Automated systems and methods for preparing biological specimens for examination
The systems and methods disclosed herein permit automated preparation of biological specimens for examination. The disclosed systems and methods provide fast, efficient, and highly uniform specimen processing using minimal quantities of fluids. The methods include at least a fixing phase for fixing a biological specimen to a substrate such as a microscope slide, a staining phase for staining the specimen, and a rinsing phase for rinsing the specimen. One or more of the fixing, staining, and rinsing phases include one or more agitation cycles for distributing reagents evenly and uniformly across the specimen. The systems can be implemented as a standalone device or as a component in a larger system for preparing and examining biological specimens.
1. A method of preparing a biological specimen on a substrate for examination, the method comprising:
(a) positioning the substrate with respect to a surface so that the biological specimen faces the surface, and so that the substrate and the surface are substantially parallel and form a separation of at least about 100 microns;
(b) sequentially dispensing (i) a first fixative solution, (ii) a first stain solution, (iii) a second stain solution, and (iv) a first rinse solution into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface; and
(c) after dispensing each one of solutions (i), (ii), (iii), and (iv) in step (b), and before dispensing the next one of solutions (i), (ii), (iii), and (iv) in step (b):
performing at least a first agitation cycle, wherein the first agitation cycle comprises changing a distance between the substrate and surface while the dispensed solution contacts the specimen for the duration of the first agitation cycle; and
removing the dispensed solution from the separation and from contacting the specimen.
2. The method of claim 1 , wherein each sequential dispensing step comprises dispensing one of the solutions in step (b) at a flow rate of at least 70 microliters per second for no more than three seconds.
3. The method of claim 1 , wherein the first agitation cycle comprises:
increasing the distance between the substrate and the surface by at least ten microns; and
decreasing the distance between the substrate and the surface by at least five microns.
4. The method of claim 1 , wherein removing the dispensed solution comprises applying a pressure of at least one pound per square inch less than an atmospheric pressure to the separation for at least two seconds.
5. A method of preparing a biological specimen on a substrate for examination, the method comprising:
(a) positioning the substrate with respect to a surface so that the specimen faces the surface, and so that the substrate is positioned to form a separation between the surface and at least a portion of the substrate of at least about 100 microns;
(b) performing a fixing phase comprising:
(i) dispensing a fixative into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface;
(ii) performing at least a first agitation phase, wherein the first agitation phase comprises changing a distance between the substrate and surface while the fixative is contacting the specimen for the duration of the first agitation phase; and
(iii) removing the fixative from the separation and the specimen;
(c) performing a first staining phase comprising:
(i) dispensing a first stain into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface;
(ii) performing at least a second agitation phase, wherein the second agitation phase comprises changing the distance between the substrate and surface while the first stain is contacting the specimen for the duration of the second agitation phase; and
(iii) removing the first stain from the separation and the specimen;
(d) performing a second staining phase comprising:
(i) dispensing a second stain into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface;
(ii) performing at least a third agitation phase, wherein the third agitation phase comprises changing the distance between the substrate and surface while the second stain is contacting the specimen for the duration of the third agitation phase; and
(iii) removing the second stain from the separation and the specimen; and
(e) performing a first rinse phase comprising:
(i) dispensing a first rinse into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface;
(ii) performing at least a fourth agitation phase, wherein the fourth agitation phase comprises changing the distance between the substrate and surface while the first rinse is contacting the specimen for the duration of the fourth agitation phase; and
(iii) removing the first rinse from the separation and the specimen.
6. The method of claim 5 , further comprising performing a second rinse phase, the second rinse phase comprising:
(i) dispensing a second rinse into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface;
(ii) performing at least a fifth agitation phase, wherein the fifth agitation phase comprises changing the distance between the substrate and surface while the second rinse is contacting the specimen for the duration of the fifth agitation phase; and
(iii) removing the second rinse from the separation and the specimen.
7. The method of claim 6 , further comprising performing a drying cycle by directing a flow of air across the specimen.
8. The method of claim 7 , wherein the combined method steps are performed in less than 70 seconds.
9. The method of claim 7 , wherein the method consumes less than 850 microliters of fixative, first stain, second stain, first rinse, and second rinse fluids.
10. The method of claim 5 , wherein the combined method steps are performed in less than 60 seconds.
11. The method of claim 5 , wherein the method consumes less than 650 microliters of fixative, first stain, second stain, and first rinse fluids.