0 1992 Wiley-Liss, Inc.

Cytometry 13561-570 (1992)

Preparation and Microscopic Visualization of Multicolor Luminescent Immunophosphors' H.B. Beverloo, A. van Schadewijk, J. Bonnet, R. van der Geest, R. Runia, N.P. Verwoerd, J. Vrolijk, J.S. Ploem, and H.J. Tanke Department of Cytochemistry and Cytometry, Sylvius Laboratory, State University of Leiden, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Received August 15, 1991; accepted February 5, 1992

The preparation of charge-stabilized suspensions of small phosphor particles (0.14.3 pm) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 pm and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-de-

INTRODUCTION Fluorescent immunocytochemical assays are increasingly utilized for a variety of applications in pathology, oncology, and genetics, mainly because fluorescent labels are very well suited for simultaneous detection of multiple antigens (1,9,15). A disadvantage of these methods, however, is the fact that their theoretical sensitivity is hardly reached because of confounding nonspecific fluorescence due to autof luorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components of the microscopic system. The contrast between specific fluorescence and autofluorescence can be enhanced by using dyes which are excited a t longer wavelengths and emit in the redhear-infra-red part of the spectrum (16), or better by applying time-resolved microscopy. Autofluorescence generally is a rapidly decaying process with fluorescence lifetimes in the range of 1100 ns, whereas phosphorescence and delayed luminescence have lifetimes in the range of hundreds of micro-

layed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads. o 1992 Wiley-Liss, Inc. Key terms: Delayed fluorescence, phosphorescence, immunophosphors, timeresolved microscopy, multiparameter immunocytochemical staining, far UV excitation fluorescence microscopy

seconds. Time-resolved microscopy is applicable if the luminescence lifetime of the label is significantly longer than the lifetime of the disturbing autofluorescence. We recently described a new type of label for this purpose, consisting of ground luminescent inorganic crystals (phosphors), widely used in cathode-ray tubes, television screens, and luminescent lamps. The luminescence of these phosphors is strong and practically non-fading and not significantly influenced by pH or temperature. It has been shown that phosphor labeled macromolecules (immunophosphors) can be used for the demonstration of membrane antigens (3,17). Oser et al. (lo), Hemmila et al. (6) and Soini et al.

'This work was supported by the Council for Medical Research (NWO-GMW) grant 900-534-059, the Netherlands Organization for Scientific Research (NWO-PGS) grant 90-129.90, and BIORES B.V., Zeist, The Netherlands.

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REVERLOO ET rlL.

(13) described soluble labels which in principle are suitable as label for time-resolved microscopy. These labels mostly contain Eu3 ' ions as luminescent center. Since the water molecules originally surrounding the Eu3+ ion reduce the Eu3+ fluorescence quantum effciency, the luminescent part has to be dissociated from the original binding site and has to be enclosed in trapping agents to acquire a sensitive assay. This release is incompatible with cytochemical staining. Soini et al. reported a europium label suitable for cytochemical purposes, but did not demonstrate the suppression of autofluorescence and background (14). Observation of delayed fluorescence or phosphorescence in a microscopic system has also been described by Zotikov and Polyakov and by Jovin et al. In the microscope described by Zotikov and Polyakov (18)one rotating disc is regulating the opening and closure of the excitation and emission light paths synchronously in order to examine the phosphorescence signals only. Jovin et al. separated the autofluorescence and the slowly decaying phosphorescence with slaved choppers on the excitation and emission sides and integrated the luminescence with a solid-state camera (7). Eosin and erythrosin and other phosphorescent and delayed luminescent labels can be used in this microscope, a s reviewed by Jovin and Vaz (8).This paper describes the preparation of various types of phosphor conjugates in order to perform simultaneous labeling of multiple antigens with two or more immunophosphors. At first the stabilizing properties of several polycarboxylic acids were evaluated. Phosphors that differ in spectral properties andior in lifetime were ground in media containing polycarboxylic acids that provided sufficient stabilization. Immunoreactive macromolecules were subsequently adsorbed to their surface. The immunocytochemical properties of immunophosphors prepared in this way were evaluated on fixed human lymphocytes. Double labeling with these immunophosphors was performed in a model system on differently haptenized latex beads. A time-resolved microscope, introduced for the visualization of immunophosphors, is described in detail. Moreover, a microscope providing excitation with deep UV light for visualization of multicolor immunophosphors (blue, green, red) is presented.

Table 1 Characteristics of Phosphors Tested for Their Suitability as Immunophosphor Excitation maximum (nm)

Type of uhosphor

Y,0,S:Eu3 Y,O2S:Tb3+ Y,03:Eu3* Y,O,S:Zn,SiO,:Mn,As ZnS:Ag (ZnCd)SCu,Al ZnS:Ag+Al+Ga ZnS:Cu + A1 + Ga +

265, 305 275 250 267 248 317, 344 345, 393 311, 338 345, 386

Emission maximum (nm) 616, 625, 704 490, 546, 588 612, 630 -

522 448 540

448 527

Decay-time, 37% 764 ps 500 ps 2 ms 11 ms 30 ks 28 FS 260 ps -

with a diameter of 850-1,230 pm (Ballotine, lead glass; Tamson, Zoetermeer, The Netherlands) were added to obtain a bead layer of 2.0-2.5 cm in height. The suspension was then ground with a mechanical stirrer until the desired particle size was obtained; this occurred usually after 24 h, a s controlled by microscopic observation. The suspension was decanted and stored at 4°C. The polycarboxylic acids which were used as stabilizing agents are given in Table 2. Generally, yttriumoxisulfide activated with europium, Y,0,S:Eu3 , was used to determine the optimal stabilization and grinding conditions, since microscopic control of the particle size of this phosphor could be easily done with standard fluorescence microscopy using excitation with near UV light. Grinding of the phosphors was continued until a stable suspension of small particles with sufficient luminescence output was achieved. The phosphor suspension was washed three times with Hepes buffered saline (HBS: 10 mM Hepes (Serva, Heidelberg, Germany), 13.5 mM NaC1; pH 7.4) by centrifugation (4,000 rpm, 30 min), to remove all non-reacted polycarboxylic acids and sonicated (20 s, 40 W, on ice, Branson sonifier B-12 equipped with a microtip, Danbury, CT). The stability of the phosphor suspension was evaluated by judging the Brownian movement of the negatively charged phosphor particles, indicating good stabilization, and by the amount of aggregation, indicating instability. Electron microscopy was performed to examine the size and size variation of the particles obtained. Y,02S:Eu3 was also ground in distilled water with a pH adjusted to 9.0 with 0.01 N NaOH. The grinding procedure was the same as described above except for the fact that polycarboxylic acids were omitted. Polycarboxylic acids were used to stabilize the different kinds of phosphor. +

+

MATERIALS AND METHODS Preparation of Phosphor Suspensions Inorganic crystals, mostly the so-called characteristic phosphors, were used as starting material. These phosphors (Table l j , kindly provided by the Nederlandse Philips Bedrijven (Dept. ELCOMA, Eindhoven, The Netherlands), have a n original size of 6-10 pm, and were ground to a size of 0.1-0.3 pm in a beaker. The general grinding procedure was the following. The phosphors (0.8% wiv) were suspended in 100 ml 5% polycarboxylic acid (vivj in distilled water and the pH was adjusted to pH 9.0 with 0.1 N NaOH. Glass beads

Preparation of Phosphor Conjugates Proteins were conjugated to the surface of the phosphor by physical adsorption using the following protocol. The phosphor suspension (0.4 mg phosphor particles per ml suspension) was washed twice with HBS, pH 7.4, and sonicated. The suspension ground in distilled water was either washed with HBS, pH 7.4, or

PREPARATION AND VISUALIZATION O F MULTICOLOR IMMUNOPHOSPHORS

Table 2 Molecular Weight and Chemical Composition of Polycarboxylic Acids Used for Stabilization Polycarboxylic acids Additol XW330 (PA-30)

30,000-50,000

Tamol 731

10,000

10

Tamol N micro

10,000

9.6

Versicol E5 Versicol E7 Versical E 11

3,500 30,000

1-2 1-2 1-2

MW

PH 7-8

Compound

Ammonium salt of polyacrylic

of

563

Phosphor Suspensions

Source Hoechst, Frankfurt, Germany

acid

250.000

Sodium salt of carboxylate polyelectrolyte Sodium salt of a sulfonated naphtalene formaldehyde condensate Polyacrylic acid Polyacrylic acid Polvacrvlic acid

with distilled water of neutral pH. Proteins were added to the various phosphor suspensions in defined concentrations (Table 3). Suspensions were agitated overnight a t 4°C or for 2 h at room temperature, and washed three times with HBS, pH 7.4, in order to remove unadsorbed proteins. The conjugates were then washed once with 0.1% BSNHBS, pH 7.4 (BSA, purity 98-99%, Sigma, St. Louis, MO), resuspended in this medium by sonication, and stored at 4°C. Adsorption of the macromolecule or antibody to the various polycarboxylic acid stabilized phosphors was validated and in some cases optimized with FITC labeled macromolecules and antibodies. In the case of the phosphors ground in distilled water, they were conjugated to macromolecules either in distilled water or in HBS, pH 7.4. After conjugation, both suspensions were stabilized in 0.1% BSAIHBS; pH 7.4.

Rohm and Haas, Brussels, Belgium

Allied Colloids, Bradford, UK

three times with 0.1% BSAIHBS, pH 7.4, and the beads were stored in 0.1% BSAIHBS, pH 7.4, at 4°C. All immunocytochemical staining and washing steps were performed in 0.1% BSA/HBS, pH 7.4, a t room temperature. The polyclonal antibodies used were all IgG fractions of the antiserum against the species IgG (Sigma, St. Louis, MO) unless otherwise stated.

ImmunocytochemicalApplications Immunocytochemical incubations of 5% paraformaldehyde fixed human lymphocytes were executed as formerly described (3).The lymphocytes were stained for the presence of the CD4 epitope on their membranes, using a mouse anti-leu3a monoclonal antibody (Becton Dickinson, Mountain View, CA) in a 1500 dilution of the monoclonal, followed by incubation with a second layer of antibodies, consisting of either a 1:250 dilution of goat anti-mouse or a 1 : l O O dilution of biotinylated Evaluation of the Phosphor Conjugates goat anti-mouse antibodies (Sigma, St. Louis, MO) for The phosphor conjugates were cytochemically evalu- 45 min. After the cells were washed three times, they ated in three systems: fixed human peripheral blood were incubated with a 1 5 0 dilution of matching phoslymphocytes and fixed human erythrocytes, and latex phor conjugates. Microscopic preparations were made beads with a defined amount of haptenized antibodies by pipetting 10 pl of labeled cell suspension on a glass attached to their surface. slide, covered with a coverslip, and mounted a t the Lymphocytes were isolated by density gradient cen- edges with Fluoromount mountant (Gurr, High Wytrifugation using Ficoll-Isopaque. The cells were fixed combe, UK). with 5% paraformaldehyde in PBS for 1h at room temIn case of immunocytochemical labeling of erythroperature, washed with PBS, and stored in PBS a t 4°C. cytes, rabbit anti-human erythrocyte membrane antiErythrocytes were fixed with 0.1% glutaraldehyde in bodies (Dakopatts, Glostrup, Denmark) at a dilution of PBS for 10 min, washed three times with PBS, and 1:6,000 were used a s primary antibodies followed by stored at 4°C. application of a 1:25 dilution of a goat anti-rabbit imBlank latex beads with a diameter of 5.0 Fm or 9.0 munophosphor conjugate or a protein A (Pharmacia, p m (Flow Cytometry Standards Corp, NC) were hap- Uppsala, Sweden) immunophosphor conjugate. The tenized by covalent conjugation of biotinylated sheep erythrocytes and the immunophosphors were allowed anti-mouse, digoxigenin labeled sheep anti-mouse Ig to react in a reaction tube (Eppendorf: 1.5 ml) without (F(abj, fragments (Boehringer Mannheim Gmb, Ger- centrifugation, for 1 h. many) or rabbit anti-goat (FITC) Ig to the surface of the Multiple labeling of antigens was evaluated, using latex beads. These antibodies were conjugated by add- latex beads with (hapten labeled) polyclonal antibodies ing the antibodies (50 pg antibody per ml latex bead attached to their surface, as model system. For multisuspension) and 0.52 M EDC (l-ethyl-3-(3-dimethyl ple labeling the differently haptenized latex beads aminopropyl carbodiimide HC1; Serva, Heidelberg, were mixed in equal amounts, and the latex beads were Germany) simultaneously (5)to the latex bead suspen- firstly incubated with, e.g., avidin d immunophosphor sion which was, prior to conjugation, washed twice conjugate. Generally the progression of the immunocywith HBS, pH 6.0, spun down (250g, 5 min) and resus- tochemical labeling was monitored microscopically afpended. After 2 h of reaction, the mixture was washed ter taking a small sample from the reaction mixture.

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BEVEKLOO ET AL.

Table 3 Concentrations and Type of Macromolecules Used for Adsorption to the Surface o f the Phosphors Protein Avidin d

Concentration (u.e/ml) 1, 10, 50, 100

PhosDhor Y,0,S:Eu3+ Y,0,S:Tb3 Y,0,:Eu3 Zn,SiO,:Mn,As Y,0,S:Eu3 + Y,0,S:Eu3+ Y,0,S:Tb3 Y2O3:Eu3+ Zn,SiO,:Mn,As Y,0,S:Eu3 Y,0,S:Tb3 ' Zn,SiO,:Mn,As Y,0,S:Eu3+ Y,O,S:Tb"+ Zn,SiO,:Mn,As Y,O,S:Eu" Zn,SiO,:Mn,As

--

ROTATING DIS

COOLED CCD CAMERA

+

+

Streptavidine

Goat IgG anti-mouse IgG (FITC) Goat IgG anti-rabbit IgG (FITC) Rabbit IgG anti-mouse IgG (FITC) Rabbit IgG anti-goat IgG (FITC) Rabbit anti-human erythrocyte membrane

1, 30 0.01, 0.05, 0.1, 0.5, 1.4, 10,50, 100 0.1, 1, 10, 50, 100

0.1, 1, 10, 50, 100 0.1, 1, 5

+

-

I

+

+

1.5, 50

Y,0,S:Eu3 Zn,SiO,:Mn,As

1, 10,50

Y,0,S:Eu3 ' Zn,SiO,:Mn,As

FILTER SLIDE

I

/

a

OBJECTIVE

LAMP

+

FIG.1. Schematic representation of a time-resolved microscope

electronic pulse delay, resulting in a trigger signal to the Strobex power supply (model 236, Strobex) to flash After the immunocytochemical labeling had proceeded the lamp. The velocity of the disc and the delay were enough, the latex beads were rinsed twice with 0.1% chosen such t h a t during and shortly after the excitaBSA/HBS, pH 7.4, followed by a n incubation for 60 min tion pulse the emission light pathway was blocked by with, e.g., a 1 : l O O sheep anti-digoxigenin immunophos- one of the non-transparent segments, to ascertain supphor conjugate of different emission color. The latex pression of fast decaying fluorescence. The flash rate beads were rinsed twice with HBS prior to microscopic and intensity were regulated by the power supply and examination. Microscopic preparations were made by the electronic pulse delay. In the system described pipetting the labeled latex beads suspensions on a glass here, the moment of flashing was not only determined slide and covering the suspensions with a quartz cov- by the IR light interrupter, but also by the adjustable erslip. electronic pulse delay. The velocity of the disc was approximately 6,000 rpm, producing 200 trigger pulses Microscopy for Visualization per second. Nevertheless, only one quarter of all availof Immunophosphors able trigger pulses actually led to a flash, because the Time-resolved microscope. Time-resolved mi- electronic pulse delay overruled the command, given croscopy was performed on a modified Leitz Orthoplan by the trigger pulses, to avoid overloading of the flashepi-fluorescence microscope (Leitz, Wetzlar, Germany) lamp. Normal flash rates were 50 flashes per second, (Fig. 1).The microscope was equipped with a Xenon resulting in a n apparently continuous fluorescence imflash lamp (model 355, Strobex, Chadwick-Helmuth age for the human eye. The delay of flashing the lamp, CO., Inc., El Monte, CA) and a filterblock A for UV regulated by the electronic pulse delay was adjustable excitation (band pass filter 340-380 nm, dichroic mir- from 100 to 2,000 ps, in order to cope with the disc ror TK400, and a long wave pass filter, LP 430 nm, rotation speeds from 1,500 to 6,000 rpm. UV microscope. A Zeiss Universal Microscope Leitz, Wetzlar, Germany) to provide the excitation pulses. A plexiglass disc, with a diameter of 185 mm, stand (Zeiss, Oberkochen, Germany) was used to conwith transparent and non-transparent segments was struct a microscope with far UV excitation (Fig. 2). The mounted a t the emission side of the microscope. The microscope was modified after a microscope allowing two non-transparent segments of the disc were 19 mm the visualization of UV fluorescence upon excitation long and they were placed opposite to each other (180") with far UV light, developed by Ploem (JS Ploem, pera t the edge of the disc, intersecting a 15 mm (diameter) sonal communication and internal report, University light path. The position of the disc was monitored by an of Amsterdam, Fig. 3). All optics in the excitation light IR light interrupter, which gave trigger pulses to a n path were made of quartz. The microscope was

PREPARATION AND VISUALIZBTION O F MULTICOLOR IMMUNOPHOSPHORS

565

CHROMATIC BE AF1 SPL ITTEH

RJECTIVE ULTRAFLU AR

LAMP

FIG.2. Schematic representation of an epi-fluorescence microscope suitable for excitation with far UV light.

FIG.3. Photograph of original microscope constructed in 1968 by JS Ploem for epi-fluoresccnce microscopy using far UV light (1).High pressure Xenon UV light source (2). Housing for UVR mirrors for the selection of UV excitation (3). Epi-illuminator for UV fluorescence microscopy.

equipped with a Xenon high pressure lamp (XBO 450 W, Osram, Munchen, Germany) as illumination source. To select the desired excitation wavelengths, the excitation light was passed onto two UV reflection mirrors, both placed a t a n angle of 45". The first one (type 323, thickness: 2 mm, Schott, Mainz, Germany) was used a s UV band pass reflector, reflecting 230 to 310 nm, and transmitting the rest of the spectrum to a heat absorber. This filter was positioned in the light pathway to avoid damage of the third filter by too much heat of the lamp. The second reflector directed the remaining UV light

into the microscope (Melles Griot B.V., Zevenaar, The Netherlands). A 220 to 290 n m band pass filter was used as excitation filter (UV-R-250, Schott, Mainz, Germany). A TK350 filter (type 323, thickness: 0.8 mm, Schott, Mainz, Germany) was used as chromatic beam splitter. Microscopic observations were made with a Zeiss U1trafluar 32 x 1N.A.0.40 glycerin immersion lens or with a n Zeiss Ultrafluar 100 x /N.A.1.25 glycerin immersion lens, which resulted in a total magnification of respectively 100 x and 312.5 x to the photo-camera. Furthermore a long wave pass filter LP 410 nm was used as barrier filter. For observations, the preparations were covered with a quartz coverslip. The coverslips were recovered after examination of the preparations, and reused after thoroughly washing and cleaning. Photomicrographs were taken with a n Olympus PM 10 A D with automatic exposure time control (Olympus, Tokyo, Japan) using a Scotch chrome 640T film or a Kodak Ektachrome 400 ASA film.

RESULTS Stabilization of Ground Phosphor Particles The phosphor particles were ground until the particles had roughly the same particle size a s a control suspension of phosphor particles with known diameter, 0.1-0.3 pm, as defined by electron microscopy. Both suspensions were compared with each other using phase-contrast microscopy and fluorescence microscopy. This grinding process normally lasted 20 to 24 h. Proper grinding and stabilization was not possible when Versicol E5 and D7 were applied for stabilization. Although some stabilization was observed during the first 2-3 h, prolonged grinding resulted in destabilization and severe aggregation. The other polycar-

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BEVERLOO ET AL.

Table 4 Influence of Polycarboxylic Acid on the Stability of the Phosphor Suspension and the Performance of the Immunoconjugate Tested on Y202S:Eu3' and Y20,S:Tb3 and Zn,SiO,:Mn,As

Phosphor

Polycarboxylic acid

Y,0,S:Eu3

+

Stability Performance of t h e of the phosphor immunosuspension conjugate

Additol XW330 5% + + + + i Additol XW330 10% Versicol E5 Versicol E 7 Versicol E 11 +++ Tam01731 (T 731) ++ Tamol N micro (T) +++ T 731lT 3.5/0.5% T 731lT 2.5/5.2% ++

+++

+++

-

Y,03:Eu3

+

Y,0zS:Tb3+ Zn,SiO,:Mn,As ZnS:Ag

Additol XW330 5% Additol XW330 5% Additol XW330 10% Tam01731 5% Tam01731 5% Additol XW330 5%

+

++

++++ ++++ +++ ++++ +++ ++++

+++++ + +a+ -

++ +++ + ++ + + ++++ ++++ +++ ++++ -b

-b

asuspension not stable a f t e r grinding; n o t used for conjugation. bPhosphor suspension w a s effected by grinding, only p a r t of

the suspension luminesces.

boxylic acids (Table 4) stabilized the phosphor particles, although some polycarboxylic acids, such as Tamol N micro or combinations of Tamol N micro and Tamol 731, caused some aggregation and destabilization. Single particle suspensions could again be obtained if these phosphor suspensions were sonicated before use. Stabilization was also achieved for those phosphors that were ground in distilled water.

FIG.4. One step immunocytochemical labeling with immunophosphor conjugates on latex beads haptenized with sheep anti-mouse biotin, or sheep anti-mouse digoxigenin or rabbit anti-goat (50 pg/ml). Fluorescent image obtained after continuous excitation with far UV light. a: lmmunocytochemical labeling on latex beads haptenized with digoxigenin labeled sheep anti-mouse antibodies on their surface. Hapten was demonstrated by application of sheep anti-digoxigenin phosphor conjugate (100 pgiml). Phosphor conjugates consisted of yttriumoxisulfide activated with terbium. Phosphor particles were stabilized with 5% Additol XW330. b: Immunocytochemical labeling on latex beads haptenized with biotin labeled sheep anti-mouse antibodies with avidin phosphor conjugate (100 pgiml). Phosphor conjugates consisted of zincsilicate activated with manganese and arsenic. Phosphor particles were stabilized with 5% Tamol 731. c: Immunocytochemical labeling on latex beads haptenized with rabbit anti-goat antibodies with goat anti-rabbit phosphor conjugate (100 pgiml). Phosphor conjugates consisted of yttriumoxisulfide activated with europium. Phosphor particles were stabilized with 5% Additol XW330. FIG.5. Multiple immunocytochemical labeling on a mixture of latex beads carrying various haptens. Beads haptenized with digoxigenin are shown with red luminescent sheep anti-digoxigenin phosphor conjugate (100 bg/ml), and beads haptenized with biotin are shown with green luminescent avidin d phosphor conjugate (100pgiml). Flu-

All ground phosphors remained luminescing after grinding and charge stabilization except for one type: the zincsulfides and related compounds could be ground until the desired proportions, but part of the acquired suspension did not show luminescence anymore, at least not visible for the human eye.

Phosphor Conjugates Cytochemical properties of the phosphor conjugates. The differently stabilized phosphor conjugates were tested as potential immunocytochemical reagents to demonstrate specifically antigens on the surface of lymphocytes after coupling antibodies and macromolecules to the various stabilized phosphor particles (Table 4). Mouse anti-leu3a (CD4) was selected because the antigen is present on the membrane of approximately 50% of the peripheral blood lymphocytes, thereby creating a n internal control. It was also possible to adsorb (fluorochromated) proteins and macromolecules to the surface of the phosphor particles which were ground in distilled water. The resulting immunophosphor showed less green fluorescence than the normally ground and conjugated immunophosphors. Nonspecific interactions, labeling intensity and tendency toward aggregation during cell labeling were the main criteria to judge the suitability of the various stabilized immunophosphors. Intense specific labeling was observed when immunophosphor conjugates stabilized in 5% or 10% Additol XW330 were used a s third layer in the detection of CD4 epitopes on the surface of the lymphocytes; phosphors stabilized in 10%Additol XW330 showed some nonspecific labeling a s was indicated by a n increased positive population in the positive controls. Negative control experiments which consisted of omission of the second layer antibodies, or application of non reacting anti-

orescence image obtained after continuous excitation with far UV light.

FIG.6. Multiple immunocytochemical labeling on a mixture of latex beads haptenized with digoxigenin or biotin. Beads haptenized with digoxigenin are shown with blue luminescent sheep anti-digoxigenin phosphor conjugate (100pgiml). Beads haptenized with biotin are shown with green luminescent avidin phosphor conjugate (100 pg/ml). Fluorescence image obtained with continuous excitation with far UV light. FIG. 7. Beads demonstrated in Figure 4 were mixed in equal amounts, to demonstrate the ability of observation of the three colors, i.e., the three differently emitting phosphor conjugates simultaneously.

FIG.8. Demonstration of autofluorescence suppression by time-resolved microscopy. Two-step labeling on 0.1% glutaraldehyde fixed human erythrocytes. Incubation with rabbit anti-human erythrocyte membrane, followed by goat anti-rabbit phosphor conjugate (100pg/ ml). a: Fluorescence image obtained with continuous excitation with UV-light. b: Image of same field obtained by time-resolved microscopy resulting in visualization of the luminescence of the phosphor particles only.

FIGS.4-8

568

BEVERLOO ET AL.

bodies with the avidin d or protein A immunophosphor arsenic and Figure 4c shows beads haptenized with conjugate, confirmed this observation. Phosphors sta- rabbit anti-goat antibodies visualized with goat antibilized in Versicol E l l resulted in intense labeling, but rabbit phosphor conjugate consisting yttriumoxisulfide also showed persisting, substantial nonspecific label- activated with europium. ing. All phosphor conjugates stabilized in Tamol 731 Double labeling. Double labeling of haptenized laand both mixtures of Tamol 731 and Tamol N micro tex beads was performed by using previous mentioned showed abundant labeling of the object. Unfortunately, blue, green, and red luminescent immunophosphors. negative control experiments of conjugates of phos- As expected, two stained populations were observed afphors stabilized in Tamol N micro or a mixture with ter immunocytochemical staining when latex beads this polycarboxylic acid showed nearly as much stain- haptenized with biotin and digoxigenin were mixed in ing intensity. These control experiments consisted of equal amounts. Figure 5 shows the latex beads hapapplication of a non-biotinylated second layer, when a n tenized with digoxigenin brightly stained with red luavidin immunophosphor conjugate was applied as im- minescent sheep anti-digoxigenin immunophosphors munocytochemical last step. Conjugates of phosphor and the latex beads haptenized with biotin stained particles stabilized in Versicol E l l or Tamol N micro with green luminescent avidin immunophosphors. In also formed small aggregates during incubation of the Figure 6 beads haptenized with either biotin or rabbit lymphocytes. These aggregates seriously hampered the anti-goat are shown that were immunocytochemically interpretation of the observed staining. The immuno- stained with blue luminescent avidin immunophosphor phosphors which were ground in distilled water showed conjugates and green luminescent goat anti-rabbit imonly weak staining in the assay described for lympho- munophosphor. The different immunophosphor conjucytes. Phosphor particles stabilized in Additol XW330 gates could better be applied sequentially since mixed were found optimally suited because grinding and sta- colors arose when the different immunophosphors were bilization in this polycarboxylic acid and subsequent applied as a mixture. adsorption of immunoreactive macromolecules to the The ability to observe all three labels simultasurface of the particles were superior with respect to neously, when excited with far UV light, is demonstability, staining intensity and nonspecific interac- strated in Figure 7. tions; Tamol 731 also gave satisfiable results. Time-resolved microscopy. For observations by Considerable differences were observed with respect time-resolved microscopy, 50 flashes per second were to the amount of antibody that was needed to obtain normally used resulting in a n apparently continuous immunocytochemical staining of the biological object. image. At pulse rates significantly below 50 Hz, the For instance rabbit anti-human erythrocyte membrane resulting image appeared to flicker. The flashes used antibodies, used in direct labeling experiments, had to had a n energy of 1.44 J per flash and a flash duration be coupled in large quantities before labeling was ob- of 15 ps. served. Others, such as, e.g., protein A, already showed The presence of aforementioned segments on the disc labeling at a concentration of 1 pg protein per ml phos- resulted in closing the emission light path way in 258 phor suspension. However, most immunophosphors ps,and in a totally closed emission light path for 17 ps, showed augmented staining intensity (CD4-staining), assuming a velocity of approximately 6,000 rpm. The if increased amounts of macromolecules were adsorbed opening of the light path took another 258 ps; the open to their surface. In summary, mostly 1pg antibody per position of the emission light path maintained for 4.46 ml phosphor suspension was sufficient to obtain label- ms. During the closure of the emission light path, the ing, but often 100 pg macromoleculesiml phosphor sus- fast decaying autofluorescence had become extinct; pension were conjugated. Avidin d conjugates per- therefore, only the specific label luminescence was obformed better when 10 kg avidin d per ml phosphor served after opening of the emission light path. Runsuspension was adsorbed. When higher amounts of avi- ning the disk at maximum velocity was found optimal, din d were supplied, the conjugates were not stable and since opening and closing times were then as short as tended to aggregate during cell labeling experiments, possible. Longer opening and closing times led to a deprobably because of the positive charge of avidin d at crease in signal. pH 7.4, which partially neutralizes the negative charge Glutaraldehyde fixed human erythrocytes, showing of the polycarboxylic acids, thereby reducing the repul- abundant fixative induced autofluorescence, were sive force of the phosphor particles. stained with rabbit anti-human erythrocyte membrane Figure 4 shows latex beads that were labeled with antibodies ending with a protein A immunophosphor immunophosphor conjugates that differ in spectral conjugate or a goat anti-rabbit immunophosphor concharacteristics. Figure 4a shows beads haptenized with jugate and were examined with time-resolved microsdigoxigenin labeled sheep anti-mouse antibodies la- copy. Figure 8a shows the dominating blue fixative inbeled with sheep anti-digoxigenin phosphor conjugates duced autofluorescence and the dim red luminescence consisting of yttriumoxisulfide activated with terbium. of the immunophosphor, when excited with continuous Figure 4b shows beads haptenized with biotinylated UV light at 360 nm. However, this unwanted fluoressheep anti-mouse antibodies with avidin phosphor con- cence was effectively suppressed upon excitation with sisting of zincsilicate activated with manganese and pulsed excitation of the time-resolved microscope, re-

PREPARATION AND VISUALIZATION O F MULTICOLOR IMMUNOPHOSYHORS

sulting in visualization of the red luminescence of the phosphors only (Fig. 8b).

DISCUSSION This study demonstrates that most phosphors can be ground to obtain particles of small size and sufficient luminescence. The stability of the prepared suspensions depends on the type of polycarboxylic acid used for stabilization and to some extent on the amount of glass beads. Predicting the stabilizing properties of a particular polycarboxylic acid beforehand is difficult, as can be concluded from Table 4. Neither the molecular weight nor the chemical composition of the polycarboxylic acids, nor the pH of the polycarboxylic acid (difference caused by counter ions), could be assigned as the dominating stabilizing factor. Most macromolecules can be conjugated by physical adsorption at the pH at which the conjugation is performed, similar to immunogold labeling (4,121. However, molecules of low isoelectric point are less suited for coupling as, at the pH of coupling, they do not adhere to the phosphor particles because of their net negative charge. Although i t was possible to conjugate these macromolecules to the phosphor particles at lower pH, e.g., pH 6.0, the macromolecules-phosphor conjugates dissociated when the pH was increased to 7.4. Covalent coupling of the macromolecules can be performed to circumvent this problem (5). This was done by means of EDC at pH 5.0. The carboxylic acid stabilized phosphor particles tend to aggregate below pH 6.0; therefore, polycarboxylic acids with a sulfonic acid group were chosen for stabilization, mixed together with carboxylic polycarboxylic acids for attachment of EDC. The pKa of these sulfonic groups is lower than t h a t of the carboxylic groups, causing the phosphor suspension to be stable at lower pH. They indeed formed stable suspensions over a longer pH-range than the exclusively carboxylic acid stabilized phosphor particles. Unfortunately the sulfonicicarboxylic acid stabilized immunophosphors, coupled by physical adsorption with basic macromolecules such as avidin, showed nonspecific binding to the biological objects, which is especially cumbersome in case of dim specific staining. Covalent coupling was not attempted, because of this nonspecific staining. Grinding of the phosphor in distilled water followed by conjugation of macromolecules in distilled water or in HBS was also found feasible. Somewhat surprisingly, it appeared that stable and immunocytochemically reactive immunophosphor conjugates could be prepared, although the staining intensity of these conjugates is not as strong as the polycarboxylic acid stabilized immunophosphor conjugates. Apparently the phosphor particles themselves carry a net charge, which causes the macromolecules to adsorb on their surface. It can be concluded that all tested phosphors are grindable and that they can be adsorbed with macromolecules, dependent on the PI of the macromolecules.

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The systematic investigation of the grinding and coupling conditions has resulted in a large range of phosphors as potential labels for (time-resolved) fluorescence microscopy. A disadvantage is the fact t h a t most of these phosphors require excitation with far UV light, like the phosphors applied in luminescent lamps. Yttriumoxisulfide activated with europium or terbium shows red or blue fluorescence, and zincsilicate activated with manganese and arsenic shows green fluorescence upon excitation with far UV light. These phosphors have narrow band emission spectra, so t h a t good spectral separation is feasible. Analogously, this property is used in color television screens to obtain sufficient image contrast. Multiple color emission upon excitation with one wavelength is therefore a realistic possibility. Nonetheless, excitation of multiple immunophosphors with near UV light or visible light would be easier because adaption of normal fluorescence microscopes for excitation with deep UV light is not a practical proposition. In this respect zincsulfides were interesting candidates, since they can be excited with near UV light or even with visible light. In their original size (6-10 pm) these phosphors luminesce brightly upon excitation with near UV light or visible light (violet light). A disadvantage is the fact that they belong to the so called “soft” phosphors (2).This group should better not be ground since damage of their lattice results in a considerable drop in lumen output. Part of the suspension did not show luminescence anymore, after grinding. The nonluminescent and luminescent part were not separable in any way, because the particles of both groups showed about the same size distribution. This reduced lumen output is a major disadvantage with regard to application of multicolor emission at single color excitation, since most phosphors excitable with visible or near UV light belong to this group. The applications of the various phosphor conjugates shown in this paper are related to the demonstration of membrane antigens. The relatively large size of the phosphor particles is limiting for many other applications on cells. Although they can be made smaller by prolonged grinding, application of smaller particles is not recommended because imperfections of the crystal, introduced by grinding, influence the lumen output negatively. The luminescence of the particles is no longer visible by the human eye. Furthermore, for immunogold staining it was found (11)that only 1nm immunogold particles, compared to immunogold particles with a diameter of 5-10 nm, resulted in increased penetration of the section and therefore resulted in increased labeling density of the section. Analogous to this finding it is expected that phosphor particles as small as one nanometer have to be made, to allow intracellular penetrations as required for instance for in situ hybridization. This is not a practical proposition since a particle of such size is not expected to show significant amounts of luminescence. However, the penetration aspect does not hold

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for the detection of macromolecules on filters and on blots. Here the accessibility is large because a large amount of proteins and nucleic acids will be present on the surface of the blots and filters. We have recently shown that immunophosphors can indeed be used to detect macromolecules on blots and filters. Detection of small amounts of DNA and proteins (picogram range) was found feasible. Nevertheless, there is a need to develop soluble timeresolved dyes with long luminescence life times with high quantum efficiency which do not significantly fade upon excitation. There is also a need for development of time-resolved microscopes, or better for development of time-resolved modules which can easily be built in to normal fluorescence microscopes without the necessity to integrate for long times. Preferably the modules will be able to manage decay times varying from a few microseconds up to several milliseconds in order to cope with the different decay times of the delayed labels. Such labels and microscopes are expected to contribute significantly to increase the sensitivity of fluorescence based detection assays.

ACKNOWLEDGMENTS The authors thank Ing W.A.P. Reynen (Philips Eindhoven) for his enthusiastic help with preparing the phosphor suspensions; Dr. J.L.M. Leunissen (Aurion B.V., Wageningen, The Netherlands) for stimulating discussions. Philips Lighting (Eindhoven), Drs. H.P.J.M. Dekkers and R.B. Rexwinkel (Dept. of Organic Chemistry, University of Leiden) for their help with measuring the luminescent characteristics of the inorganic crystals. LITERATURE CITED 1. Bakkus, MHC, Brakel-van Peer KMJ, Adriaansen HJ, WierengaWolf AF, Akker TW van den, Dicke-Evinger MJ, Benner R: Detection of oncogene expression by fluorescent in situ hybridization in combination with immunof luorescent staining of cell surface markers. Oncogene 4:1255-1262, 1989. 2. Butler KH: Fluorescent lamp phosphors. Pennsylvania State University Press, University Park and London, 1980. 3. Beverloo HB, van Schadewijk A, van Gelderen-Boele S, Tanke

HJ: Inorganic phosphors as new labels for immunocytocbemistry and time-resolved microscopy. Cytometry 11:784-792, 1990. 4. Geoghegan WD, Ackerman GA: Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold. J Histochcm Cytochem 251187-1200, 1977. 5. Goodfriend TL, Levine L, Fasman GD: Antibodies to bradykin and angiotension: a use of carbodiimides in immunology. Science 144:1344-1346, 1964. 6. Hemmila I: Lanthanides as probes for time-resolved fluorometric immunoassays. Scan J Clin Lab Invest 48:389-400, 1988. 7. Jovin TM, Marriott G, Clegg RM, Arndt-Jovin DJ: Photophysical processes exploited in digital imaging microscopy: Fluorescence resonance transfer and delayed luminescence. Ber Bunsen-Ges Phys Chem 93:387-391, 1989. 8. Jovin TM, Vaz WLC: Rotational and translational diffusion in membranes measured by fluorescence and phosphorescence methods. Methods Enzymol 172:471-513, 1989. 9. Nederlof PM, Flier S van der, Wiegant J, Raap AK, Tanke HJ, Ploem JS, Ploeg M van der: Multiple fluorescence in situ hybridization. Cytometry 11:126-131, 1990. 10. Oser A, Roth WK, Valet G Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence. Nucleic Acid Research 16:1181-1196, 1988. 1. Plas PFEM van der, Leunissen JLM: Immunocytochemical detection of tubulin in whole mount preparations of PtK2-cells: improved penetration characteristics of Auroprobe one. AuroFile 2, Janssen Life Sciences, Beerse, Belgium, 1989. 2. Slot JW. Geuze HJ: A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 38:87-93, 1985. 3. Soini E, Kojola H: Time-resolved fluorometry for lanthanide chelates-A new generation of nonisotopic immunoassays. Clin Chem 29:65-68, 1983. 14. Soini EJ, Pelliniemi U,Hemmila IA, Mukkala V-M, Kankare J J , Frojdman K: Lanthanide chelates as new fluorochrome labels for cytochemistry. J Histochem Cytochem 36:1449-1451, 1988. 15. Staines WA, Meister B, Melander T, Nagy J I , Hokfelt T: Threecolor immunofluorescence histochemistry allowing triple labeling within a single section. J Histochem Cytochem 36:141-151, 1988. 16. Southwick PL, Ernst LA, Tauriello EW, Parker SR, Mujumbar RB, Mujumbar SR, Clever HA, Waggoner AS: Cyanine dye labeling reagents: Carboxymethylindocyanine succinimyl esters. Cytometry 11:418-430, 1990. 17. Tanke HJ, Slats JCM, Ploem JS: Labeled macromolecules: a process for their preparation and their use for immunological or immunocytochemical assays. International Patent No. 8502187, 1986. 18. Zotikov AA, Polyakov Y S : The use of the phosphorescence microscope for the study of the phosphorescence of varoius cells. Microscopica Acta 79:415-418, 1977.

Preparation and microscopic visualization of multicolor luminescent immunophosphors.

The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive con...
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