, conjugation with boron polyhedra improves the efficacy of chlorin e₆ derivatives as antitumor photosensitizers. To obtain the compounds that may possess several clinically favorable characteristics, we synthesized a series of metal chlorin e₆ conjugates with 1-carba-closo-dodecaborate anion that contain Pd(II), Sn(IV) or Zn(II) in the coordination sphere of the chlorin macrocycle. The compounds were synthesized by alkylation of amino group in chlorin e₆ metal complexes with 1-trifluoromethanesulfonylmethyl-1-carba-closo-dodecaborate cesium. The water soluble Pd(II) complex of chlorin e₆ 13(1)-N-{2-[N-(1-carba-closo-dodecaboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester (compound 6) evoked low dark cytotoxicity; in striking contrast, 6 potently sensitized human tumor cells to illumination with monochromatic red light. Confocal microscopic studies demonstrated that photoactivation of 6 rapidly (within minutes) changed the patterns of intracellular drug distribution from diffuse cytoplasmic to clustered perinuclear. Co-localization experiments revealed that 6 associated with lysosomes in illuminated cells. These events were paralleled by alteration of mitochondrial shape, a decrease of mitochondrial transmembrane electric potential and the loss of plasma membrane impermeability for propidium iodide, the latter being a hallmark of cell necrosis. Similar mechanisms of cell photodamage were found for structurally close Pd(II) complex of chlorin with neutral carborane and for Sn(IV) chlorin conjugated with the anionic carborane. Thus, metal complexes of carboranylchlorins are efficient photosensitizers capable of triggering rapid necrosis. These compounds are promising for further development as multipotent agents in which each moiety, i.e., metal, the chlorin macrocycle and the boron substituent, as well as the entire complex, can be useful in cancer diagnostics and treatment.     

FLUORESCENCE SPECTRAL CHANGES OF HEMATOPORPHYRIN DERIVATIVE UPON BINDING TO LIPID VESICLES, Staphylococcus aureus AND Escherichia coli CELLS

Abstract— The binding of hematoporphyrin derivated (Hpd) to lipid vesicles and bacterial membranes was determined by fluorescence spectroscopy. The fluorescence measurements of Hpd in aqueous solutions showed two bands at 613 and 677 nm. In lipid environments of lecithin vesicles the fluorescence spectrum was shifted to 631 and 692 nm, respectively. Hpd was rapidly bound to the cell membrane of Staphylococcus aureus while much less binding occurred in the presence of Escherichia coli. At the same time, spheroplasts of both bacteria were shown to bind Hpd to a similar extent. These results are well correlated with the photoinactivation of the gram positive bacteria with Hpd while the gram negative cells were shown to be resistant. The pH dependence of both Hpd binding to S. aureus as well as the photodynamic inhibitory effect of the same bacteria are similar. It is concluded that the segregation of Hpd to the cell membrane is a prerequisite for its photodynamic effect.     

Structural Factors and Mechanisms Underlying the Improved Photodynamic Cell Killing with Silicon Phthalocyanine Photosensitizers Directed to Lysosomes Versus Mitochondria

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.     

PHOTODYNAMIC RELEASE OF PROTOPORPHYRIN FROM INTACT ERYTHROCYTES IN ERYTHROPOIETIC PROTOPORPHYRIA: THE EFFECT OF SMALL REPETITIVE LIGHT DOSES

Abstract— Erythrocytes from patients with erythropoietic protoporphyria contain large amounts of protoporphyrin bound to (hemo)globin. Irradiation of these cells causes a shift in fluorescence emission maximum and a decreased fluorescence intensity which is consistent with transfer of protoporphyrin from (hemo)globin to the cell membrane. When the erythrocytes were irradiated intermittently, nearly 70% of the protoporphyrin was released and the hemolysis was less than 3%. Giving the total light dose as a single pulse, resulted in 84% protoporphyrin release and 16% hemolysis.
In vivo the erythrocytes obtain small, repetitive light doses when circulating in the dermal capillaries. We suggest the possibility that in patients with erythropoietic protoporphyria these small light pulses could be sufficient to photodamage the binding place of protoporphyrin on (hemo)globin. In the dark, protoporphyrin can then move from (hemo)globin through the cell membrane and bind to albumin in the serum. Our findings indicate that if protoporphyrin is not present in the cell membrane during irradiation, no photohemolysis will occur. This may explain why patients with erythropoietic protoporphyria have no abnormal hemolysis. The effect of intermittent light pulses may also contribute to the understanding of the protoporphyrin release from erythrocytes in patients with erythropoietic protoporphyria.     

Effects of photoinduced membrane rigidification on the lysosomal permeability to potassium ions

Mechanism for the photoinduced increase in the lysosomal K+ permeability is still unknown. In this study, we investigated the effect of photodamage-induced membrane rigidification on the lysosomal K+ permeability by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and by monitoring proton leakage with p-nitrophenol. Membrane fluidity was measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Methylene blue-mediated photodamage to lysosomes decreased their membrane fluidity and increased their K+ permeability. The photoinduced increase in the K+ permeability can be reversed by fluidizing the rigidified lysosomal membranes with benzyl alcohol. The results suggest that the membrane rigidification induced by photodamage may increase lysosomal K+ permeability. This conclusion is supported by the observation that rigidifying lysosomal membranes by the treatment with membrane rigidifier cholesteryl hemisuccinate also enhanced the lysosomal K+ permeability.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction.

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: 1) In both cases the cells are arrested in the G1 portion of the cell cycle; 2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Analysis of plasma membrane protein changes in Dictyostelium discoideum during concanavalin A induced receptor redistribution using two-dimensional gel electrophoresis

The 127 major polypeptides obtained from the purified plasma membrane of Dictyostelium discoideum were examined using two-dimensional gel electrophoresis and a microcomputer-based videodensitometer. Plasma membrane proteins were analyzed at four discrete stages of concanavalin A induced cell surface capping; (i) the cell surface in the absence of ligand (unbound), (ii) the surface immediately after ligand binding (bound), (iii) the cell surface after receptors had patched (patched) and (iv) the cell surface after receptors had capped (capped). Plasma membranes were obtained at various stages of capping by using a colloidal silica density perturbation technique which immediately immobilized the proteins, preserving their lateral distribution in the bilayer during the isolation. Proteins were characterized with respect to post-translational modification changes resulting from the capping process as well as changes in their association with the plasma membrane fraction. Posttranslational changes of plasma membrane proteins, such as phosphorylation, methylation and proteolytic cleavage, were not observed during the four stages of capping. Myosin heavy chain phosphorylation, however, decreased almost twofold during patching and capping. Actin, which is known to colocalize directly underneath capped receptors did not appear to be recruited to the cap from the cytoplasm.     

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.     

SITES and EFFICACY OF PHOTODAMAGE BY TIN ETIOPURPURIN in vitro USING DIFFERENT DELIVERY SYSTEMS

Photosensitization by tin etiopurpurin (SnET2) was determined in cell culture using sensitizer dissolved in ethanol or solubilized via three different delivery systems: Cremophor EL, gamma-cyclodextrin or Molecusol (a more water-soluble cyclodextrin derivative). Sensitizer uptake was substantially more efficient when the delivery systems were employed, in terms of intracellular level needed to lethally photosensitize cells. We observed photodamage at both membrane and mitochondrial loci, but the former was better correlated with loss of viability.     

Strategies for selective cancer photochemotherapy: antibody-targeted and selective carcinoma cell photolysis

A principle objective in chemotherapy is the development of modalities capable of selectively destroying malignant cells while sparing normal tissues. One new approach to selective photochemotherapy, antibody-targeted photolysis (ATPL) uses photosensitizers (PS) coupled to monoclonal antibodies (MAbs) which bind to cell surface antigens on malignant cells. Selective destruction of human T leukemia cells (HBP-ALL) was accomplished by coupling the efficient PS chlorin e(6) to an anti-T cell MAb using dextran carriers. Conjugates with chlorin: MAb ratios of 30:1 retained > 85% MA b binding activity, and had a quantum yield for singlet oxygen production of 0.7 +/- 0.1, the same as that of free chlorin e(6). Cell killing was dependent on the doses of both MAb-PS and 630-670 nm light and occurred only in target cell populations which bound the MAb. On the order of 10(10) singlet oxygen molecules were necessary to kill a cell. A second approach to specific photochemotherapy, selective carcinoma cell photolysis (SCCP), relies on preferential accumulation of certain cationic PS by carcinoma cell mitochondria. We have evaluated several classes of cationic dyes, and in the case of N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC) and some of its analogs, have demonstrated highly selective killing of human squamous cell, bladder and colon carcinoma cells in vitro. In isolated mitochondria, EDKC uptake and fluorescence depended on membrane potential, and the dye specifically photosensitized damage to Complex I in the electron transport chain. N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine and some of its analogs accumulated within subcutaneous xenografts of human tumors in nude mice with tumor:skin ratios > 8. Photoirradiation caused significant inhibition of tumor growth, without cutaneous phototoxicity.     

Interaction of chlorin p6 with bovine serum albumin and photodynamic oxidation of protein

The binding of chlorin p6, a photosensitizer having basic tetrapyrrole structure, to bovine serum albumin (BSA) and oxidation of the protein following photodynamic treatment is studied. The Stern-Volmer plot indicates that binding of chlorin p6 to BSA was of single class. Binding parameters, binding association constant and number of binding sites, were found to be 1.62+/-0.27 x 10(5)M(-1) and 1.086+/-.019, respectively. Photodynamic oxidation of protein was studied by (i) loss of intrinsic fluorescence of protein, (ii) protein carbonyl formation, (iii) protein hydroperoxide (iv) formation of TCA soluble amino groups and (v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Intrinsic protein fluorescence was observed to decrease almost linearly as a function of irradiation time at a fixed concentration of chlorin p6 and with increasing concentration of chlorin p6 at fixed time of irradiation. Protein carbonyl and hydroperoxide formation was found to increase with increasing photodynamic treatment. No significant increase in 5% TCA soluble amino groups was observed. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that photodynamic treatment of BSA in presence of chlorin p6, rose bengal and riboflavin causes non-specific fragmentation of protein. Photodynamic carbonyl formation by chlorin p6 was not inhibited by sodium formate (100 mM) or mannitol (25 mM) but was significantly inhibited by sodium azide (2 mM). Protein carbonyl formation increased almost 90% when H2O was replaced by D2O. The results show that chlorin p6 induced photodynamic oxidation of BSA was mainly mediated by singlet oxygen.     

Structure and mobility of lipid membranes on a thermoplastic substrate

Supported lipid membranes constitute one of the most important model systems for cell membranes. The properties of lipid membranes supported by the hydrophobic solid polymer cyclic olefin copolymer (COC) were investigated. Lipid layers consisting of varying amounts of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP, cationic) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, neutral) prepared by vesicle fusion and solvent exchange were compared. All lipid mixtures coated the COC surface homogeneously forming a fluid membrane as verified by fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). The exact structure of the supported membranes was determined by synchrotron reflectivity experiments using a microfluidic chamber. The X-ray data are in agreement with a compressed (head-to-head distance = 29 angstroms) and less densely packed bilayer.     

Low fluorescence background electroblotting membrane for DNA sequencing.

A low fluorescence background polypropylene (PP) membrane has been developed for ultimate use as an electroblotting membrane in DNA sequencing based on fluorescence detection. The DNA binding capacity of this membrane is improved by a surface modification using radio frequency plasma discharge (RFPD) in ammonia gas. The RFPD operational parameters are evaluated both in terms of membrane nitrogen content and in terms of the product's capacity for binding radioisotope-labeled DNA fragments. The surface morphologies of the derivatized membranes are examined by scanning electron microscopy; their mechanical and electrical properties, which are important for the subsequent sequencing procedures, are likewise established. Due to the goal of developing a membrane suitable for multiplex processing, in which the electroblotted DNA must withstand dozens of hybridization/stripping cycles, special attention is given the covalent attachment of DNA to the membrane. The modified PP membrane is evaluated in a multiplex sequencing application using radioisotope-labeled DNA probes, and found to yield somewhat better binding of a given amount of electroblotted DNA than the commonly used GeneScreen membrane. A tenfold repetition of the probing indicates little loss of signal; the membrane-bound DNA is stable upon storage and shows no detectable loss in probing efficiency after one month.     

Mechanisms of photosensitivity in porphyric patients with special emphasis on erythropoietic protoporphyria

In erythropoietic protoporphyria, protoporphyrin overproduction occurs mainly in erythroid tissue. Protoporphyrin can be released from erythrocytes in the dark, but the release is greatly increased if the erythrocytes are exposed to small amounts of light. Protoporphyrin can be bound in plasma either to albumin or to low density or high density lipoprotein. The cutaneous symptoms in erythropoietic protoporphyria are primarily elicited by protoporphyrin-sensitized photodamage of endothelial cells due to the presence of protoporphyrin in lipid structures. Which structures are damaged first in endothelial cells is unknown. Endothelial cells probably accumulate protoporphyrin from albumin or lipoproteins present in the plasma. A direct transfer from the erythrocyte membrane to the endothelial cell membrane can also occur. The transfer processes are probably facilitated by light exposure.

Degranulation of mast cells, invasion of neutrophus into interstitial tissue and complement activation seem to be of less importance than endothelial cell injury in the pathogenesis of erythropoietic protoporphyria. These processes may, however, participate in the final expression of the cutaneous symptoms.

Uroporphyrin and coproporphyrin are hydrophilic and are probably unbound in plasma, although weak binding to plasma proteins cannot be excluded. In the hepatic porphyrias and in erythropoietic porphyria, the clinical symptoms are probably evoked by uroporphyrin and coproporphyrin present in the interstitial tissue. Very little is known about the primary targets of uroporphyrin and coproporphyrin photodamage in these disorders, but photodamage to intercellular structures probably represents the initial event. Activation of complement may contribute to the final expression of the cutaneous symptoms.     

A method for visualization of biomolecules labeled by a single quantum dot in living cells by a combination of total internal reflection fluorescence microscopy and intracellular fluorescence microscopy

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 °C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490 nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.