WO2007138568A2

WO2007138568A2 - A device for microscopy flow-through experiments on non-adherent live cells

Info

Publication number: WO2007138568A2
Application number: PCT/IL2007/000570
Authority: WO
Inventors: Cher Ashtamker; Robert Fluhr
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2006-05-25
Filing date: 2007-05-10
Publication date: 2007-12-06
Also published as: WO2007138568A3

Abstract

A device used in microscopic applications and particularly to those used for examining specimens containing non-adherent live cells or other particles of interest suspended in a liquid medium, in flow-through experiments, includes an open mesh to be located between a slide plate and coverslip for receiving a specimen of the cells of interest. The open mesh is constructed and dimensioned to make up a multitude of small mesh openings defining a two-dimensional array of interconnecting physical traps for receiving the specimen and is effective in restricting the movement of the cells of interest therein without unduly affecting the viability of the cells of interest, or restricting the flow of the surrounding medium, thus allowing the conducting of flow-through experiments.

Description

A DEVICE FOR MICROSCOPY FLOW-THROUGH EXPERIMENTS ON NON- ADHERENT LIVE CELLS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to slides used in microscopic applications and particularly to those used for examining specimens containing cells or other particles of interest suspended in a liquid medium, in which the medium is exchanged or so- called flow-through experiments. The invention also relates to methods of malting such devices, and further, to a method of using such devices in flow-through experiments.

Many biological phenomena vary within a cell population. This fact motivated a long— standing interest in single-cell measurements and responses. A broadly used approach of real— time single-cell analysis is the imaging of fluorescent signals by confocal microscopy. This method involves observing an individual cell over a period of time and has been effectively used to monitor adherent cells. Importantly, cells that grow in suspension, such as cells from the immune system, plant cells, and other non-adherent cells, are difficult to examine under these conditions, as they cannot be immobilized without a significant loss of viability. Immobilization of cells is particularly important to achieve rapid medium exchange. To this end, several solutions have been proposed in order to overcome this difficulty. Most proposed solutions use physical or chemical forces to immobilize cells, such as optical tweezers, modifications thereof, and different coating materials.

Optical tweezers use the electromagnetic force of light in order to create "light traps" for particles and cells in liquids [1, 2]. This method was successfully used for cell sorting and for studying other cellular biological properties [3, 4]. Some modified methods use special materials that can switch their physical state from solution to gel, and vice versa, when they are exposed to certain light [5] or temperature [6], and allow entrapment of individual cells on the gel surface. Agarose gel surfaces were also applied to embed live bacterial cells for imaging [7]. Other methods use different surface coatings in order to attach cells to the slide surface. One example is the mussel adhesive protein [8]; other known adhesion substrates suitable for a large variety of cell types are collagen and poly-Lysine. There are several devices, such as the μ-slides [9], that were developed to allow flow-through and immunoassay experiments on living cells, but such devices are generally designed for adherent cells or biofilms.

However, all the foregoing known devices and methods for microscopic analysis of non-adherent live cells in flow-through experiments suffer inherently from a number of disadvantages described more particularly below with respect to their effects on the viability of the cells of interest, and/or on the conditions needed in flow-through experiments. Many of these disadvantages are also present in observing the activity of other types of particles, such as microbeads (e.g., US Patent 4,689,307) in flow-through experiments.

OBJECTS AND BRIEF SUMMARY OF THE PRESENT INVENTION

An object of the present invention is to provide a device to be used in a microscope or similar instrument that enables single-cell analysis of non-adherent live cells or other particles of interest, in flow— through experiments. Another object of the invention is to provide a method of making such a device; and a still further object of the invention is to provide a method of using such a device for flow— through experiments.

According to one aspect of the present invention, there is provided a microscopic device for examining specimens containing cells or other particles of interest suspended in a liquid medium, in flow-through experiments. The new device comprising: a supporting member; and a coverslip to be applied over the supporting member; characterized in that the device further comprises an inert open mesh to be located between the supporting member and the coverslip for receiving the specimen of interest; the open mesh being constructed and dimensioned to include a plurality of small mesh openings defining a two-dimensional array of interconnecting physical traps for receiving the specimen and effective in restricting the movement of cells or particles of interest therein, without unduly affecting the viability of the cells or disturbing their surrounding medium. In the preferred embodiments of the invention described below, the device further comprises the specimen containing cells or other particles of interest on the supporting member. According to another aspect of the present invention, there is provided a microscopic device for examining specimens containing cells or other particles of interest suspended in a liquid medium, in flow-through experiments. The new device comprising: a supporting member; and a coverslip to be applied over the supporting member with a specimen of interest to be observed inbetween; characterized in that the device further comprises an inert open mesh to be located between the supporting member and the coverslip for receiving the specimen of interest; the open mesh being constructed and dimensioned to include a plurality of small mesh openings defining a two-dimensional array of interconnecting physical traps for receiving the specimen and effective in restricting the movement of cells or particles of interest therein, without unduly affecting the viability of the cells or disturbing their surrounding medium.

As will be described more particularly below, the small mesh openings of the open mesh define a two-dimensional array of "physical traps" (as distinguished from the "light traps" created by optical tweezers) for receiving the specimen. The physical traps are interconnected by narrow passageways that while effective in imposing a restriction of cell movement, do not affect the cells viability nor confine the surrounding medium, facilitating free diffusion of chemicals and solutions used in flow-through experiments. Such a device is particularly useful for examining live cells suspended in a liquid medium, such as non-adherent cells, sperm cells, blood cells, plant cells in suspension, embryonic cells, prokaryote cells and cancer cells, and is therefore described below with respect to this application, but it will be appreciated that such a device could be used for investigating cell aggregates, cell clusters such as e.g., pollen or other particles, such as microbeads, viral particles, subcellular structures, cell organelles e.g. nuclei, etc. in flow— through experiments.

In the preferred embodiments of the invention described below, the open mesh includes interwoven filaments defining the two-dimensional array of interconnecting physical traps. The filaments create an open cage of selected depth depending on the thickness of the filaments used. In addition, the coverslip is attached to the mesh and the supporting member by adhesive applied along opposed edges of the coverslip, leaving opposite edges unbounded to permit applying the flow-through solution to one of the unbounded edges and to be drawn from the other side of the unbounded edges.

According to further features in the described preferred embodiments, the coverslip is of rectangular configuration, having a pair of opposed longitudinally- extending edges attached to the open mesh and supporting member, and a pair of opposed transversely-extending edges unbounded to the open mesh and supporting member. It will be appreciated, however, that the coverslip could be of different configuration then described.

According to further features in the described preferred embodiments, the two-dimensional array of interconnecting physical traps is in the form of rectangular, or other geometrical weave pattern matrix.

In the preferred embodiments of the invention described below, the supporting member is a slide plate. It will be appreciated, however, that the supporting member could be another coverslip. In one described preferred embodiment, the open mesh is defined by a fabric of mono or multi-type filaments.

According to another described preferred embodiment, the fabric includes mesh openings of up to 325 microns.

According to yet another described preferred embodiment, the fabric is made of monofilaments of 28-122 micron diameter.

According to yet another described preferred embodiment, the fabric includes mesh openings of 200-5,000 microns.

According to yet another described preferred embodiment, the fabric is made of monofilaments of 150-1,000 microns diameter. According to yet another described preferred embodiment, the monofilaments are of nylon.

According to yet another described preferred embodiment, the monofilaments are of polyester.

According to yet another described preferred embodiment, the fabric is a braid of said monofilaments.

In another described preferred embodiment, the laboratory device is made by applying the open mesh to the coverslip; applying the specimen containing the cells or other particles of interest to the open mesh; applying the coverslip, with the open mesh and the specimen therein, to the supporting member; and then attaching two opposed edges of the coverslip to the supporting member (slide plate or another coverslip), leaving two other opposed edges unbounded for the application of the flow-through solution application.

According to another described embodiment, the device is made by applying the open mesh to the supporting member; applying the specimen containing the cells or other particles of interest to the open mesh; applying the coverslip over the open mesh and the specimen therein on the supporting member; and then attaching two opposed edges of the coverslip to the supporting member, leaving two other opposed edges unbounded for the flow-through solution application.

According to a further aspect of the present invention, there is provided a method of using the above-described device for flow-through experiments comprising: applying the flow-through solution to one of the unbounded edges, while applying an absorbing material to the other of the unbounded edges to draw the flow- through solution through the interconnected physical traps defined by the open mesh, while the cells of interest are trapped therein.

In the preferred embodiments of the invention described below, the absorbing material is an absorbent paper sheet. Automatic regulated pumps can replace the mechanical procedure described for obtaining flow-through.

According to another described embodiment, the flow— through experiments comprise real time analysis.

As will be described more particularly below, such a device provides a number of important advantages over the previously-described devices for examining non-adherent live cells or other particles of interest, in flow— through experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described herein, by way of example only, with reference to the accompanying drawings, wherein:

Fig. 1 is an exploded view illustrating one form of the device constructed in accordance with the present invention; Fig. 2 is an enlarged fragmentary view illustrating the construction of the open mesh in the device of Fig. 1;

Fig 3 illustrates one manner of using the device of Figs. 1-2 for performing a flow-through experiment in order to examine live non-adherent cells of interest; Fig. 4 is an exploded view of Figure 3 that schematically illustrates the mechanism of action involved in the flow-through experiment using the described device;

Fig. 5 is a confocal laser scanning microscopy (CLSM) image, which illustrates the results of two flow-through experiments using live BY-2 cells and performed using such a device constructed according to Figure 3.

Fig. 6 is a CLSM image which illustrates the results of a flow-through experiment using live cells and performed using such a device constructed according to Figure 3. The same cells were stained with two probes, AR and DCF, after the addition of HaO₂. Figs. 7a-e illustrate results of flow-through experiments using live cells and performed using such a device constructed according to Figure 3. Figure 7a depicts a cell stained in a basal level, and 13 minutes following addition of an elicitor (cryptogein) and following H₂O₂ production. Figure 7b is a graph depicting level of emission with and without an inhibitor which reduces H₂O₂ production. Figure 7c depicts two cells, showing the periphery and nuclear region of the cells (top panel), the same cells stained with DCF in a basal state (middle panel) and following the addition of the elicitor (lower right panel) or H₂O₂ (lower left panel). Figure 7d depicts graphs which show the emission of the probes through time, in the cell periphery and nucleus, and following the addition of the elicitor (right panel) or H₂O₂ (left panel). Figure 7e is a graph depicting the maximal rate of signal acquisition as measured in the periphery or nucleus expressed as the time gap. Negative values were defined when the nuclear compartment reacted before the periphery compartment.

Figs. 8a-c are CLSM images which illustrate results of flow-through experiments using live cells and performed using such a device constructed according to Figure 3. Images show cells double-stained with DCF and markers specific for: membrane- FM 4-64 (Figure 8a), mitochondria- MitoTracker (Figure 8b) and ER- DPX (Figure 8c). Right inserts in each panel are enlarged sections of the cell periphery (upper insert) and the nuclear regions (lower insert). Scale bars in lower panel = 10 μm.

Figs. 9a-b are CLSM images which illustrate results of flow-through experiments using live cells and performed using such a device constructed according to Figure 3. Figure 9a shows 3D reconstructions of BFA treated (lower panels) or untreated (control, upper panels) BY-2 cells stained with subcellular-specific stains. Cells were stained separately with the ER marker DiOC₅(3) (right inserts) or DCF (left inserts). Figure 9b depicts cells double-stained with DCF and the ER-specific marker DPX. Upper insert shows non-treated cells showing DCF, DPX and merge images; Lower inserts, showing DCF, DPX and merge images were collected at 30 min (middle insert) and 60 min (lower insert) after BFA addition. Scale bar = 10 μm.

Figs. 10a-e illustrate results of several flow-through experiments using live cells and cellular organelles and performed using such a device constructed according to Figure 3. Figure 10a shows a confocal and transmission microscope image of the same purified nucleus triple stained with the DCF, the membrane specific dye FM A- 64 and with the DNA specific dye DAPI (green, red and blue colors, respectively). Figure 10b is a CLSM image showing the same cell stained with DCF before and after the addition of 1 mM Ca²⁺. Figure 10c shows confocal and transmission microscope images of the same isolated nuclei stained with DCF (upper panel) or AUR (lower panel) before and after the addition of Ca²⁺.

It is to be understood that the foregoing drawings, and the description below, are provided primarily for purposes of facilitating understanding the conceptual aspects of the invention and possible embodiments thereof, including what is presently considered to be a preferred embodiment, hi the interest of clarity and brevity, no attempt is made to provide more details than necessary to enable one skilled in the art, using routine skill and design, to understand and practice the described invention. It is to be further understood that the embodiments described are for purposes of example only, and that the invention is capable of being embodied in other forms and applications than described herein. DESCRIPTION OF PREFERRED EMBODIMENTS

Figs. 1-2 illustrate a preferred embodiment of a device constructed in accordance with the present invention for examining cells of interest in flow-through experiments. As indicated earlier, although the illustrated device can be used to observe any particle of interest, is particularly useful for observing live, non-adherent cells when subjected to the flow-through solution in a manner wherein movement of the cells is restricted by the device without duly affecting the viability of the cells of interest, and allowing free diffusion of the flow-through solutions.

Thus, in a preferred embodiment of the present invention the specimen comprises cells (i.e., cellular specimen). The cells may be prokaryotic or eukaryotic cells. Preferably, the cells are viable cells. Prokaryote cells can be for example, plant or animal pathogen, an effect of which on a specific cell may be observed using the device of the present invention. The cells may be directly isolated from an organism and subject to analysis ^"using the device of the present invention. Alternatively, primary cultures or cell lines may be included in the specimen. Cells may be unaffected healthy cells or diseased cells such as cancer cells, pathogen infected cells and the like.

Cells of the present invention may be intact cells or subcellular particles or fragments isolated therefrom (homogeneous or heterogeneous preparations thereof). Examples include, but are not limited to membranes, nuclei, mitochondria and the like. Specimens of the present invention which comprise non-adherent cells are of specific interest in accordance with the present invention since changing of solutes and washing procedures require the separation of cells from medium, which is particularly difficult when cells are not adhered to a surface. Thus, examples of non- adherent cells which may be analyzed using the teachings of the present invention include, but are not limited to, sperm, blood, stem and plant cells (in suspension). Non adherent polyploidy cells, cell aggregates or clusters can additionally be observed using the device of the present invention. For example, pollen and developing first stage zygotes may be observed and analyzed according to the present invention.

The illustrated device includes a slide plate 2 and a coverslip 3 to be applied over the slide plate with a specimen of the cells of interest inbetween. Slide plate 2 and coverslip 3 are of transparent materials, as in conventional constructions, to enable examination of the specimen by a microscope.

In accordance with the present invention, the illustrated device further include an open mesh 4 to be located between slide plate 2 and coverslip 3 for receiving the specimen to be examined. As shown particularly in Figs. 2 and 4, open mesh 4 is constructed of a plurality of interwoven filaments 4a, 4b to produce a plurality of small mesh openings defining a two-dimensional array of physical traps for receiving the specimen to be examined. In view of the interwoven arrangement of the filaments 4a, 4b, each filament 4a, 4b alternate in contact with, and spaced from, the slide plate 2 and the coverslip 3, such that the spaces between the alternating contact points of the filaments defines narrow passageways interconnecting the physical traps 5 for receiving the specimen. This will be more clearly described below with respect to Figs. 2 and 4, showing the passageways at 6.

As seen particularly in Figs. 2 and 4, the narrowing of the passageways 6 interconnecting the physical traps 5 are effective in restricting movement of the cells of interest 8 without unduly affecting the viability of such cells of interest, or disturbing the flow-through medium used in the flow— through experiments.

Open mesh 4 is preferably a fabric of monofilaments formed in a braid so as to produce the two-dimensional array or matrix of mesh openings defining the interconnecting physical traps 5. One example is a fabric of nylon monofilaments having a filament diameter of 28-122 microns, and mesh openings of 1-325 microns, supplied by A.D. Sinun, of Israel, under the trademark "Nitex". A second example is a fabric supplied by the same company under the same trademark but made of nylon monofilaments of 150-1,000 microns, and having mesh openings of 200-5,000 microns. A third example is a fabric supplied by the same company under the trademark "Petex" and constituted of polyester monofilaments having a diameter of 32-1,000 microns and mesh openings of 1-5,000 microns. All the foregoing materials are inert and stable under heat and solvent conditions.

As shown particularly in Figs. 1 and 3, coverslip 3 is preferably of substantially rectangular configuration, having a pair of opposed longitudinally- extending edges 3a, 3b and a pair of opposed transversely-extending edges 3c, 3d. Coverslip 3 is attached to the open mesh 4, and also to the slide plate 2, along the pair of opposed longitudinally-extending edges 3a, 3b; the pair of opposed transversely- extending edges 3c, 3d are unbounded. As will be described below with respect to Fig. 3, during a flow-through experiment, the flow-through solution is applied to the unbounded transversely-extending edge 3 c and, after being drawn across the full length of the open mesh 4 between the coverslip 3 and slide plate 2, is removed from the unbounded transversely-extending edge 3d.

Preferably, the attachment of edges 3a, 3b of coverslip 3 is achieved by the use of an adhesive. Any suitable adhesive may be used which does not release toxic substances to the specimen. The diameter of the monofilaments in the open mesh 4, and the size of the mesh openings defining the physical traps 5, depend on the type of cells (or other particles) to be examined, e.g., fungi, bacteria, yeast, blood cells, microbeads, nuclei, etc. Other compatible materials may be used for open mesh 4, preferably having non- fluorescent properties, such as polydimethylsaline (PDMS), etc. The new device illustrated in Figs. 1-2 may thus be made by applying the open mesh 4 either to the coverslip 3 or the slide plate 2; applying the specimen to the physical trap 5 in the open mesh 4; and applying the coverslip 3 to the slide plate 2 with the open mesh 4, containing the specimen, inbetween. The coverslip 3 may then be attached to the slide plate 2, with the specimen-containing open mesh 4 inbetween, along the two longitudinally-transversely-extending edges 3a, 3b of the coverslip, leaving the two transversely-extending edges 3c, 3d unbounded.

While in the above description the open mesh 4 is applied to the coverslip 3 it will be appreciated that the open mesh 4 may be applied to the slide plate 2 instead.

Fig. 3 illustrates a preferred method of using the above-described laboratory device for performing flow-through experiments; whereas Fig. 4 schematically illustrates the manner in which the interconnecting physical traps produced by the open mesh 4 are effective to restrict the movement of the cells of interest 8 in the specimen without unduly affecting the viability of the cells of, or unduly disturbing the progress of the flow-through solution. Thus, as shown in Fig. 3, the flow-through solution used in the experiments is applied from an applicator 10 to the slide plate 2 along the unbounded transversely- extending edge 3 c of the coverslip 3, while a liquid-absorbing material 11, such as an absorbent paper sheet, is applied to the opposite unbounded edge 3d of the coverslip. The absorbent material thus draws the liquid through the full length of the open mesh 4. As the liquid flow-through solution is thus drawn through the open mesh 4, the physical traps 5 defined by the filaments 4a, 4b of the open mesh restrict movement of the cells of interest within the specimen previously applied to the open mesh. At the same time, the interconnecting passageways 6 defined by the alternating contact points of the filaments 4a, 4b of the open mesh with the slide plate 2 and the coverslip 3, permit the flow-through solution to flow the complete length of the open mesh as shown in Fig. 4, such that the cells of interest can be observed while the flow-through solution is substantially undisturbed.

As is shown in Figures 5-10 and in Example 1 of the Examples section which follows, the articles provided herein may be used for any cellular or subcellular visual detection assays of interest, preferably in real time analyses such as for assaying cellular responses to different agents, cellular manipulation (e.g., genetic modification, cellular treatment with exogenous factors) which may be useful for drug screening, personalized medicine and research.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLEl Use of the device inflow through experiments

A device generated according to the teachings of the present invention has been successfully used to follow changes in hydrogen peroxide (H₂O₂) production in tobacco BY-2 suspension cells, in response to different elicitors. It also allowed a simpler and quicker execution of all the preparation procedures, such as probe incubation and washing steps, using minimal reaction volumes on slides. Materials and Experimental procedures

Assays preformed using the device for monitoring H₂O ₂ production in BY-2 cells - Several kinetics experiments were effected for monitoring H₂O₂ production in tobacco BY-2 cells, which demonstrate the ability of the device of the present invention to effectively immobilize cells to a confined area, while adding and changing various solutions. H₂O₂ production was monitored with the use of three different fluorescent probes: 2\ y-dichlorofluorescin diacetate, H₂DCFDA (DCF), Amplex Red (AR) which stain H₂O₂ inside cells, and Amplex Ultra Red (AUR) that reacts with H₂O₂ mainly in the surrounding medium. Cells were stained following the addition of H₂O₂, or following the addition of H₂O₂ elicitors including W7, a calmodulin antagonist, and cryptogein, a fungal pathogen.

In the basic assay, cells were monitored with the probes for 2 min in order to determine basal staining level (control). Thereafter, a solution containing H₂O₂ or an elicitor was added to the slide and monitoring was continued for an additional 13 min Other assays included:

A comparison between H₂O₂ production in the cellular periphery and nuclear regions, in individual immobilized cells. Measurements were collected from both regions every 0.3 sec for a total reaction time of 2 to 5 min, following cryptogein or H₂O₂ addition. Monitoring of specific signaling pathways involved in the response of cells to an elicitor was effected by double staining of cells with DCF, together with markers for specific subcelluar compartments, following the addition of an elicitor.

Monitoring of specific signaling pathways involved in the induced production of H₂O₂ was effected with brefeldin A (BFA), a fungal metabolite which modifies intracellular protein trafficking from the ER to the Golgi apparatus. Following the use of BFA, a change in the staining pattern induced by the elicitor, indicates an association between the DCF signal and an ER pathway which results in H₂O₂ production.

Monitoring H₂O₂ production in specific cellular organelles was effected by triple staining of purified nuclei with DCF, the membrane specific dye FM 4-64 and with the DNA specific dye DAPI. Calcium, a downstream component of the cryptogein signaling pathway was added to assess the role of the nucleus in H₂O₂ production as part of the pathway. The reaction was also effected in the presence of reaction inhibitors DPI and catalase. Results

Use of the device of the present invention for comparison of different elicitors and probes, in different time frames - Figure 5 demonstrates the change in fluorescent emission of H₂O₂ probes, DCF and AUR, before (control) and after W7 application. Cells maintained their spatial position using the device of the present invention, allowing simple monitoring of signals changes through time. Figure 6 shows complete co-localization of two fluorescent probes, AR and DCF, following the addition of H₂O₂. The same cell could be stained with different probes and held such that cellular organelles can clearly be defined. .

Use of the device for monitoring cellular processes in subcellular compartments - Figures 7a-e further demonstrate using the device of the present invention for monitoring H₂O₂ elicitation and production in subcellular compartments. Figures 7a-b show monitoring of stained cells in basal level and 13 minutes after addition of cryptogein. Figures 7c-d show the kinetics of H₂O₂ accumulation in the cellular periphery and nuclear regions. Quantitative analysis of five independent experiments showed an average of 2.6 ± 0.3 second delay between the signal measured at the periphery, that occurred first, and the signal measured at the nuclear region, that followed (Figure 7e). These results demonstrate that the device can be used for precise real-time monitoring of live cells in suspension, within split second time frames. Figures 8a-c show double staining of specific cells with DCF and specific markers for different subcellular compartments. All staining, washing, reagent supplementation, and monitoring procedures were done directly on the device. Figure 9 shows cells examined after the application of BFA, which modifies intracellular protein traffic from the ER to the Golgi apparatus. Results show that BFA influenced the DCF signal, therefore indicating that induction OfH₂O₂ with DCF is associated with the ER. In this case, signaling was monitored for time frames of 30 to 60 minutes, indicating that the device can be used for following single immobilized cells in real time for periods of at least 1 hour.

Figures 10a-e show the use of the device for monitoring real time processes in the nucleus. Figure 10a shows a fluorescent emission and transmission microscope images of the same purified nucleus triple stained with DCF, the membrane specific dye FM 4-64, and with the DNA specific dye DAPI (green, red and blue colors, respectively). DCF was shown to stain subnuclear components, and a complex of unstained substructures within and around the nucleolus could clearly be detected and documented, using the device of the present invention. Figure 10b shows the results of addition of 1 mM Ca²⁺, which was found to induce the signal in B Y-2 cells. The generation of H₂O₂ in response to calcium addition, suggests that plant nuclei are capable of generating their own calcium currents. Figure 10c shows a nucleolus localized reaction stained with DCF (upper panel) or AUR (lower panel). Figure 1Od shows that the rate of the H₂O₂-generated signal, as measured by DCF, correlated with Ca²⁺ concentrations but not with the addition of the electron donor NADPH. Figure 1Oe shows that the signal was partially inhibited by DPI (60 % inhibition) and by exogenous catalase (30 % inhibition).

The results presented herein above, as obtained using the device of the present invention and as shown in Figures 5-10, describe new data on the source of H₂O₂, and the enzymes participating in its production, during the complex cascade of reactions to the elicitor protein cryptogein, in BY— 2 plant cells. In all the experiments given herein above, the reaction of the cells to the elicitor or other reactants, as effected by the change of probe signal elevation, could easily be detected in specific cells that barely changed their spatial location. The results validate that the mesh imposes restriction on BY-2 cell movement without interfering with reagent diffusion. Moreover, H₂O₂ kinetics could be continuously monitored in single specific cells and cellular compartments and organelles, using various probes and detection devices, for the duration of at least an hour and in time frames shorter than a second. AU staining, washing, reagent supplementation, and monitoring procedures were done directly on the device without needing to move or pellet the cells under observation. The real time results presented in the examples given above could not have been achieved with such precision and ease, with the former methods used for examining non-adherent live cells in flow-through experiments.

ADVANTAGES OVER PREVIOUSLY-KNOWN METHODS The above-described device, being based on use of an open mesh for physically trapping cells of interest, can therefore be called a "MeshSlide", because it involves physical or mechanical "trapping" or "paging", it inherently provides a number of important advantages over previously-known methods of cells or particles of interest in flow-through experiments. Optical Tweezers

Optical Tweezers provide an excellent tool to study non-adherent cells by immobilizing them using electromagnetic properties of light in solution. However, optical tweezers are fragile systems that need a strong laser setup (minimum 100 mW) and specialized instruments (including infrared capability, and specialized microscopes) that are not readily available in most labs. If one would like to study the reactions in cells in response to light, it is impossible as the immobilization is done with light. The addition of external solutions can interfere with the trapping of the cells, so this method cannot be used for flow-through experiments. Optical tweezers are also not generally used in experiments utilizing fluorescent probes (because of the nature of the method, which requires light). The MeshSlide does not require special equipment, can work with a wide variety of microscopes, does not interfere with fluorescence, and allows flow-through experiments. Agarose gel Agarose gel has auto— fluorescent emission that interferes when using fluorescent probes on cells. It has an added disadvantage of coating the cell, therefore possibly interfering with some of the natural cellular processes. Cells can also react to the oligosaccharides present in the agar. The MeshSlide allows one to work with cells in their native media, with minimal interference with the natural surroundings, and also does not interfere with fluorescence emissions. Coating and Adhesive Materials

There are many adhesion substrates that are suitable for immobilizing a large variety of cell types, but all of them coat the cells, and therefore can interfere with cellular responses. They also change the surround environment of the cells so that they are no longer in their native condition. As stated above, the MeshSlide does not disturb the natural environment of the cells. The Traditional Method of Staining Suspension Cells

It is mostly done in eppendorf tubes in multiple steps, where each step involves spin-down of the cells by centrifugation, removal of the supernatant, and re- suspending of cells in the next solution. The steps include: (1) initial spin down of cells; (2) addition of probe solution; and (3) several washes. Each step in the process requires a few minutes. Any time one wants to change reagents, the whole process has to be started again. In the MeshSlide everything can be done in the unit itself, drastically cutting the time needed to take measurements from the time a change in conditions is made. The MeshSlide also requires a smaller reaction volume and results in a major reduction of costs. Summary of Advantages

The discussed device is compatible for all non-adherent cells visualization experiments; it restricts cell movement thereby simplifying the surveillance, even with the addition of different solutions; it minimizes the time lapse between sample preparation and detection methods, thereby allowing retrieval of data points at time scale previously not attainable; it is a fast and easy way of microscopy sample preparation; because it enables the use of low sample and reaction volumes, it saves reagents; it enables all manipulation procedures (pre-incubation, staining, washing) to be performed on the device; it maintains the natural media conditions of cells and enables their refreshment with minimum interference; it enables the mesh also to be applied as a grid for cell counting; it eliminates the need for additional accessories or cells modifications; and it enables volume production of the laboratory device at low cost.

While the invention has been described with reference to several preferred embodiments, it will be appreciated that these are set forth merely for purposes of example, and that many other variations, modifications and applications of the invention may be made.

REFERENCES

1. Ashkin, A.,, J.M. Dziedzic, and T. Yamane, Optical trappings and manipulation of smgle cells using infrared laser beams. Nature, 1987.,

330(6150): p.769-771.

2. Grier, D.G., A revolution in optical manipulation. Nature, 2003. 424(6950): p.810-816.

3. MacDonald, M.P., G. (C) Spalding, and K. Dholakia, Microfluidic sorting in an optical lattice. Letters to Nature, 2003. 426(6965): p.421-424. 4. Ashkin, A., Optical trappings and manipulation of neutral particles using lasers. PNAS 1997. 94(10): p.4853^860.

5. Maruyama H, A.Fig. , Fukuda T, Katsuragi T.₅ Immobilization of individual cells by local photo-polymerization on a chip. Analyst, 2005. 130(3): p.304-310.

6. Arai, F, N.(C), Maruyama H. Ichikawa A, El-Shimy H, Fukuda T., On chip single-cell separation and immobilization using optical tweezers and thermosensitive hydrogel. Lab Chip, 2005. f(12): p.1399-1403.

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Biointerfaces, 2004. 34(4); p.205-212.

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Claims

What is claimed is:

1. A microscopic device for examining a specimen containing cells or other particles of interest suspended in a liquid medium, in flow-through experiments, said device comprising: a supporting member; and a coverslip to be applied over said supporting member; characterized in that said microscopic device further comprises an inert open mesh to be located between said supporting member and coverslip for receiving the specimen of the cells or other particles of interest; said open mesh being constructed and dimensioned to include a multitude of small mesh openings defining a two-dimensional array of interconnecting physical traps for receiving said specimen and effective to restrict movement of the cells or other particles of interest therein without unduly affecting the viability of the cells or disturbing their surrounding medium, and allowing relatively free passageway of flow-through solutions.

2. The microscopic device of claim 1 further comprising the specimen containing cells or other particles of interest on said supporting member.

3. A microscopic device for examining specimens containing cells or other particles of interest suspended in a liquid medium, in flow— through experiments, said device comprising: a supporting member; and a coverslip to be applied over said supporting member with a specimen of interest to be observed inbetween; characterized in that said laboratory device further comprises an inert open mesh to be located between said supporting member and coverslip for receiving said specimen of the cells or particles of interest; said open mesh being constructed and dimensioned to include a multitude of small mesh openings defining a two-dimensional array of interconnecting physical traps for receiving said specimen and effective to restrict movement of said cells or other particles of interest therein without unduly affecting the viability of said cells of interest or disturbing their surrounding medium, and allowing relatively free passageway of flow-through solutions.

4. The microscopic device according to claims 1 or 3, wherein said open mesh includes interwoven filaments defining said two-dimensional array of interconnecting physical traps.

5. The microscopic device according to claims 1 or 3, wherein said coverslip is attached to said mesh and said supporting member by adhesive applied along opposed edges of the coverslip, leaving opposite edges unbounded, to permit applying the flow— through solution to one of said unbounded edges and to be removed from the other of said unbounded edges.

6. The microscopic device according to claims 1 or 3, wherein said coverslip is of substantially rectangular configuration, having a pair of opposed longitudinally-extending edges attached to said open mesh and supporting member, and a pair of opposed transversely-extending edges unbounded to said open mesh and supporting member.

7. The microscopic device according to claims 1 or 3, wherein said open mesh is defined by a fabric of mono or multi-type filaments.

8. The microscopic device according to claim 7, wherein said fabric includes mesh openings of up to 325 microns.

9. The microscopic device according to claim 8, wherein said fabric is made of monofilaments of 28-122 micron diameter.

10. The microscopic device according to claim 7, wherein said fabric includes mesh openings of 200-5,000 microns.

11. The microscopic device according to claim 10, wherein said fabric is made of monofilaments of 150-1,000 microns diameter.

12. The microscopic device according to claim 7, wherein said monofilaments are of nylon.

13. The microscopic device according to claim 7, wherein said monofilaments are of polyester.

14. The microscopic device according to claim 7, wherein said fabric is a braid of said monofilaments.

15. The microscopic device according to claims 1 or 3, wherein said two- dimensional array of interconnecting physical traps is in the form of rectangular, or other geometrical weave pattern matrix.

16. The microscopic device according to claims 1 or 3, wherein said supporting member is a slide plate.

17. The microscopic device according to claims 1 or 3, wherein said supporting member is another coverslip.

18. A method of making the microscopic device of claims 2 or 3, comprising: applying said open mesh to said coverslip; applying said specimen containing said cells or other particles of interest to said open mesh; applying said coverslip, with said open mesh and said specimen therein, to said supporting member; and then attaching two opposed edges of the coverslip to the supporting member, leaving two other opposed edges unbounded for said flow-through solution application.

19. A method of making the microscopic device of claims 2 or 3, comprising: applying said open mesh to said supporting member; applying said specimen containing said cells or other particles of interest to said open mesh; applying said coverslip over said open mesh and said specimen therein on the supporting member; and then attaching two opposed edges of the coverslip to the supporting member, leaving two other opposed edges unbounded for said flow-through solution application.

20. The microscopic device or method of any of claims 2, 3, 18 and 19, wherein said cells or other particles of interest comprise live cells.

21. The microscopic device or method of any of claims 2, 3, 18 and 19, wherein said cells comprise non-adherent cells.

22. The microscopic device or method of claim 21, wherein said nonadherent cells are selected from the group consisting of sperm cells, blood cells, plant cells in suspension, embryonic cells and cancer cells.

23. The microscopic device or method of any of claims 2, 3, 18 and 19, wherein said particles of interest comprise isolated subcellular structures.

24. The microscopic device or method of any of claims 2, 3, 18 and 19, wherein said cells of interest comprise prokaryote cells.

25. The microscopic device or method of any of claims 2, 3, 18 and 19, wherein said cells comprise pollen.

26. The microscopic device or method of any of claims 2, 3, 18 and 19, wherein said particles of interest comprise viral particles.

27. The microscopic device or method of any of claims 2, 3, 18 and 19, wherein the cells comprise cell aggregates or cell clusters.

28. A method of using the microscopic device of claims 1, 2 or 3 for flow- through experiments, comprising: applying the flow-through solution to one of said unbounded edges while applying an absorbing material to the other of said unbounded edges to draw said flow-through solution longitudinally through the open mesh.

29. The method according to claim 28, wherein said absorbing material is an absorbent paper sheet.

30. The method according to claim 28, wherein said flow-through experiments comprise real time analysis.