Fluorescence lifetime imaging (FLIM) of rhodamine 123 in livingcells

Herbert Schneckenburger,*a,b Karl Stock,b Marco Lyttek,a Wolfgang S. L. Strauss b andReinhard Sailer b

a Fachhochschule Aalen, Institut für Angewandte Forschung, Beethovenstr. 1, 73430 Aalen,Germany. E-mail: herbert.schneckenburger@fh-aalen.de

b Institut für Lasertechnologien in der Medizin und Messtechnik an der Universität Ulm,Helmholtzstr. 12, 89081 Ulm

Received 30th May 2003, Accepted 26th August 2003First published as an Advance Article on the web 9th September 2003

A novel setup for fluorescence intensity and lifetime imaging (FLIM) of living cells is reported. Time-resolvingtechniques are combined with total internal reflection fluorescence microscopy (TIRFM), which permits opticalexcitation of either plasma membranes or whole cells depending on whether the angle of incidence of the excitationlight is greater or smaller than the critical angle for total internal reflection. The method is applied to BKEz-7endothelial cells incubated with various concentrations of the well established mitochondrial marker rhodamine123 (R123). Measurements show that only at low concentrations this dye is mainly located within the mitochondria,whereas at higher concentrations an accumulation within the plasma membrane occurs as well. Concomitantly,fluorescence quenching in the mitochondria is observed at high concentrations, probably due to aggregation of theR123 molecules. Therefore, for diagnostic applications the concentration of R123 in the incubation medium shouldnot be above 25 µM.

IntroductionThe application of time-resolving fluorescence techniques inbiomedical research has been a challenge for many years. Sinceradiative and non-radiative transitions from the excited molecu-lar state to the ground state are competing, the fluorescencelifetime (given as the reciprocal of the sum of all transitionrates) of a molecule is sensitive to numerous parameters, e.g.degree of aggregation or conformation. Therefore, lifetimemeasurements are appropriate to probe the microenvironmentof a fluorescent molecule. Fluorescence lifetime measurementswith spatial resolution date back to the 1980s, when cells ortissue samples were scanned first in one 1,2 and later in twodimensions.3 Since that time the implementation of time-reso-lution in laser scanning microscopy (LSM) has been improvedcontinuously,4 and one of the most recent developments con-sists of a detection device, where fluorescence decay curves aremeasured for each pixel of a LSM image.5 Parallel develop-ments include spatially resolved phase fluorometry 6–8 as well astime-gated image intensifying camera systems. The time-reso-lution of image intensifiers has recently been increased fromabout 5 ns 9,10 to the picosecond range.11 Concomitantly, detec-tion sensitivities of image intensifying camera systems havebeen improved, since the detection efficiency decreases whenlowering the time gate.

Microscopic (lateral and axial) resolution in fluorescence life-time imaging (FLIM) has so far been in the range of somehundreds of nanometers using either multiphoton micro-scopy 12 or optical sectioning by a structured illumination tech-nique.11 Very recently the axial resolution in time-resolvedfluorescence microscopy has been improved using evanescentwaves for selective excitation of fluorophores in close proximityto the plasma membrane. Time-gated spectroscopy upon excit-ation by evanescent waves was applied to single cells,13 but nofluorescence lifetime images have been reported, so far.

Rhodamine 123 (R123) is a probe of the transmembranepotential 14 and is accumulated within the inner mitochondrialmembrane.15 In addition to its properties of staining mito-chondria, a preferential accumulation of R123 in tumourcells 16 as well as some photosensitizing properties 17 have been

described. R123 has also been used to probe the function therespiratory chain located within the inner mitochondrialmembrane.18 Following optical excitation of the coenzymenicotinamide adenine dinucleotide in its reduced form(NADH), the non-radiative energy transfer from NADH to theR123 molecules was determined. According to the Förstermechanism 19 this energy transfer is limited to intermoleculardistances below 10 nm. Therefore, R123 fluorescence wasrelated to the amount of mitochondrial NADH, which wasused as a parameter to probe malfunction of enzyme complexesof the respiratory chain. A superpostion by cytoplasmicNADH or further fluorophores showing a similar emissionspectrum could thus be avoided.

In addition to mitochondrial accumulation of R123, someamounts of this dye may be located in further intracellularcompartments as well as in the plasma membrane.20 Therefore,the method of total internal reflection fluorescence microscopy(TIRFM) 21 was used to excite R123 selectively within theplasma membrane (and adjacent cellular sites) by an evanescentelectromagnetic wave, and to compare fluorescence arising fromthe plasma membrane with R123 fluorescence after illumin-ation of the whole cell. Fluorescence intensity and lifetimeimages (FLIM) of cultivated cells incubated with various con-centrations of R123 were therefore recorded using both typesof illumination, to our knowledge for the first time.

Materials and methodsBKEz-7 endothelial cells from calf aorta 22 were routinely culti-vated in Eagle’s minimum essential medium (MEM) sup-plemented with 10% fetal calf serum (FCS), glutamine (2 mM),and antibiotics (penicillin, streptomycin) at 37 �C and 5% CO2.150 cells mm�2 were seeded on microscope object slides andgrown for 48 h. After that period cells were incubated for 30min in MEM (same as above, but only with 5% FCS) contain-ing various concentrations of R123 (5, 10, 25, 50 or 100 µM)prior to rinsing with Earle’s balanced salt solution (EBSS).When using an incubation period of 30 min R123 concen-trations up to 25 µM were reported to be non-cytotoxic.18 ForD

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microscopic experiments an open chamber (filled with EBSS)and a 63 × /0.90 water immersion objective lens were used.

A fluorescence microscope (Axioplan 1, Carl Zeiss Jena,Germany) was equipped with a novel illumination device,23

which allowed the cells to be illuminated under dark field condi-tions with a variable angle of incidence Θ. Total internal reflec-tion (TIR) occurred at Θ ≥ 64.6�. Therefore, when using theangles Θ = 62� and Θ = 66� in the experiment, whole cells wereilluminated in the first case, whereas the plasma membrane andadjacent cellular sites were illuminated selectively in the secondcase (with a penetration depth d ≈ 160 nm of the evanescentwave). In all cases the electric field vector was polarized per-pendicular to the plane of incidence. As a picosecond lightsource a modelocked argon ion laser (Innova 90, Coherent,Palo Alto, USA) was used at a wavelength of 476 nm, a pulseduration of 200 ps and a repetition rate of 99 MHz. The repeti-tion rate was reduced to 2.5 MHz by a pulse picker (Pockelscell, LM 0202, LINOS Photonics, Planegg, Germany). A mono-mode quartz fiber with collimation optics at its entrance andexit (KineFLEX-P-3; Point Source, Southampton, UK) wasused to couple excitation light to the illumination device,permitting a small divergence angle of ±0.25� in the planeof the sample. Light pulses with an energy of 40 pJ each(corresponding to an average power of 100 µW) were used toirradiate an elliptic object field with a major axis of about 1 mmand a minor axis of 400 µm. No phototoxicity and almost nophotobleaching were observed under these conditions. The elec-trical output of the modelocker was used to trigger the Pockelscell as well as the time gate of the image intensifier (PicostarHR 12, LaVision, Göttingen) which was coupled to a cooledICCD camera with 640 × 480 pixels. Time gates between 200 psand 1000 ps as well as variable delay times with respect to thetrigger pulse could be adjusted. The whole setup is depicted inFig. 1.

Fluorescence decay kinetics and images were recorded fromobject fields of 220 × 160 µm, which typically contained 5–10individual cells in each case. Since the fluorescence maximum ofR123 was around 530 nm, a long pass filter for 515 nm was usedto avoid the detection of scattered light as well as autofluores-cence from the cells. Fluorescence decay kinetics were measuredby shifting the time gate in intervals of 200 ps over a scale of10–20 ns (dwell time: 1 s for each interval), and lifetimes wereobtained from mono- or biexponential curve fitting using aleast-square fitting algorithm (without deconvolution). Foreach R123 concentration between 7 and 14 fluorescence decaycurves were recorded, and the mean values as well as the stand-ard deviations of fluorescence lifetimes were calculated. In add-ition, for biexponential decays, which are described by thefluorescence intensity IF(t) = A1e

�t/τ1 � A2e�t/τ2, the relative

intensity of the component i (i = 1, 2) with fluorescence lifetimeτi and amplitude Ai was calculated according to the formula:

Fig. 1 Experimental setup for fluorescence lifetime imaging (FLIM)with variable-angle excitation.

Fluorescence images were recorded within the time intervalsof 0.5–1.0 ns (A) and 4.5–5.0 ns (B) after the pulse maximum(see Fig. 2) The acquisition time of each image was 1 s at Θ = 62�and 10 s at Θ = 66�. In addition to the fluorescence intensities IA

and IB, the effective fluorescence lifetime τeff could be displayedas an image. The value of τeff was calculated for each pixel fromthe intensities IA and IB, and the time shift ∆t = 4 ns between theintervals A and B using eqn. (2):

Only when the fluorescence decay curve was mono-exponential, τeff corresponded to the real fluorescence lifetime(as measured by decay kinetics); if this curve was biexponential,τeff depended on both lifetimes. For all fluorescence images thebackground (recorded without laser illumination) wassubtracted.

In an additional experiment, the fluorescence intensity IF

within a certain time interval close to the maximum of thefluorescence decay curve (width: 1 ns) was determined as afunction of the angle Θ of light incidence between 65� and 72�in steps of 1�. As previously shown,23 the intensity of a fluor-escence marker IF excited by an evanescent electromagneticwave can be described by eqn. (3):

if this marker is located within a thin layer (e.g. plasma mem-brane) of thickness t, or by eqn. (4):

if the marker is distributed homogenously above this layer (e.g.within the cytoplasm). In eqns. (3) and (4) F represents anexperimental constant, c the concentration of the fluorophore,∆ the average distance of the layer from the glass surface, d(Θ) =(λ/4π)(n1

2 sin2Θ � n22)�1/2 the penetration depth of the evan-

escent wave and T(Θ) = 4 cos2Θ/ [1 � (n2/n1)2] the transmission

factor through the cell/substrate interface, if the electric fieldvector is polarized perpendicular to the plane of incidence.24

For fitting the experimental data according to a two-layermodel (with refractive indices n1 = 1.53 for the glass substrateand n2 = 1.38 for the cytoplasm 23), eqns. (3) and (4) were used,and ∆ was calculated by a least-square fitting algorithm. Vari-able-angle experiments were performed using R123 concen-trations of 10 or 50 µM.

Ii = Aiτi/(A1τ1 � A2τ2) (1)

Fig. 2 Fluorescence decay curve of BKEz-7 endothelial cellsincubated with R123 (50 µM, 30 min) obtained from an image of 220 ×160 µm. Excitation wavelength: λex = 476 nm, detection range: λd ≥ 515nm, angles of incidence: Θ = 62� (whole cell illumination; upper curve)or Θ = 66� (TIR illumination; lower curve). Fluorescence intensitiesIA and IB (measured within the time gates A and B) as well as the timeshift ∆t used for fluorescence lifetime imaging are indicated.

τeff = ∆t/ln(IA/IB) (2)

IF(Θ) = FcT(Θ)te�∆/d(Θ) (3)

IF(Θ) = FcT(Θ)d(Θ)e�∆/d(Θ) (4)

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ResultsFluorescence decay curves of BKEZ-7 endothelial cells incu-bated with 50 µM R123 are depicted in Fig. 2. Either whole cells(Θ = 62�) or only plasma membranes (Θ = 66�) were illumin-ated. The time gates A and B (with intensities IA and IB) as wellas the time shift ∆t = 4 ns are also indicated. During illumin-ation of whole cells, decay curves were monoexponential with afluorescence lifetime τ = (2.7 ± 0.3) ns, if R123 concentrationsof 5 or 10 µM were used. For high R123 concentrations of 50 or100 µM, decay curves were biexponential with τ1 = (0.55 ± 0.1)ns and τ2 = (2.7 ± 0.3) ns. Relative fluorescence intensites were I1

= 0.4 ± 0.2 and I2 = 0.6 ± 0.2 for both concentrations. For aR123 concentration of 25 µM only part of the decay profilescould be fitted by two exponentials whereas all other profileswere monoexponential with the decay time τ2. In contrast, whenthe plasma membrane was illuminated selectively, the decayprofiles were always monoexponential with τ = (2.8 ± 0.2) nsat R123 concentrations between 10 and 100 µM. However,at a R123 concentration of 5 µM the fluorescence intensity atΘ = 66� was very low, and decay profiles could not be evalu-ated reliably. Differences between whole cell illumination(biexponential decay) and TIR illumination (monoexponentialdecay) can be deduced from Fig. 2.

Fig. 3 shows the patterns of fluorescence intensity (measuredin the interval A; top) and effective fluorescence lifetime (bot-tom) of BKEz-7 endothelial cells incubated with 10 µM R123upon illumination of whole cells. The intensity image showsdark cell nuclei and brightly fluorescent mitochondria, whereasthe lifetime image shows an almost homogenous distribution ofthe effective fluorescence lifetimes all over the cells. Upon select-ive illumination of the plasma membrane and its adjacent cellu-lar parts, a rather diffuse fluorescence pattern and a few brightspots can be deduced from the intensity image (Fig. 4, top). The

Fig. 3 Fluorescence intensity IA (top) and fluorescence lifetime τeff

(bottom; scale: 0–8 ns) of BKEz-7 endothelial cells incubated with 10µM R123 measured at Θ = 62� (λd ≥ 515 nm; image size: 220 × 160 µm).

bright spots probably arise from parts of those mitochondria,which are in close proximity to the plasma membrane. Again,fluorescence lifetimes (Fig. 4, bottom) were rather homogenousall over the cells with a large noise level resulting from thequotient of low intensities which were used to calculate theeffective lifetime according to eqn. (2).

After incubation of BKEz-7 cells with 50 µM R123,illumination of the whole cells results in brightly fluorescentmitochondria superimposed by some diffuse fluorescence dis-tributed all over the cells (Fig. 5, top). The corresponding life-time image (Fig. 5, bottom) shows a homogenous distributionof fluorescence lifetimes in the cytoplasm (and the cell nucleus),but a lower effective lifetime within the mitochondria. This canbe deduced from the dark spots in the lifetime image. Uponselective illumination of the plasma membrane (and adjacentcellular sites), fluorescence was distributed all over the cellsurface and was considerably higher than after incubation of10 µM R123 (Fig. 6, top). Again, bright fluorescent spots aris-ing from mitochondria in close proximity to the plasma mem-brane were identified. The fluorescence lifetime image in thiscase was homogenous all over the cell surface (Fig. 6, bottom).

In addition, the fluorescence intensity excited by the evan-escent electromagnetic wave was integrated over imagesrecorded at variable angles of incidence 65 ≤ Θ ≤ 72�. The resultfor a sample of BKEz-7 cells incubated with 50 µM R123 isdepicted in Fig. 7. This Figure also shows the fitted curves for afluorescence marker of the plasma membrane according to eqn.(3) and for a marker of the cytoplasm according to eqn. (4).Only eqn. (3) provides a reasonable fit with an average dis-tance between the plasma membrane and the glass substrate of∆ = 90 nm. For cells incubated with 10 µM R123 the angulardependence of the fluorescence signal was similar for Θ ≥ 66�,but the fluorescence intensity was considerably lower than forcells incubated with 50 µM R123 (data not shown).

Fig. 4 Fluorescence intensity IA (top) and fluorescence lifetime τeff

(bottom; scale: 0–8 ns) of BKEz-7 endothelial cells incubated with 10µM R123 measured at Θ = 66� (λd ≥ 515 nm; image size: 220 × 160 µm).Same object field as in Fig. 3.

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DiscussionFluorescence lifetime imaging revealed to be a valuable methodto probe both, the cellular location and the microenvironmentof fluorescent molecules. Precision of this method depends ontime resolution as well as on the signal/noise ratio of the detec-tion device. After background subtraction the noise level withinindividual pixels varied between 0 and 20 counts in comparisonwith measured signals within the time gate A around 4000counts (whole cell illumination), 400 counts (TIR illumination)or 40 counts (extremely low value in TIR images at low R123concentrations). Corresponding signals within the time gate Bwere about 1000, 100 or 10 counts. Without any backgrounda fluorescence lifetime τeff = 2.88 ns is calculated according toeqn. (2) when using the values mentioned above. A maximumbackground of 20 counts would result in an effective fluor-escence lifetime of 2.92, 3.19 or 5.77 ns for the three cases. Inother words, the influence of the background on effective fluor-escence lifetime is rather low for most of the experimentsreported above. However, at very low fluorescence intensities anincrease of the mean value and strong fluctuations of τeff areobserved, e.g. in the plasma membrane (at low R123 amounts,see Fig. 4) or at cell edges (Fig. 5).

As shown above, R123 is preferentially accumulated withinthe mitochondria of BKEz-7 endothelial cells after incubationwith rather low concentrations (5–10 µM). At higher con-centrations R123 is increasingly found in further intra-cellular compartments as well as within or close to the plasmamembrane. Location of this dye in further intracellularcompartments is deduced from measurements by laser scanningmicroscopy showing pronounced mitochondrial fluorescence inaddition to some diffuse fluorescence all over the cytoplasm(data not shown). Location within the plasma membrane or its

Fig. 5 Fluorescence intensity IA (top) and fluorescence lifetime τeff

(bottom; scale: 0–8 ns) of BKEz-7 endothelial cells incubated with 50µM R123 measured at Θ = 62� (λd ≥ 515 nm; image size: 220 × 160 µm).

close vicinity can be deduced from the angular dependenceof fluorescence intensity when using an evanescent electro-magnetic wave for excitation. Fluorescence lifetimes are distri-buted homogenously over the plasma membrane with a timeconstant around 2.8 ns. The lifetime remains unchanged, whenR123 concentrations are increased up to 100 µM. Fluorescencelifetimes measured inside the cells are about the same, but atconcentrations above 25 µM, an additional short-lived com-ponent (τ ≈ 0.6 ns) is detected. Short-lived fluorescence mainlyarises from the mitochondria, as shown by the lifetime image inFig. 5. Since the fluorescence lifetime τ corresponds to the

Fig. 6 Fluorescence intensity IA (top) and fluorescence lifetime τeff

(bottom; scale: 0–8 ns) of BKEz-7 endothelial cells incubated with 50µM R123 measured at Θ = 66� (λd ≥ 515 nm; image size: 220 × 160 µm).Same object field as in Fig. 5.

Fig. 7 Angular dependence of fluorescence intensity of BKEz-7endothelial cells incubated with R123 (50 µM) and fitted curves for amarker of either the plasma membrane (according to eqn. (3)) or thecytoplasm (according to eqn. (4)).

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reciprocal rate of radiative (kr) and non-radiative (knr) transi-tions from the excited molecular state to the ground stateaccording to eqn. (5):

the contribution of non-radiative transitions increases withinmitochondria with increasing R123 concentration. In otherwords, the fluorescence quantum yield η = kr/(kr � knr) isdecreased within the mitochondria at concentrations above25 µM. This kind of fluorescence quenching is often a con-sequence of aggregation and causes non-linearity betweenfluorescence intensity and fluorophore concentration. Somedecrease of the fluorescence lifetime may also result from alter-ations of the pH value or intermolecular energy transfer toadjacent molecules. However, at present, there is no indicationfor those latter cases.

Quenching of R123 fluorescence in mitochondria andaccumulation of R123 in further cell membranes (in particularthe plasma membrane) at high concentration limits the appli-cation of this dye for in vitro diagnostics. For example,for measurements of mitochondrial malfunction using non-radiative energy transfer from the mitochondrial coenzymeNADH to R123 molecules,18,25 the concentration of R123 in theincubation medium should not be above 25 µM.

Selective fluorescence lifetime imaging (FLIM) of the plasmamembrane of living cells became possible by a combination ofFLIM and total internal fluorescence microscopy (TIRFM). Inthe near future this combination may become promising fornumerous studies, e.g. for measuring dynamics of membranelipids 26 or proteins,27 for the detection of membrane-proximalion fluxes,28 for imaging of endocytosis or exocytosis,29,30 aswell as for the investigation of photosensitizers (with tumour-localizing properties) in close proximity to the plasmamembrane.31,32

Acknowledgements

The authors thank C. Hintze, M. Wagner and M. Kretzschmarfor technical assistance. The project was supported by the Bun-desministerium für Bildung und Forschung (BMBF), grants no.1706698 and 13N7514.

References1 H. Schneckenburger, F. Pauker, E. Unsöld and D. Jocham, Intra-

cellular distribution and retention of the fluorescent components ofPhotofrin II, Photobiochem. Photobiophys., 1985, 10, 61–67.

2 I. Bugiel, K. König and H. Wabnitz, Investigation of cells byfluorescence laser scanning microscopy with subnanosecondresolution, Lasers Life Sci., 1989, 3, 47–53.

3 E. P. Buurman, R. Sanders, A. Draijer, H. C. Gerritsen, J. J. F. vanVeen, P. M. Houpt and Y. K. Levine, Fluorescence lifetime imagingusing a confocal laser scanning microscope, Scanning, 1992, 14, 155–159.

4 H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia and W. G. vanSark, Fluorescence lifetime imaging in scanning microscopes:acquisition, speed, photon economy and lifetime resolution,J. Microsc., 2002, 206, 218–224.

5 W. Becker, A. Bergmann, C. Biskup, L. Kelbaukas, T. Zimmer andN. Klöcker, K. Benndorf, High resolution TCSPC lifetime imaging,in Multiphoton Microscopy in the Biomedical Sciences III, eds. A.Periasamy and P. T. C. So, Proc. SPIE, Vol. 4963, Bellingham, USA,2003, in press.

6 J. R. Lakowicz and K. Berndt, Lifetime-selective fluorescenceimaging using an rf phase-sensitive camera, Rev. Sci. Instrum., 1991,62, 1727–1734.

7 T. W. J. Gadella, T. M. Jovin and R. M. Clegg, Fluorescence lifetimeimaging microscopy (FLIM): Spatial resolution of microstructureson the nanosecond time scale, Biophys. Chem., 1993, 48, 221–239.

8 A. Squire, P. J. Verveer and P. I. H. Bastiaens, Multiple frequencyfluorescence lifetime imaging microscopy, J. Microsc., 2000, 197,136–149.

9 M. Kohl, J. Neukammer, U. Sukowski, H. Rinneberg, D. Wöhrle,H.-J. Sinn and A. Friedrich, Delayed observation of laser-induced

τ = (kr � knr)�1 (5)

fluorescence for imaging of tumours, Appl. Phys. B, 1993, 56, 131–138.

10 H. Schneckenburger, K. König, T. Dienersberger and R. Hahn,Time-gated microscopic imaging and spectroscopy in medicaldiagnosis and photobiology, Opt. Eng., 1994, 33, 2600–2606.

11 M. J. Cole, J. Siegel, S. E. Webb, R. Jones, K. Dowling, M. J. Dayel,D. Parsons-Karavassilis, P. M. French, M. J. Lever, L. O. Sucharov,M. A. Neil, R. Juskaitis and T. Wilson, Time-domain whole-fieldfluorescence lifetime imaging with optical sectioning, J. Microsc.,2001, 203, 246–257.

12 M. Straub and S. W. Hell, Fluorescence lifetime three-dimensionalmicroscopy with picosecond precision using a multifocalmultiphoton microscope, Appl. Phys. Lett., 1998, 73, 1769–1771.

13 H. Schneckenburger, K. Stock, W. S. L. Strauss, J. Eickholz and R.Sailer, Time-gated total internal reflection fluorescence spectroscopy(TG-TIRFS): application to the membrane marker laurdan,J. Microsc., 2003, 211, 30–36.

14 R. K. Emaus, R. Grunwalg and J. LeMasters, Rhodamine 123 as aprobe of transmembrane potential in isolated rat liver mito-chondria: spectral and metabiolic properties, Biochim. Biophys.Acta, 1986, 850, 436–448.

15 L. V. Johnson, M. L. Walsh and L. B. Chen, Localization ofmitochondria in living cells with rhodamine 123, Proc. Natl. Sci.USA, 1980, 77, 990–994.

16 K. Nadakavukaren, J. Nadakavukaren and L. B. Chen, Increasedrhodamine 123 uptake by carcinoma cells, Cancer Res., 1985, 45,6093–6099.

17 P. Moliè, R. Santus, M. Bazin, E. Kohen, V. Carillet, F. Bon,J. Rainasse and L. Dubertret, Is Rhodamine 123 a photosensitizer?,Photochem. Photobiol., 1990, 52, 703–710.

18 H. Schneckenburger, M. H. Gschwend, W. S. L. Strauss, R. Sailer,M. Kron, U. Steeb and R. Steiner, Energy transfer spectroscopy formeasuring mitochondrial metabolism in living cells, Photochem.Photobiol., 1997, 66, 33–41.

19 T. Förster, Zwischenmolekularer Übergang von Elektronenanr-egungsenergie, Z. Elektrochem., 1960, 64, 157–164.

20 J. L. Weaver, P. S. Pine, A. Aszalos, P. V. Schoenlein, S. J. Currier,R. Padmanabhan and M. M. Gottesman, Laser-scanning andconfocal microscopy of daunorubicin, doxorubicin and rhodamine123 in multidrug-resistant cells, Exp. Cell Res., 1991, 196, 323–329.

21 D. Axelrod, Cell-substrate contacts illuminated by total internalreflection fluorescence, J. Cell Biol., 1981, 89, 141–145.

22 W. Halle, W.-E. Siems, K. D. Jentzsch, E. Teuscher and E. Göres,Die in vitro kultivierte Aorten-Endothelzelle in der Wirkstoffor-schung – Zellphysiologische Charakterisierung und Einsatzmöglich-keiten der Zellinie BKEz-7, Pharmazie, 1984, 39, 77–81.

23 K. Stock, R. Sailer, W. S. L. Strauss, M. Lyttek, R. Steiner andH. Schneckenburger, Variable-angle total internal reflectionfluorescence microscopy (VA-TIRFM): realization and applicationof a compact illumination device, J. Microsc., 2003, 211, 19–29.

24 W. M. Reichert and G. A. Truskey, Total internal reflectionfluorescence (TIRF) microscopy (I) Modelling cell contact regionfluorescence, J. Cell Sci., 1990, 96, 219–230.

25 M. H. Gschwend, R. Rüdel, W. S. L. Strauss, R. Sailer,H. Brinkmeier and H. Schneckenburger, Optical detection ofmitochondrial NADH content in human myotubes, Cell. Mol. Biol.,2001, 47, OL95–OL104.

26 T. Parasassi, E. K. Krasnowska, L. Bagatolli and E. Gratton,Laurdan and prodan as polarity-sensitive fluorescent membraneprobes, J. Fluoresc., 1998, 4, 65–373.

27 S. E. Sund and D. Axelrod, Actin dynamics at the living cellsubmembrane imaged by total internal reflection fluorescencephotobleaching, Biophys. J., 2000, 79, 1655–1669.

28 G. M. Omann and D. Axelrod, Membrane-proximal calciumtransients in stimulated neutrophils detected by total internalreflection fluorescence, Biophys. J., 1996, 71, 2885–2891.

29 W. J. Betz, F. Mao and C. B. Smith, Imaging exocytosis andendocytosis, Curr. Opin. Neurobiol., 1996, 6, 365–371.

30 M. Oheim, D. Loerke, W. Stühmer and R. H. Chow, The last fewmilliseconds in the life of a secretory granule, Eur. J. Biophys., 1998,27, 83–98.

31 W. S. L. Strauss, R. Sailer, M. H. Gschwend, H. Emmert, R. Steinerand H. Schneckenburger, Selective examination of plasma associ-ated photosensitizers using total internal reflection fluorescencespectroscopy (TIRFS) – correlation between photobleaching andphotodynamic efficacy of protoporphyrin IX, Photochem.Photobiol., 1998, 67, 363–369.

32 R. Sailer, W. S. L. Strauss, H. Emmert, K. Stock, R. Steiner and H.Schneckenburger, Plasma membrane associated location ofsulfonated meso-tetraphenylporphyrins of different hydrophilicityprobed by total internal reflection fluorescence spectroscopy,Photochem. Photobiol., 2000, 71, 460–465.

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