FLUORESCENCE MICROSCOPY SAMPLE MOUNTING METHOD AND STRUCTURE

Abstract: A method and structure for mounting fluorescent samples, e.g., fluorescent microbeads, are based on immobilizing the samples in a gel which preserves the spectral properties of the samples and mounting the gel containing the samples on a slide and beneath a coverslip.

Description: Field of Invention This invention relates to a method of and structure for mounting fluorescent samples to immobilize the samples without altering their spectral properties. More specifically, the invention relates to fluorescent microbead standards that are mounted on microscope slides in such a way that they are immobilized while maintaining their spectral properties as when in free suspension. Background Art Microbeads can be produced which not only have the size of biological cells, but also have the fluorescent excitation and emission properties, i.e., the spectral properties of stained cells. Maintaining these properties is extremely important when using these microbeads as standards for performing quantitative fluorescence measurements. However to retain these properties, it is necessary to keep the microbeads in the same environment as that in which the stained cells are held. This is because the fluorescent dyes associated with the microbeads, e.g. fluorescein, are very sensitive to environmental conditions such as solvent and pH. Moreover, when observing a suspension of these microbeads which have been placed on a glass slide with a cover slip under a microscope they are found to be highly mobile within the aqueous suspending solution. This mobility make these microbeads very difficult to photograph and to quantitatively measure their size and fluorescence intensities. In US-A-3736042, a method for preparing a microscope slide assembly, a fluorocarbon layer is coated onto the slide, wells provided in the layer contain an adhesive layer which retains organisms under test during microscope examination. Furthermore with time, these mounts dry out leaving the microbeads in air which changes their spectral properties. Disclosure of the Invention The invention provides a mounting media and mounting procedure which allows fluorescent samples, specifically microbeads, on a microscope slide to be immobilized while still retaining the spectral properties they possess when in free solution. To the mounting media in which the microbeads are normally suspended is added a small percentage of non-fluorescent material which causes the media containing the microbeads to gel under certain described conditions. When suspended in the gel, the mircobeads are immobilized but are still in contact with the same aqueous environment as they were when in free suspension and thus exhibit the same spectral properties. Accordingly the present invention provides a method of mounting fluorescent microbeads (10) on a microscope slide (12) so that the fluorescent microbeads may be utilized as fluorescent microbeads standards for performing quantitative fluorescence measurements characterized in that, (a) a non-fluorescent mounting medium in which the fluorescent microbeads exhibit defined spectral properties when suspended therein is provided; (b) a gelation material which is effective to cause the non-fluorescent mounting medium to gel is added to the non-fluorescent mounting medium; (c) while the gelation-material-containing mounting medium is in ungelled form, a selected quantity of fluorescent microbeads is added thereto so that the fluorescent microbeads are suspended therein; (d) the ungelled gelation-material-containing mounting medium containing suspended microbeads is placed on a microscope slide beneath a cover slip (14); and (e) the ungelled gelation-material-containing mounting medium containing the fluorescent microbeads is caused to gel thus immobilizing the fluorescent microbeads therein with the spectral properties of the fluorescent microbeads being the same as when the fluorescent microbeads are suspended in the ungelled mounting medium. The present invention also provides a fluorescent microbead sample structure which comprises a transparent slide (12) and a cover slip (14) characterized in that it additionally comprises a gelled mounting medium (16) containing dispersed fluorescent microbeads (10), said cover slip (14) overlying the gelled mounting medium. Description of Drawings Figure 1 is a plan view of a slide mounted according to the invention. Figure 2 is an elevation view of the slide shown in Figure 1. Best Mode for Carrying Out the Invention Gelation materials suitable for the invention must be colorless when in solution, non-fluorescent at the wavelengths used during observation and measurement, completely miscible in the suspending solution, and not have any physical or chemical effect on the microbeads. Materials suitable for such use are special polymers of high molecular weight. For aqueous solutions such high polymers include gelatin, starch, algenate, and dextran. Concentrations of .1 to 5% by weight of these materials in solution have been found to be useful in the invention. To form the solutions, it is desirable to heat the solution until the mentioned polymer materials are completely dissolved, i.e., such that there is a very little tindle light scattering in the solution. The microbeads are mixed into the warm solution, placed on a microscope slide, coverslipped and allowed to cool. While warm, the mounting media containing the microbeads flows freely. During this process it has been observed that all of the microbeads settle to the surface of the glass slide thus placing them in the same focal plane, i.e., on top of the glass slide. Upon cooling to room temperature the media gels and immobilizes the microbeads. The resulting slide structure is illustrated in Figures 1 and 2. It has been found that the gel can be prevented from drying out over time by sealing the edges of the cover slip with a material which is impervious to water vapor. A bead of either epoxy resin or silicone rubber is deemed suitable for establishing a vapor barrier (18) to water loss from the gel. Once the sealent material is dried, the slides can be used as with any normal slide mount with the exception of keeping them in the dark when not in use. This prevents possible photo-bleaching, i.e., a decrease in fluorescent intensity of the fluorescent samples. A specific example is next given. EXAMPLE To a solution at pH 7.2 containing 0.1mol NaCl,0.05mol phosphate, 0.5% bovine serum albumin, and 0.5% Na Azide was added 5% gelatin by weight and the solution stirred and heated to 60°C for 10 minutes. The resulting clear solution was allowed to cool to 45°C into which 7.5 microns fluorescein and R-phycoerythrin microbeads were dispersed. Ten microliters of this warm microbead suspension were placed on a glass microscope slide and coverslipped as illustrated in Figures 1 and 2. The suspension completely spread under the coverslip and gelled within 30 minutes at room temperature. The edges of the coverslip were sealed with a bead of epoxy resin and allowed to set overnight. Upon examination under a fluorescence microscope, the microbeads were found to be completely immobilized and the fluorescein and R-phycoerythrin microbeads were found to have the same spectral properties they have in free aqueous solution, i.e., under blue excitation, the fluorescein microbeads were green and the R-phycoerythrin microbeads were orange. Thus, the object of the invention was achieved.

Claims: A method of mounting fluorescent microbeads (10) on a microscope slide (12) so that the fluorescent microbeads may be utilized as fluorescent microbeads standards for performing quantitative fluorescence measurements, characterized in that (a) a non-fluorescent mounting medium in which the fluorescent microbeads exhibit defined spectral properties when suspended therein is provided; (b) a gelation material which is effective to cause the non-fluorescent mounting medium to gel is added to the non-fluorescent mounting medium; (c) while the gelation-material-containing mounting medium is in ungelled form, a selected quantity of fluorescent microbeads is added thereto so that the fluorescent microbeads are suspended therein; (d) the ungelled gelation-material-containing mounting medium containing suspended microbeads is placed on a microscope slide beneath a cover slip (14); and (e) the ungelled gelation-material-containing mounting medium containing the fluorescent microbeads is caused to gel thus imbolizing the fluorescent microbeads therein with the spectral properties of the fluorescent microbeads being the same as when the fluorescent microbeads are suspended in the ungelled mounting medium. A method as claimed in claim 1 characterized in that the edges of the coverslip are sealed to the slide after the mounting medium has gelled, with a vapour barrier material (18). A method as claimed in claim 1 or 2 characterized in that the gelation material comprises a gelatin and the mounting medium is caused to gel by cooling the medium. A fluorescent microbead sample structure which comprises a transparent slide (12) and a cover slip (14) characterized in that it additionally comprises a gelled mounting medium (16) containing dispersed fluorescent microbeads (10), said cover slip (14) overlying the gelled mounting medium. A structure as claimed in claim 4 characterized in that it includes a vapor barrier sealing the edges of the coverslip to the slide. A fluorescent microbead sample structure as claimed in claim 4 or 5, characterized in that the gelled mounting medium comprises a mounting medium wherein the fluorescent microbeads have defined fluorescent spectra when suspended therein, and wherein the fluorescent microbeads have the same fluorescent spectra when dispersed in the gelled mounting medium. A fluorescent microbead sample structure as claimed in any of claims 4 to 6, characterized in that the gelled mounting medium is derived from a solution which has been gelled by addition to the aqueous solution of a polymeric material selected from gelatin, starch, algenate or dextran.

Inventors: SCHWARTZ, ABRAHAM
Applicants: CARIBBEAN MICROPARTICLES CORPORATION
Application year: 1987
Main classification: G01N1
IPC codes: G01N1