Invited Paper
Fluorescence intensity, lifetime and anisotropy screening of living cells based on total internal reflection techniques Thomas Bruns1,*, Brigitte Angres2, Heiko Steuer2, Wolfgang S. L. Strauss3, and Herbert Schneckenburger1,3 1 Hochschule Aalen, Institut für Angewandte Forschung, Anton-Huber-Str. 21, D-73430 Aalen, Germany; 2 NMI - Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen, Abteilung Zelluläre Testsysteme, Markwiesenstrasse 55, D-72770 Reutlingen, Germany; 3 ILM - Institut für Lasertechnologien in der Medizin und Meßtechnik an der Universität Ulm, Helmholtzstr. 12, D-89081 Ulm, Germany * correspondence to:
[email protected]; Tel: +49-7361-576-3425; Fax: +49-7361-576-3318
ABSTRACT A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Two different screening procedures for the detection of fluorescence arising from the plasma membrane of living cells by High Throughput Screening (HTS) and High Content Screening (HCS), are distinguished. In the first case a rapid measurement of large sample numbers based on fluorescence intensity, and in the second case a high content of information from a single sample based on the parameters fluorescence lifetime (Fluorescence Lifetime Screening, FLiS) and fluorescence anisotropy (Fluorescence Lifetime Polarization Screening, FLiPS) is achieved. Both screening systems were validated using cultivated cells incubated with different fluorescent markers (e. g. NBDcholesterol) as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved, and energy transfer is disrupted, as proven by changes in fluorescence intensity and decay times. Keywords and subject terms: fluorescence screening, fluorescence lifetime, fluorescence anisotropy, total internal reflection (TIR), protein kinase c, caspase sensor, apoptosis, Förster resonance energy transfer (FRET), living cells
1. INTRODUCTION For more than 20 years1 total internal reflection (TIR) of laser light has been used to study cell-substrate interfaces in order to get more detailed information on plasma membranes of living cells. When a light beam propagating through a medium of refractive index n1 (e.g. glass) meets an interface with a second medium of refractive index n2 < n1 (e.g. cytoplasm), total internal reflection occurs at all angles of incidence Θ which are greater than a critical angle Θc = arcsin(n2/n1). While being totally reflected, the incident beam establishes an evanescent electromagnetic field that penetrates into the second medium and decays exponentially with the distance z from the interface. According to the relation
(
d = (λ 4π ) ⋅ n1 sin 2 Θ − n 2 2
)
2 −1 2
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII, edited by Daniel L. Farkas, Dan V. Nicolau, Robert C. Leif, Proc. of SPIE Vol. 7182, 718208 · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.807963
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penetration depths d between about 60 nm and more than 300 nm are attained depending on the wavelength λ and the angle of incidence Θ. Therefore, fluorophores located within or close to the plasma membrane can be examined almost selectively in living cells. So far, total internal reflection fluorescence microscopy (TIRFM) has been applied for measuring the topography of cell-substrate contacts1−3, membrane4 or protein5 dynamics, membrane-proximal ion fluxes6,7 as well as endocytosis or exocytosis8−10. With regard to potential diagnostic or pharmaceutical applications total internal reflection (TIR) techniques appear rather promising for measurements of signal transduction, intracellular translocation of molecules or membrane dynamics. Therefore, screening of a larger number of samples is desirable. For these purposes, we developed a microtiter plate reader system based on multiple TIR excitation of a laser beam on up to 96 samples in parallel. For High Throughput Screening (HTS), fluorescence arising from the plasma membrane of living cells is detected simultaneously from all samples using an integrating charge-coupled device (CCD) camera. For basic validation of the system, cultivated U373MG glioblastoma cells incubated with fluorescent membrane markers, as well as T47D breast cancer cells stably transfected with a plasmid encoding for a plasma membrane-associating yellow fluorescent protein (EYFP-Mem), were used. In addition, as a biological model system for kinetic measurements, we used T47D breast cancer cells stably transfected with a plasmid encoding for a fusion protein of protein kinase c and green fluorescent protein (PKCα-EGFP), since activation of PKC by phorbol-12-myristate-13-acetate (PMA)11 results in translocation of the protein towards the plasma membrane. However, in some cases, e.g measurements of membrane stiffness and fluidity, additional data of individual samples are needed. Therefore, an optical setup for analysis of fluorescence lifetime and fluorescence anisotropy (High Content Screening, HCS)12 was recently developed and combined with the HTS reader system13, such that individual samples selected by HTS can be examined in detail by HCS. While fluorescence lifetime represents a general measure for the interaction of a marker molecule with its microenvironment, the rotational diffusion time corresponds to the time of rotation of a molecule from a position with defined orientation into a position with arbitrary orientation reflecting directly the viscosity of the microenvironment, i.e. membrane fluidity in the case of living cells. For basic validation of the HCS system, again stably transfected T47D-EYFP-Mem breast cancer cells were used. Further validation of fluorescence lifetime and fluorescence anisotropy was accomplished with the membrane marker 22-(N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol) applied to the human glioblastoma cell line U373-MG. Resonant transfer of optical excitation energy via dipole-dipole interaction has been described by T. Förster about 60 years ago14. After successful cloning of the gene encoding for green fluorescent protein (GFP; naturally produced by the jellyfish Aequorea Victoria)15 and its variants emitting light in the blue, yellow or red spectral ranges, Förster resonance energy transfer (FRET) from an optically excited donor to an acceptor molecule has become a well established and broadly used analytical technique. This technique has also been applied for the detection of programmed cell death (apoptosis) by fusing cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP) with a caspase-3 sensitive peptide (DEVD) used as a linker16,17. Upon induction of apoptosis this linker was cleaved by caspase-3 activity, and FRET was interrupted. So far, FRET has been deduced from stationary measurements of donor (CFP) and acceptor (YFP) fluorescence18. In view of a further enhancement of FRET sensitivity as well as suppression of background fluorescence enhanced cyan fluorescent protein (ECFP) was anchored to the plasma membrane of HeLa cervical carcinoma cells, as depicted in Figure 1, and membrane associated fluorescence was excited selectively by an evanescent electromagnetic field21. So far, energy transfer from the donor (ECFP) to the acceptor (enhanced yellow fluorescent protein, EYFP) has been determined from measurements of individual cells, where apoptosis could be visualized by cell morphology18 via deduction from the ratio of acceptor / donor fluorescence as well as from the fluorescence lifetime of the donor. In contrast, larger cell collectives without any visual control were used in the present experiment, i.e. mixed populations of apoptotic and non-apoptotic cells were examined, and apoptosis (of part of the cells) was deduced from changes in fluorescence lifetimes and intensities of the donor ECFP. In view of potential applications in high content screening it was a main purpose of this experiment to excite a larger number of individual samples simultaneously upon TIR.
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EYFP Cytosol
ECFP
Caspase 3
DE VD
Induction of Apoptosis
DE VD
FRET DE VD
FRET
Plasma Membrane Glass Slide
Fig. 1. Membrane associated test system for apoptosis based on FRET and its interruption.
2. MATERIALS AND METHODS 2.1 Cell culture and cell incubation U373-MG human glioblastoma cells obtained from the European Collection of Cell Cultures (ECACC No. 89081403) were routinely grown in RPMI 1640 culture medium supplemented with 10% fetal calf serum (FCS), glutamine and gentamycin at 37°C and 5% CO2. After seeding of 500 cells/mm² within single cavities of a microtiter plate and a growth phase of 48 h (in order to obtain a sub-confluent cell monolayer), cells were incubated with RPMI medium (same as above) containing either the membrane marker 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO) at a concentration of 8 µM (20 min.) or the mitochondrial marker rhodamine 123 (R123) at variable concentrations between 5 µM and 15 µM (30 min.). It has previously been shown that in this range of concentrations R123 molecules are partly accumulated within the plasma membrane19. At the end of the incubation time cells were washed and reincubated with phosphate buffered saline (PBS) prior to fluorometric measurements. Furthermore, U373-MG human glioblastoma cells routinely grown and seeded under the conditions described above were incubated for 60 min with Earle’s Balanced Salt Solution (EBSS) containing NBD-cholesterol at a concentration of 4 µM. At the end of the incubation time cells were washed with EBSS prior to fluorometric measurements. Solutions of 4 µM NBD-cholesterol in EBSS were used for control measurements. T47D breast cancer cells obtained from the American Type Culture Collection (ATTC, Rockville, MD, USA) and stably transfected with the living coloursTM subcellular localization vector pEYFP-Mem (Clontech, Palo Alto, USA) were also routinely cultivated in RPMI 1640 medium supplemented with 10% FCS and antibiotics at 37°C and 5% CO2. Cells were seeded at different densities in single cavities of a microtiter plate and grown for 48 h prior to fluorometric measurements. Furthermore, T47D breast cancer cells were stably transfected with a plasmid encoding for a fluorescent PKCα fusion protein (pPKCα-EGFP, Clontech, Palo Alto, USA), which could be activated within the cell by phorbol12-myristate-13-acetate (PMA). PMA was used at concentrations of 1, 10 or 100 nM in the culture medium (same as above) and incubated for up to 90 min.. Subconfluent cell monolayers were obtained after a 48 h growth period. A membrane bound caspase-3-sensitive FRET probe was generated which contains the DEVD linker sequence described by Nagai and Miyawaki20 for the SCAT3.1 construct. In brief, using standard PCR techniques and the plasmids pECFPMem and pEYFP-C1 (both from BD Biosciences Clontech, Palo Alto, CA) as templates two PCR fragments were generated and fused to yield cDNA encoding the N-terminal signal sequence for posttranslational palmitoylation, followed by ECFP linked to EYFP by the SCAT3.1 DEVD sequence. This cDNA was cloned into the backbone of the vector pMemECFP to yield the plasmid MemECFP-DEVD-EYFP. A similar construct was generated containing the non-cleavable sequence DEVG instead of DEVD for control experiments (MemECFP-DEVG-EYFP). The same plasmids were also used and further described by Angres et al.21. HeLa cervical carcinoma cells were grown at 37° C, 5% CO2, in MEM growth medium containing MEM medium supplemented with 10% fetal calf serum (FCS), non essential amino acids, antibiotics and 2 mM glutamine. Transient cell transfections with plasmids were performed by
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lipofection using the FuGene transfection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. In addition, stable cell lines were prepared as described elsewhere22. For all FRET-based experiments on the TIR reader stable cell lines expressing either MemECFP-DEVD-EYFP or MemECFP-DEVG-EYFP were used. Cells were seeded at a density of 500 cells/mm2 within specific cavities on glass slides (s. below). After a growth phase of 48 h (to obtain the subconfluent monolayer), cells were incubated for 4 h with 2 µM staurosporine (S6942, Sigma-Aldrich Chemie GmbH, Germany) diluted in cultivation medium in order to induce apoptosis. Fluorescence of all samples was measured before and after incubation with staurosporine. 2.2 Total internal reflection (TIR) reader Cavities
Cavities
Microtiter plate
Microtiter plate
Mirror
Collimator
Glass Rod
Glass Rod
Fiber
8x
Microscope Objective Lens
Filter
Beam Splitter
Pol.||
Polarizing beam splitter 1x
PMT
Filter Objective
Ar+-Laser
Pulsed laser diode
Pol. ⊥
CCD Camera
PMT
Synchronisation
Fig. 2. High throughput screening (HTS) setup
Time-Correlated Single Photon Counting
Fig. 3. High content screening (HCS) setup
The basic concept is similar for both HTS and HCS setups using a microtiter plate and an evanescent electromagnetic field. As a light source an air-cooled argon ion laser or a polarized picosecond laser diode was used together with a single mode fibre-optic system including collimating optics (kineFlex, Point Source, Southampton, UK) as well as a beam splitter unit (consisting of a combination of 3 beam splitters and 3 mirrors) which divided the original laser beam into 8 beams of almost identical intensity, 1 mm in diameter and a distance of 9 mm between each other. As depicted in Figures 2 and 3, each beam was incident on a rectangular glass rod which was optically coupled (by immersion oil) to the glass bottom of a microtiter plate. 96-well cell culture plates (without bottom and with a distance s = 9 mm between adjacent wells) were obtained from Greiner GmbH (Frickenhausen, Germany). Optiwhite glass bottoms of a thickness d = 2 mm and low absorbance (optical density D = 0.045 over a light path of 12 cm; Glaswerke Haller GmbH, Kirchlengern, Germany) were fixed on the plates using a non-cytotoxic silicon adhesive (ALPA-SIL Extra, Alpina Technische Produkte GmbH, Germany). Cytotoxicity was assessed microscopically by comparison of cell growth when using either glass slides with various adhesives or control slides without adhesive. Adhesives were regarded to be noncytotoxic, if after a growth period of 48 h neither cell number nor cell morphology differed from that of control cells. Multiple total internal reflection occurs within this glass bottom, if the angle of incidence Θ is above the critical angle Θc = 63.9° (resulting from the refractive indices n1 = 1.525 for the glass bottom and n2 = 1.37 for the cells). In the present setup Θ can be calculated from the distance s = 9 mm between two cavities of the plate and the thickness d = 2 mm of the glass bottom18 according to Θ = arctan (s/2d) = 66°. Therefore, the condition of total internal reflection is fulfilled for all cavities, and a penetration depth of the evanescent field of about 150 nm within the cells is calculated according to Eq. 1. The exciting laser light is coupled out of the glass bottom using a second glass rod of identical shape in order to avoid uncontrolled reflections.
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2.2.1 High throughput screening As a light source an air-cooled argon ion laser (Mod. 5400B, Ion Lasers Technology, Salt Lake City, USA) operated at λ = 488 nm and P = 3 mW was used. Since the Gaussian profile of the laser beam is preserved in the glass bottom, all samples of the microtiter plate exposed to the evanescent field are illuminated by an ellipse of 2.5 mm ×1 mm in diameter. Fluorescence of the microtiter plate is detected simultaneously using an integrating digital CCD camera (ProgRes C10, Jenoptik GmbH, Jena, Germany) together with a wide-angular objective lens (CINEGON 1.4/12 – 0515, Schneider GmbH, Bad Kreuznach, Germany) and a long pass filter for λ ≥ 515 nm, as depicted in Figure 2. In some cases an additional band pass filter for λ = 529 ± 25 nm was used to reduce background luminescence of the glass plate. Exposure times varied between 2 s and 60 s. Cavities without any cells or any dyes served as controls, and an average intensity obtained from those control wells was subtracted from the fluorescence values of all samples. For analyzing fluorescence images of the microtiter plate (2080 x 1542 pixels), the whole images are split into matrices for individual samples of approximately 4000 elements each. Then the intensity values of each matrix element are summed up such that the fluorescence of each sample is represented by a single value. Presently, 8 samples on the left side and 8 samples on the right side of the microtiter plate are excluded from evaluation, since these samples are partly covered by the glass rods. Fluorescence of all other 80 samples is analyzed considering an adjustment of the slight differences in light intensity between the 8 individual laser beams after splitting as well as background subtraction (see above), but no further algorithms for data correction. This implies that currently no corrections are made concerning light absorption within the glass bottom or optical aberrations within the detection path. 2.2.2 High content screening The experimental setup developed for fluorescence lifetime and fluorescence anisotropy screening is depicted in Figure 3. As light sources picosecond laser diodes (LDH-P-C-400 or LDH-P-C-470 with driver PDL 800-B, PicoQuant GmbH, Berlin, Germany; pulse duration: 50-70 ps; repetition rate: up to 40 MHz) emitting light at 391 nm (average power: 65 µW) or 470 nm (average power: 100 µW) are used. Via a polarization maintaining single mode fiber and a glass rod of rectangular shape collimated laser light is coupled directly into the glass bottom of the microtiter plate for excitation of a pre-selected single row of cavities. In all cases the electric field vector is polarized perpendicular to the plane of incidence. Due to the Gaussian laser beam profile (beam diameter: 700 µm) the illumination spot on each cavity of the microtiter plate is of elliptical shape with an area of about 1 mm2. Fluorescence arising from about 2000 cells of each cavity is collected by a detection unit consisting of a microscope objective lens with 10x magnification (numerical aperture: NA = 0.30), an additional focusing lens (f = 25 mm) and a long pass filter for λ ≥ 515 nm. After passing a polarizing beam splitter, fluorescence polarized parallel [I||(t)] and perpendicular [I⊥(t)] to the exciting laser light is detected by two photomultiplier tubes (H5783-01, Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany). The signals from these photomultipliers are collected by a router (NRT 400, PicoQuant GmbH), synchronized with the laser pulses and fed to a time-correlated single photon counting device (TimeHarp 200, PicoQuant GmbH). In addition, unpolarized fluorescence I(t) = I||(t) + 2 I⊥(t) is calculated and further evaluated. The whole equipment consisting of lenses, beam splitter and photomultipliers can be moved on a programmable scanning table and positioned below each cavity of the microtiter plate. The two photomultipliers have been tuned for identical sensitivities in the two detection paths, i.e. deviations from 50:50 beam splitting have been compensated electronically. A test experiment with polarized laser light showed that cross detection of the “wrong” polarization is below 4% for both photomultipliers. Unpolarized fluorescence decay curves I(t) are fitted as a sum of exponential terms with re-convolution by the instrumental response function (IRF) corresponding to t
I (t ) = ∫ IRF (t ′)∑ Ai exp− (t − t ′) / τ i dt ′ −∞
i
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(2)
with fluorescence lifetimes τi and pre-exponential factors Ai representing the fractional contributions of the component i. The decay parameters are iteratively recovered with a nonlinear least-squares error minimization based on the Levenberg-Marquardt algorithm23. The reduced χ2 ratios and their autocorrelation functions are used to facilitate the assessment of the fit quality. In addition, the weighted residuals – corresponding to the deviations between measured and fitted values (divided by the square root of photon counts) − are calculated. Moreover, the anisotropy function r(t) is calculated from fluorescence intensities parallel [I||(t)] and perpendicular [I⊥(t)] to the exciting electric field vector according to
r (t ) =
I || (t ) − I ⊥ (t ) I || (t ) + 2 I ⊥ (t )
.
(3)
r(t) is again fitted as a sum of exponential components according to
r (t ) = ∑ Ri exp(−t / τ r ,i )
(4)
i
where τr,i correspond to the rotational relaxation times (with respect to different molecular axes) and Ri to the initial anisotropy of each component. For a comparison of fluorescence lifetimes and rotational diffusion times of whole cells (upon epi-illumination) and plasma membranes (upon TIR illumination) a fluorescence microscope was used as described elsewhere3,4. 2.3 FRET-based screening setup for apoptosis using HTS and HCS For excitation of the membrane associated donor ECFP or for direct excitation of the acceptor EYFP collimated laser beams from the previously described polarized laser diodes were coupled into a conventional microscope glass slide of 1 mm thickness (with superimposed cavities for individual samples instead of a microtiter plate) via polarization maintaining single-mode fibres and a glass rod of rectangular shape. The polarized light propagation within the glass and established evanescent field is analogue to the HCS-setup described above (see Fig. 4). In the present setup the familiar angle of light incidence Θ = 66° was chosen, resulting in a distance s = 4.5 mm between individual samples and a penetration depth d = 120−150 nm of the evanescent electromagnetic field within the cells (depending on the excitation wavelength) according to Equation 1. Due to the Gaussian shaped laser beam profile of 500 µm diameter each illumination spot on up to 12 samples (of about 1000 cells each) was of elliptical shape with an area around 0.5 mm2. For HTS measurements fluorescence intensities of all samples were detected simultaneously by the integrating digital CCD camera. ECFP fluorescence (excited at 391 nm) was measured with a band pass filter at 475 ± 20 nm, whereas EYFP fluorescence (excited directly at 470 nm) was detected with a long pass filter at λ ≥ 515 nm. Exposure times were 10 s at 470 nm and 20 s at 391 nm. Measurements of EYFP fluorescence at an excitation wavelength of 391 nm (ECFP excitation with subsequent energy transfer ECFP → EYFP) were omitted, since in this case a spectral overlap by ECFP was unavoidable. After measurement of fluorescence intensities13 prior (Ip) and after (Ia) incubation with staurosporine, the ratio Ia/Ip of all samples was calculated, and median values ± median absolute deviation (MADs) of this ratio were determined for 20−30 individual measurements of HeLa-MemECFP-DEVD-EYFP as well as HeLa-MemECFP-DEVGEYFP cells. Control measurements were carried out with cells prior and after a time interval of 4h, but without application of staurosporine. Since possible errors due to light absorption in the glass slide, variations in the number of cells per sample or optical aberrations within the detection path affected Ia and Ip in the same way, these errors were eliminated by calculation of the ratio Ia/Ip. Therefore, no further correction was needed. In addition to fluorescence intensities, we determined fluorescence lifetimes of ECFP using the HCS-setup as reported above. Fluorescence decay curves I(t) were fitted without instrumental response function as a sum of exponential terms corresponding to Equation 2.
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2.4 Microscopic measurements In addition, for a microscopic assessment of the cell models, U373-MG cells, T47D-EYFP-Mem cells or T47D-pPKCαEGFP cells were seeded on microscope slides (at a density of 150 cells/mm2) and grown for 48 h under the same conditions as mentioned before. U373-MG cells were incubated with DiO or R123 as reported above. Fluorescence was collected using a 40x/1.3 oil immersion objective lens and a long pass filter for λ ≥ 515 nm. For illumination, again an argon ion laser operated at 488 nm was used in combination with single mode fibre optics as well as a custom made dark field condenser permitting variable angles of illumination below or above the critical angle Θc 3. For detection of fluorescence images an image intensifying camera system (Picostar HR 12; LaVision, Göttingen, Germany)4 operated in continuous wave mode was used. To visualize membrane associated fluorescence of ECFP and EYFP in single cells, a fluorescence microscope (Axioplan 1, Carl Zeiss Jena, Germany) equipped with a TIR condenser (coupled to the laser diodes via single-mode fibres)3, a 40×/1.30 (oil) objective lens and an electron multiplying (EM-) CCD camera24 were used. The same system had already been used to measure fluorescence spectra and decay kinetics of individual cells21. Excitation wavelengths and spectral filtering were the same as mentioned above.
3. RESULTS AND DISCUSSION 3.1 High throughput screening fluorescence reader 3.1.1 Microscopic assessment of the cell models
(a)
(b)
Fig. 4. Fluorescence microscopy of T47D-EYFP-Mem cells illuminated at Θ = 62° (a: whole cell illumination) or at Θ = 66° (b: TIR illumination) by an argon ion laser (488 nm); fluorescence was measured at λ ≥ 515 nm (image size: 140 µm × 140 µm). Reproduced from Ref. 13 with modifications.
For fluorescence microscopy the angles of incidence of the illuminating laser beam were fixed at Θ = 62° for transillumination (whole cell measurements) and Θ = 66° for TIRFM (selective measurements of the plasma membrane and adjacent parts of the cytoplasm). Images of U373-MG cells incubated with DiO showed fluorescent intracellular filaments upon transillumination and a fluorescent membrane area with some bright spots (supposed to be cell-substrate contacts) upon TIR-illumination. Fluorescence images of U373-MG cells incubated with R123 showed a mitochondrial pattern at Θ = 62° and again some membrane-associated fluorescence at Θ = 66° (data not shown). Fluorescence images of T47D-EYFP-Mem cells are depicted in Figure 4 using again angles of incidence of 62° (a) and 66° (b) for illumination. In both cases, fluorescence mainly arises from the cell surface (plasma membrane), which appears as a sharp contour surrounding the cells at Θ = 62° and as a fluorescent area at Θ = 66°. Intracellular fluorescence of untreated T47D-PKCα-EGFP cells (excited at Θ = 62°) was rather pronounced, whereas membrane-associated
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fluorescence (excited at Θ = 66°) appeared very week and diffuse (data not shown). However, a considerable increase of membrane-associated fluorescence was observed upon application of PMA, resulting in brightly fluorescent cell surfaces at PMA concentrations of 100 nM and incubation times above 30 min.. Therefore, TIR illumination appeared to be appropriate to examine the translocation of PKCα-EGFP from the cytoplasm towards the plasma membrane upon PKC activation. 3.1.2 Experiments for validation 2.25
Fluorescence Intensity [a.u.]
2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 0
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2
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(a)
250
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Cell Density [cells/mm ]
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(b)
Fig. 5. TIR fluorescence of T47D-EYFP-Mem breast cancer cells seeded at different densities of 0, 125, 250 or 500 cells/mm² in a microtiter plate (excitation wavelength: 488 nm; fluorescence detection: 515−555 nm; recording time: 60 s); (a) image of the microtiter plate; (b) quantitative evaluation: mean values ± standard deviations of 24 measurements (250 cells/mm² and 500 cells/mm²) or 16 measurements (125 cells/mm²). Reproduced from Ref. 13 with modifications.
For validation of the TIR reader, the same cell models as reported above, i.e. incubated glioblastoma cells and stably transfected breast cancer cells, were used. Following incubation of U373-MG glioblastoma cells with the membrane marker DiO (8 µM) in 80 cavities of the microtiter plate, the fluorescence intensity was the same for all samples with a standard deviation of about ± 10%. For U373-MG cells incubated with R123, the fluorescence signal increased linearly with R123 concentration within the measured range of 5−15 µM (correlation coefficient: 0.9995). Again standard deviations determined at 5, 10 and 15 µM (with 24 samples in each case) were around ± 10% (data not shown). Furthermore, T47D-EYFP-Mem cells were seeded at different densities (125 or 250 or 500 cells/mm²) into individual cavities of the microtiter plate. Cavities without any cells served as controls and were used for background correction, as mentioned above. As depicted in Figure 5, the fluorescence intensity arising from the individual cavities reflects the number of seeded cells. This can be deduced from the fluorescence image of the microtiter plate (Fig. 5a) as well as from a quantitative evaluation of mean values and standard deviations of the samples of this plate (Fig. 5b) showing a virtually perfect linear relationship between the average fluorescence intensity and the number of cells (correlation coefficient: 0.9994). 3.1.3 Kinetic measurements Similar to microscopic measurements, fluorescence of untreated T47D-PKCα-EGFP breast cancer cells appeared rather weak upon TIR illumination at Θ = 66°. Following PKC activation with PMA at a concentration of 10 nM or 100 nM, however, an increase of fluorescence intensity was observed due to translocation of the fluorescent fusion proteins from the cytosol towards the plasma membrane. In comparison with a PMA concentration of 10 nM fluorescence increase was more pronounced at a concentration of 100 nM. Mean values and standard deviations obtained for the higher PMA concentration are depicted in Figure 6 (resulting from 8 wells of the microtiter plate and determined in intervals of 5 min. up to 50 min. as well as in intervals of 10 min. between 50 min. and 90 min. after application of PMA). Fluorescence
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intensities were normalized for the values measured at 80 minutes, since at that time the absolute intensities were most similar and differed by less than 20% between each other. After some delay time which is likely to reflect cellular uptake of the drug, TIR fluorescence shows a monotonous increase by about a factor 8 during a time period of 10−60 min. after PMA application and an almost constant fluorescence signal thereafter. The time course of fluorescence intensity can fairly well be fitted by the function
I F (t ) = I 0 + (I max − I 0 ) ⋅ [1 + exp− (t − t0 ) / τ ]
−1
(5)
with the minimum intensity I0, the maximum intensity Imax, the time constant τ as well the time t0 at the point of inflexion when IF (t) is equal to (I0 + Imax) / 2. This fitting function is also depicted in Figure 6. According to Equation 5, t0 = 24.03 min. is calculated, and a fluorescence intensity corresponding to 90% of the maximum is attained at t = 53 min. after PMA application.
Fluorescence Intensity [a.u.]
1.2
1.0
0.8
0.6
0.4
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0.0 0
10
20
30
40
50
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70
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Incubation Time [min.]
Fig. 6. Time course of TIR fluorescence intensity of T47D-PKCα-EGFP breast cancer cells after incubation with 100 nM PMA in a microtiter plate (excitation wavelength: 488 nm, fluorescence detection: ≥ 515 nm; recording time: 60 s; density of seeded cells: 500 cells/mm2); mean values ± standard deviations of 8 samples as well as an approximation by the fitting function derived from Equation 5. Reproduced from Ref. 13 with modifications.
3.1.4 Discussion (HTS) Simultaneous excitation of surfaces of living cells by an evanescent electromagnetic field became possible by splitting a laser beam and by multiple total internal reflections (TIR) within a glass bottom of a microtiter plate. In each case the angle of incidence Θ must be larger than the critical angle Θc. This implies that at a given distance between individual samples, the glass bottom must have a defined thickness, e.g. 2 mm for a 96-well plate or 1 mm for a 384-well plate. In addition, light absorbance and scattering along the plate should be small, such that all samples are illuminated by similar intensities. In our case (using Optiwhite glass) an optical density of 0.045 was determined for a path length l = 12 cm. Thus, laser intensity is reduced by about 10% when the beam is propagating along the whole plate and around 8% between the 2nd and the 11th cavity of each row (as used in the present setup), respectively. Therefore, differences in laser irradiation up to ± 4% presently contribute to the standard deviation of the measured values of about ± 10%. However, in the future differences of irradiation between individual samples could be eliminated applying an appropriate correction algorithm. In comparison with common window glass, where light intensity is reduced by about 45% over an optical path length of 12 cm (corresponding to an optical density of 0.26) light attenuation in Optiwhite glass is rather small and appears acceptable for the TIR reader. Special glass, quartz or plastic materials of even lower attenuation are available, but seem to be either too expensive for routine applications or less favourable for TIR due to their comparably low refractive index. Attenuation of the propagating laser beam due to absorption of evanescent light by the samples is rather small. When assuming for each sample an absorber concentration of c = 10-4 M, an extinction coefficient ε = 105 L / (mol × cm)
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and an absorbing layer which is thick compared with the penetration depth d of the evanescent field, the portion of absorbed light is 1.2 × 10-3 per sample or 1.44% for 12 samples within one array. The influence of attenuation can be further reduced by reflection of laser light at the end of the glass plate, such that each sample is illuminated by two totally reflected laser beams with decreasing intensity from left to right for the incident and from right to left for the reflected beam. Beam reflection, however, needs very precise optical alignment, and multiple reflections may easily cause erroneous results. As shown in the present results, TIR fluorescence was proportional to the concentration of fluorescent dyes used for incubation of the cells in the micromolar range as well as to the number of illuminated cells with membrane-associated fluorescent proteins. Assuming a doubling time of the T47D-EYFP-Mem cells of 24 h, about 1000, 2000 or 4000 cells were exposed to TIR illumination within each spot of the microtiter plate upon seeding of 125, 250 or 500 cells/mm², respectively, whereas about 17500, 35000 or 70000 cells were contained within the whole cavities (6.5 mm in diameter). The fluorescence signal arising from about 1000 T47D-EYFP-Mem cells was by a factor 4 larger than background luminescence, such that a minimum of a few 100 cells may be measured reliably. For quantification of the fluorescence signals a rather homogenous distribution of cells in the cavities is necessary. Sub-confluent cells layers may result in higher statistical spreading of the measured values than confluent cell layers, since the portion of illuminated cells within the wells may vary. Further problems arise, if cells are growing as 3-dimensional clusters or multi-layers which are not accessible to TIR illumination. The biological experiments reported above, however, indicate that those situations may be avoided, if cell measurements are performed at standardized conditions, i.e. control of cell densities upon seeding and growth times between seeding and fluorescence measurements. A further possibility to reduce statistical spreading in fluorescence measurements would be beam expansion such that larger parts (up to about 50%) of the individual wells are illuminated. However, illumination by parallel light beams with a homogenous profile should be maintained, and light scattering at the edges of the wells has to be avoided in any case. This is achieved e.g. by using lasers or laser diodes with single mode fibres and appropriate (possibly aspheric) telescope optics. Detection devices should be sensitive enough for low photon fluxes. Assuming incident laser powers around 0.3 mW per well, about 0.3 µW are absorbed by each sample (corresponding to a flux of 7.5 × 1011 photons per second). Assuming further a fluorescence quantum yield η = 0.1 and a solid angle of detection Ω = 0.1 sr, the flux of fluorescence photons on the detector (arising from one well) is about 6 × 108 s-1 (corresponding to a power of about 0.2 nW). Those low level signals can be detected when using charge coupled device (CCD) cameras in an integrating mode with recording times of several seconds up to about 1 minute. Image distortion by the wide-angular objective lens of the camera can be almost avoided, if the aperture of this lens (ratio of pupil diameter and focal length) is kept smaller than 0.5. Determination of the kinetics of intracellular translocation of PKCα after stimulation with PMA proved that the TIR fluorescence reader can also be used for dynamic measurements, e.g. for assessment of cellular uptake of pharmaceutical agents, activation of signal transduction or cytotoxic reactions. Signal acquisition and evaluation can be automated in these cases, such that the limiting factor will be the recording time for an individual image. When using an integrating CCD camera (as described above), a few seconds are needed for each image, such that the time resolution of kinetic measurements is presently limited to about 5−10 seconds. Further advances in camera technology may improve this time resolution in the near future. In view of future applications of the TIR fluorescence reader for high throughput screening (HTS) it is essential to mention that microtiter plates can be easily exchanged without readjustment of the optical setup. In addition, due to the low penetration depth of the evanescent field only very small signals arising from cell culture media or supernatants are registered when using TIR illumination. Therefore, samples will become measurable in microtiter plates without any time-consuming washing procedures. 3.2 High content screening fluorescence reader More specific fluorescence parameters, i.e. lifetimes or polarizations, can be obtained by replacing the CCD camera by appropriate detectors. Although this may require a scanning of the samples with a prolonged recording time, it results in
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a higher degree of information (high content screening, HCS). A combination of HTS and HCS appears valuable for the measurement of membrane dynamics based on spectral, polarization or lifetime data, as already reported for fluorescence microscopy4. 3.2.1 Fluorescence lifetime screening For validation of the HCS-system time-correlated fluorescence of individual samples with about 2000 cells each was registered in unpolarized and polarized mode. Unpolarized fluorescence decay kinetics are depicted in Figure 7 for U373-MG glioblastoma cells incubated with NBD-cholesterol as well as for T47D-EYFP-Mem transfectants expressing a membrane-associating fluorescent protein. The instrumental response curve (IRF) of a cavity without any cells (also depicted in this Figure) served as a control and was used for re-convolution fitting. Fluorescence decay curves were fairly biexponential, and corresponding fitting curves as well as weighted residuals are included in Fig. 7. 1
U373-MG w. NBD-cholesterol (4µM) 0.1
Weighted Residuals
Amplitude [norm.]
T47D-EYFP-Mem
IRF 0.01
0
5
10
15
20
25
30
35
40
45
6 3 0 -3 -6
U373-MG w. NBD-cholesterol (4µM) 0
5
10
15
20
25
30
0
5
10
15
20
25
30
6 3 0 -3 -6
35
40
45
T47D-EYFP-Mem 35
40
45
Time [ns]
Time [ns]
(a)
(b)
Fig. 7. (a) Normalized fluorescence intensity I(t) of U373-MG glioblastoma cells incubated with NBD-cholesterol (4 µM, 60 min) and of stably transfected T47D-EYFP-Mem breast cancer cells together with bi-exponential fitting curves and instrumental response function (IRF). (b) Weighted residuals. Excitation wavelength: 470 nm; detection range : λ ≥ 515 nm. Reproduced from Ref. 12 with modifications. Table 1. Lifetimes τi and normalized amplitudes Ai of membrane associated fluorescence of U373-MG glioblastoma cells incubated with NBD-cholesterol (4 µM; 60 min) as well as of T47D-EYFP-Mem cells (excitation wavelength: 470 nm; detection range: λ ≥ 515 nm). Values represent medians and median absolute deviations (MADs) of 40 individual samples of the microtiter plate in each case. U373-MG with NBD-cholesterol (4µM) T47D-EYFP-Mem A1 [norm.]
0.52
0.75
τ1 [ns]
7.27 (± 0.07)
2.89 (± 0.04)
A2 [norm.]
0.48
0.25
τ2 [ns]
1.60 (± 0.04)
0.93 (± 0.11)
3.04
1.42
2
χ
The parameters (Ai, τi) obtained from bi-exponential curve fitting (with re-convolution) are summarized in Table 1. Values represent medians and median absolute deviations (MADs) of 40 individual measurements of different samples of the microtiter plate. The amplitude of the short-lived component was almost similar (NBD-cholesterol) or about 3 times lower (EYFP-Mem) than that of the long-lived component. The low MAD values demonstrate that the fluorescence lifetimes of individual samples are highly reproducible. Fluorescence lifetimes of NBD-cholesterol in U373-MG cells are about twice as high as those measured for NBD-cholesterol solutions. Additional measurements in a fluorescence microscope proved that fluorescence lifetimes of whole cells (upon epi-illumination) and plasma membranes (upon TIR illumination) differ by less than 10%.
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3.2.2 Fluorescence anisotropy screening 22500 20000
I(t)
Amplitude [counts]
17500
III(t)
15000 12500
I (t)
10000 7500 5000 2500 0 0
2
4
6
8
10
12
14
16
18
20
Time [ns]
Fig. 8. Fluorescence intensities I||(t), I⊥(t) and I(t) = I||(t) + 2 I⊥(t) of stably transfected T47D-EYFP-Mem breast cancer cells. Excitation wavelength: 470 nm; detection range : λ ≥ 515 nm.
Fluorescence kinetics I||(t) and I⊥(t) (polarized) as well as I(t) = I||(t) +2 I⊥(t) (unpolarized) of T47D-EYFP-Mem cells are depicted in Figure 8. I||(t) and I⊥(t) result from both, fluorescence decay and rotational diffusion. The anisotropy functions r(t) calculated from Equation 3 for T47D-EYFP-Mem cells and U373-MG glioblastoma cells incubated with NBD-cholesterol are depicted in Figure 9. Both anisotropy curves decrease monotonously from an initial value R0 and are fitted as mono-exponential functions (also depicted in Fig. 9a), resulting in a rotational relaxation time τr = 7.88 ± 0.81 ns, an initial anisotropy R0 = 0.275 ± 0.019 and χ2 = 1.026 for U373-MG glioblastoma cells incubated with NBD-cholesterol as well as τr = 6.17 ± 0.26 ns, R0 = 0.348 ± 0.020 and χ2 = 1.439 for T47D-EYFP-Mem cells. Values represent medians ± MADs of 20 individual samples in each case. χ2 values as well as the weighted residuals depicted in Fig. 9b indicate a rather good exponential fit for NBD-cholesterol and some deviations from the monoexponential behaviour for EYFP-Mem. Rotational relaxation times of NBD-cholesterol in U373-MG cells exceeded those measured for NBD-cholesterol solutions by a factor 2.3, and also the initial anisotropy R0 was considerably lower in solution (around 0.17). Again, the values of rotational relaxation times of whole cells and plasma membranes differ by less than 10% when measured in the fluorescence microscope. 0.40 0.35
T47D-EYFP-Mem
0.25
U373-MG w. NBD-cholesterol (4μM)
Weighted Residuals
Anisotropy
0.30
0.20 0.15 0.10 0.05 0.00 0
5
10
15
20
25
30
35
40
45
6 3 0 -3 -6
U373-MG w. NBD-cholesterol (4µM) 0
5
10
15
20
25
30
0
5
10
15
20
25
30
6 3 0 -3 -6
40
45
T47D-EYFP-Mem 35
40
45
Time [ns]
Time [ns]
(a)
35
(b)
Fig. 9. (a) Anisotropy functions r(t) of stably transfected T47D-EYFP-Mem breast cancer cells and U373-MG glioblastoma cells incubated with NBD-cholesterol (4 µM, 60 min) including mono-exponential fitting curves and (b) weighted residuals. Excitation wavelength: 470 nm; detection range: λ ≥ 515 nm. Reproduced from Ref. 12 with modifications.
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3.2.3 Discussion (HCS) A High Content Screening System (HCS) for the parameters fluorescence lifetime and fluorescence anisotropy has been established with TIR excitation. The system has been validated with cultivated cells either incubated with a fluorescent membrane marker or expressing a membrane-associating fluorescent protein. Highly reproducible fluorescence lifetimes and rotational diffusion times have been measured for a larger number of samples. Therefore, plasma membrane associated parameters, e.g. membrane fluidity or protein dynamics can be measured reliably. In addition, the HCS system has been combined with the TIR fluorescence reader13 described above, so that a larger number of samples can be screened rapidly, whereas a smaller number of selected samples is examined for fluorescence lifetime and anisotropy parameters. Fluorescence decay curves of NBD-cholesterol in U373-MG cells as well as of T47D-EYFP-Mem cells showed a biexponential behaviour, indicating that different molecular conformations or locations of the fluorophores might co-exist. This seems to be similar in the plasma membrane and in intracellular membranes, since fluorescence lifetimes are almost identical upon TIR and epi-illumination. In contrast, fluorescence lifetimes of NBD-cholesterol in solution are smaller by a factor 2 in comparison with cell measurements, which again demonstrates the important role of its micro-environment. For NBD-cholesterol as well as T47D-EYFP-Mem cells the relative fluorescence intensity − corresponding to the normalized product of relative amplitude and fluorescence lifetime A2 τ2 / (A1 τ1 + A2 τ2) of the short-lived component − is comparably small. This may explain why only one rotational diffusion time can be resolved in each case. Rotational diffusion times of NBD-cholesterol in U373-MG cells and T47D-EYFP-Mem cells are similar, but only in the case of NBD-cholesterol the clear mono-exponential behaviour of r(t) indicates rather free molecular rotation, whereas in the case of EYFP-Mem deviations from this mono-exponential behaviour may result from some restriction in molecular rotation due to membrane binding. The initial anisotropy R0 mainly reflects the angle β between optical excitation and emission dipoles of the fluorophores which according to the relation25 R0 = 2/5 [(3cos2β-1)/2] is about 17° for EYFPMem and 27° for NBD-cholesterol. TIR measurements of fluorescence anisotropy may largely contribute to studies of membrane dynamics of living cells. Presently, membrane stiffness and fluidity are investigated as a function of temperature, cell age and intracellular cholesterol4,26. Changes of cholesterol amounts in cell membranes have been related to various diseases27-29 and may have some influence on the uptake of pharmaceutical agents. Therefore, in the future fluorescence anisotropy screening using TIR illumination may also have some potential for clinical studies. 3.3 FRET-based screening setup for apoptosis using HTS and HCS TIR images of HeLa-MemECFP-DEVD-EYFP cells under the fluorescence microscope are depicted in Fig. 10 upon optical excitation of donor (a) or acceptor (b) molecules. Donor fluorescence in Fig. 10a was selected by an interference filter at 475 ± 20 nm, whereas acceptor fluorescence in Fig. 10b was measured exclusively at λ ≥ 515 nm. Similarity of both images proves co-localization of ECFP and EYFP molecules within the MemECFP-DEVD-EYFP complex. Intensitiy ratios Ia/Ip of the donor ECFP in TIR reader experiments after (Ia) and prior (Ip) to the application of staurosporine are given in Table 2 for HeLa-MemECFP-DEVD-EYFP as well as for HeLa-MemECFP-DEVG-EYFP cells (the latter containing the non-cleavable linker DEVG). This table shows that the intensity of ECFP fluorescence in HeLa-MemECFP-DEVD-EYFP cells increased upon apoptosis, when energy transfer to EYFP was disrupted, but remained constant in HeLa-MemECFP-DEVG-EYFP cells as well as in control cells (without incubation with staurosporine). In contrast to ECFP, EYFP fluorescence decreased in HeLa-MemECFP-DEVD-EYFP as well as in HeLa-MemECFP-DEVG-EYFP cells. In the case of the DEVD construct one would expect that this was due to cleavage of EYFP from ECFP and subsequent EYFP movement out of the evanescent field of illumination. However, EYFP decreased also in cells containing the DEVG control construct where cleavage was not expected to occur21 (and where consequently ECFP fluorescence did not increase). Looking for an explanation of this finding we observed in microscopic experiments that part of the cells were detached from the surface upon incubation with staurosporine. Their escape from the evanescent electromagnetic field might be a dominating effect in the TIR reader for both cell lines and could explain a general decrease of fluorescence which appeared to be more pronounced for EYFP than for ECFP. Evaluation of EYFP fluorescence may be further complicated by some fluorescence fading observed in control
Proc. of SPIE Vol. 7182 718208-13
measurements of HeLa-MemECFP-DEVG-EYFP cells (without incubation with staurosporine). Therefore, up to now only the fluorescence intensity of MemECFP, but not that of EYFP can be used as an indicator of apoptosis.
(a)
(b)
Fig. 10. TIR images of HeLa cells transiently transfected with the MemECFP-DEVD-EYFP plasmid upon excitation of the donor (ECFP) at 391 nm and its detection at 475 ± 20 nm (a) as well as upon excitation of the acceptor (EYFP) at 470 nm and its detection at λ ≥ 515 nm (b). Image size: 140 µm × 140 µm. 1.0
prior to apoptosis after apoptosis
0.9
Amplitude [norm.]
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
2
4
6
8
10
12
14
16
18
20
Time [ns]
Fig. 11. Fluorescence decay curves of ECFP in HeLa-MemECFP-DEVD-EYFP cells prior and after application of staurosporine (individual samples). Excitation wavelength: 391 nm; detection range: 475 ± 20 nm. Reproduced from Ref. 22 with modifications.
Decay curves of ECFP fluorescence in HeLa-MemECFP-DEVD-EYFP cells after picosecond laser pulse excitation are depicted in Fig. 11 prior and after application of staurosporine. Fluorescence decrease appears slightly longer after incubation with staurosporine. This is confirmed by the median values ± MADs of 21 individual experiments depicted in Table 3. All decay curves were fairly tri-exponential with fluorescence lifetimes around 0.65 ns, 2.6 ns and 10.6 ns, relative amplitudes of about 0.52, 0.41 and 0.07, and χ² values around 1.15. Upon addition of staurosporine almost no changes of relative amplitudes were observed, whereas all fluorescence lifetimes increased in HeLa-MemECFP-DEVDEYFP and remained constant in HeLa-MemECFP-DEVG-EYFP cells. Energy transfer from donor (ECFP) to acceptor (enhanced yellow fluorescent protein, EYFP) molecules was deduced from the ratio of acceptor / donor fluorescence as well as from the fluorescence lifetime of the donor which according to
1 τ − 1 τ 0 = k ET
(6)
permits to calculate the energy transfer rate kET (τ , τ0 = fluorescence lifetimes in presence and absence of the acceptor, respectively). This indicates that according to Equation 6 energy transfer was interrupted or lowered when the cleavable
Proc. of SPIE Vol. 7182 718208-14
linker DEVD, but not when the non-cleavable linker DEVG was used and shows that apoptosis can be evaluated in principle on the basis of fluorescence lifetimes of a membrane associated fluorescent protein (ECFP). A prolongation of the fluorescence lifetime τ2 of ECFP upon interruption of energy transfer ECFP → EYFP has already been detected by fluorescence microscopy of individual apoptotic cells21. Since the induction of apoptosis by staurosporine after four hours is never homogeneous in a cell population30, it should be emphasized that a similar prolongation is now measured for a mixed population of apoptotic and non-apoptotic cells without any visual control. This prolongation now includes the fluorescence lifetimes τ1 and τ3, as depicted in Table 3 and predicted by Equation 6. Only the two shorter components (τ1, τ2), but not the comparably weak long-lived component (τ3) can be correlated with literature data of (E)CFP fluorescence31,32. Table 2. Intensity ratio of ECFP and EYFP fluorescence after (Ia) and prior (Ip) to incubation with staurosporine (4 h) in two transfected HeLa cell lines (medians ± MADs). ECFP was excited at 391 nm and measured at 475 ± 20 nm, whereas EYFP was excited at 470nm and measured at λ ≥ 515 nm. In control experiments Ia was also determined after 4 h, but without staurosporine. In parenthesis the number of measurements is indicated.
HeLa-MemECFP-DEVD-EYFP ECFP EYFP
HeLa-MemECFP-DEVG-EYFP ECFP EYFP
Ia/Ip
1.1185 ± 0.0395 [30] 0.7731 ± 0.0384 [20] 0.9838 ± 0.0267 [20] 0.6608 ± 0.0616 [20]
Ia//Ip
0.9864 ± 0.0179 [30] 0.9705 ± 0.0170 [20] 0.9789 ± 0.0138 [20] 0.8526 ± 0.0263 [20]
control
Table 3. Decay times, relevant amplitudes (normalized to 1) and χ2 values of ECFP fluorescence after and prior to incubation with staurosporine (4 h) in two transfected HeLa cell lines (medians ± MADs). ECFP was excited at 391 nm and measured at 475 ± 20 nm. In parenthesis the number of measurements is indicated. HeLa-MemECFP-DEVD-EYFP prior to incubation +2µM staurosporine
HeLa-MemECFP-DEVG-EYFP prior to incubation +2µM staurosporine
τ1 [ns]
0.656 ± 0.018 [21]
0.684 ± 0.023 [21]
0.626 ± 0.036 [21]
0.623 ± 0.022 [21]
τ2 [ns]
2.626 ± 0.070 [21]
2.746 ± 0.056 [21]
2.617 ± 0.058 [21]
2.614 ± 0.041 [21]
τ3 [ns]
10.64 ± 0.30 [21]
11.43 ± 0.15 [21]
11.52 ± 0.46 [21]
11.17 ± 0.22 [21]
A1 [norm.]
0.522 [21]
0.516 [21]
0.510 [21]
0.535 [21]
A2 [norm.]
0.405 [21]
0.413 [21]
0.421 [21]
0.400 [21]
A3 [norm.]
0.073 [21]
0.071 [21]
0.069 [21]
0.065 [21]
1.157 [21]
1.128 [21]
1.132 [21]
1.152 [21]
2
χ
Commonly, fluorescence readers in clinical and pharmaceutical laboratories include parallel detection of larger cell collectives in microtiter plates, e.g. for evaluation of drug effects on cells. Those plate readers have previously also been used for stationary measurements of FRET including its application to apoptosis33,34. The main advantages of the present reader system over existing systems are the involvement of fluorescence lifetime measurements as well as sample excitation by an evanescent electromagnetic field with multiple TIR of an incident laser beam. This permits versatile measurements of membrane associated fluorophores within nanometre ranges offering perfect suppression of background fluorescence from inner parts of the cells as well as from the surrounding medium. Therefore, the present system appears to be a sensitive device, not only for detection of apoptosis, but more generally for drug screening and in vitro diagnostics in a nanometre scale.
Proc. of SPIE Vol. 7182 718208-15
ACKNOWLEDGMENTS The two projects about HTS and HCS were supported by the Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg. The project about the FRET-based caspase sensor for detection of apoptosis was funded by the Landesstiftung Baden-Württemberg GmbH. In this regard we thank the staff members of the Core Facilities of the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) of the Universitätsklinikum Tübingen for conducting the cell sorting. Technical assistance in all projects by Claudia Hintze is gratefully acknowledged.
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