Patent Application
20140055853
The invention relates generally to an open top microfluidic device for multiplex staining and imaging which provides a method of encapsulating a mounted biological sample to allow for sequential in situ multiplexing analysis of the sample based on the concept of dye cycling, without the need for coverslipping and de-coverslipping during the staining and imaging process.
For sequential in situ multiplexing analysis, a biological sample such as a tissue samples or tissue microarrays (TMA) need to be stained with multiple molecular probes to investigate protein expression or spatial distribution quantitatively or qualitatively. The staining process may be performed manually or using an automated slide stainer. In both methods coverslipping is performed to allow for imaging of the stained sample. And provides a means of protecting the mounted sample. Coverslipping is a time consuming operation and often a source of error related to slide-to-slide variation, manipulation of the sample, and leakage of excess mounting media. In cases where multiple staining protocols and imaging modalities are used, the coverslip may be removed between processes. For example running combinations of H&E (hematoxylin and eosin), FISH (Fluorescent in Situ Hybridization), or ISH (In Situ Hybridization) processes may require the sample to be exposed to reagents in between the various process steps. As such further variation in imaging may be seen as de-coverslipping in particular may result in tissue loss.
Thus, a need therefore exists for a more robust system that can address the needs of sequential in situ multiplexing analysis and imaging without the variations introduced by coverslipping process.
The present invention overcomes the aforementioned drawbacks by providing an open top microfluidic slide carrier which may allow sequential staining and imaging without the need for using or removing a coverslip.
According to one aspect of the present invention an open top microfluidic device is presented comprising a slide carrier and one or more multiplexing stations. The slide carrier comprises a base layer having at least one attachment point positioned on the top surface and at least one attachment point position on the bottom surface of the base layer, a frame adapted to attach to the base layer, at least one central opening defined by the frame body and having four sidewalls wherein the four sidewalls are capable of retaining a fluid in contact with the base layer and is further configured to overlay a portion of a mounted biological sample, positioned against the top surface of the base layer and to retain the tissue slide against the base layer in a horizontal position; and a clamping mechanism to align and attach the frame to the base layer via said attachment points. The multiplexing stations comprise at least one attachment point position to align with at least one of the attachment points of the bottom of the base layer, a reagent delivery device configured to deliver one or more reagents into the fluidic chamber, and a reagent removal device configured to remove one or more reagents out of the fluidic chamber.
In another embodiment, a heater may be positioned below the base layer and configured to apply heat to a tissue slide positioned within the slide carrier.
Various other features and advantages of the present invention will be made apparent from the following detailed description and the drawings.
The drawings illustrate an embodiment presently contemplated for carrying out the invention.
To more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provide for specific terms, which are used in the following description and the appended claims.
The singular forms “a” “an” and “the” include plural referents unless the context clearly dictates otherwise. Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term such as “about” is not to be limited to the precise value specified. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques
As used herein, the term “biological sample” refers to a sample obtained from a biological subject, including sample of biological tissue or fluid origin obtained in vivo or in vitro. Such samples may be, but are not limited to, tissues, fractions, and cells isolated from mammals including, humans. The biological sample may be mounted or fixed onto a solid support for example a tissue section fixed to a microscope slide, or a tissue micro array.
In certain embodiments, the open top microfluidic slide carrier provides a means of containing and positioning a mounted biological sample, so that it can sequentially be: stained with a dye, imaged with any high resolution microscope or fluorescent reporter, bleached or quenched, then the cycle repeated without requiring the sample be protected with a coverslip. As traditionally used, a coverslip covers mounted sample as a way to protect both the sample and the microscope objective and to introduce optical correction consistent with a microscope objective requirements.
As such, in certain embodiments, the slide carrier is designed to enable all aspects of tissue preparation, staining, imaging, and bleaching to occur without the need to apply a coverslip to the slide. The slide carrier is designed for easy transport by simple robotics to move it between a staining station and an imaging station. Furthermore, the open design of the carrier allows imaging from either below the sample, through an optical port or aperture in the base, for example through glass, or above the sample via an immersion fluid applied and the use of an immersion objective lens objective, or through a temporary lifter slip which does not come in contact with the surface of the tissue sample.
The side walls of the frame's central opening and the base layer creates an open top fluidic chamber around the slide in such a manner to allow reagents to be dispensed above the slide. As long as the slide is held in an upright or semi-upright orientation, the walls around the slide will prevent reagent from running out or leaking from the slide carrier. In certain embodiments the fluidic chamber has a volume capacity in the range of 1 μL to 1000 μL. While in other embodiments, the fluidic chamber has a volume capacity in the range of 25 μL to 250 μL.
As shown further in
The slide carrier forms an open top microfluidic flow chamber around the mounted sample. The height of the sample carrier may be determined based on the mounted sample and the volume of reagent to be applied. For example, where the sample is a tissue section, on a microscope slide having the internal cavity may have a dimension of approximately 55 mm by 20 mm and an internal chamber volume or holding capacity of the chamber in the range of 10 μL to 1000 μL, preferably, 50 μL to 200 μL determined by the carrier's dimensions.
In certain embodiments, the frame may have more than one opening. In such a configuration, multiple mounted biological samples may be place on the base and covered with the frame in a manner to create a fluid chamber around each individual sample for multiplexing and imaging.
As such, in certain embodiments, the slide carrier may be used for multiplexed tissue staining and imaging as described in U.S. Pat. No. 7,629,125 and U.S. Pat. No. 7,741,046.
The slide carrier provides a means of holding and positioning the biological sample mounted on slide. The slide may be configured similar to a standard glass pathology slide allowing the sample to be moved between multiplexing stations wherein the various process steps associated with multiplexing may occur. For example one multiplexing stations may be configured wherein individual stations are arranged to provide for one or more method steps in the process or arranged such that each station is configured for a single step. The steps include, but are not limited to, deparaffinization, multiplex staining step including, an antigen retrieval step, an incubation step, and dye bleaching, and an imaging step. Each step may be repeated, and depending on the protocol or need be repeated, performed in different order, or be excluded. In each case the stations are configured maintain control of spatial location and integrity of the sample.
In certain embodiments, the multiplexing station has an attachment point that corresponds to an attachment point on the base layer (20) of the slide carrier. In this manner the slide carrier may be positioned and temporarily fixed onto the multiplexing station. The slide carrier is configured to be transported from one multiplexing station to another; as such transport points or guides may be designed into the base layer or frame. In certain embodiments, a robotic device may be configured to engage with the slide carrier to transfer between stations and to control the position of the slide carrier on, or relative to, the one or more multiplexing staining station.
Multiplexing reagents may be dispersed from a dispenser in fluid communication with the slide carrier via a reagent deliver device. In other embodiments, one or more reagents may be dispensed directly into the open chamber using a device that comprises capillary tubing or pipetting action. Such a configuration is shown in
In still other embodiments,
In certain embodiments, as shown in
For example as shown in
Multiple dispensers may be used and connected to the slide carrier through a common port including configured including, but not limited to, a capillary type system or a plug cover type system. In certain molecular pathology application, reagents may be a specified panel of pre-packaged biomarkers for a particular test. The dispenser may also be designed to allow the addition of custom reagents by the user.
In certain embodiments, the slide carrier may further comprise a spring loaded top. The spring loaded top is configured in such a way that it may be opened during staining and closed during imaging to prevent imaging media evaporation.
In certain embodiments, a multiplexing station may be configured as an imaging device. In certain embodiments, the slide for mounting the biological sample is optically transparent in a specified range of wavelengths. The slide may be imaged in an inverted fashion by having the imaging device, such as a microscope objective, positioned below the slide. As such, optical analysis of materials/structures maybe accomplished by either epi-illumination or transmitted illumination, if both are transparent. In embodiment wherein the sample carrier may be used for multiplexed tissue staining and analysis, using transparent substrate and solid support will allows for both epi-fluorescence imaging and transmitted brightfield imaging. This enables analysis of fluorescence-based molecular pathology as well as conventional tissue analysis based on, for example, hematoxylin and eosin stain (H&E) chromogenic staining.
In other embodiments, the imaging station may be configures such that the sample can be imaged from the top either as the sole means of imaging or in combination with imaging in an inverted fashion as described above. In one such embodiment, imaging may be achieved by a fluid immersion objective to achieve high magnification and high numerical aperture. The fluid medium may include, but is not limited to water, glycerol, silicone oil, or mineral oil, or a combination thereof. Water immersion objectives are known to have slightly lower numerical aperture than comparable oil immersion lenses but enable high-resolution imaging. As such, the choice of fluidic medium and objective may vary based on the specific application.
In certain embodiments, the fluid medium may be a glycerol and water solution. The solution may be from approximately 50 to 95% aqueous glycerol solution. In certain embodiments the percentage of glycerol may be approximately 50-70%. In still other embodiments, other materials may be added to the solution. For example an anti-fade reagent may be added such as agent such as a 4% addition of 1,4-diazabicyclooctane (DABCO). For example an anti-fade reagent may be used such as an aqueous solution of 90% glycerol/4% DABCO. Other additives such as buffers or stabilizers may be used such as a phosphate buffered saline (PBS) as well as commercial products (Vectashield®, Vector Laboratories, Burlingame, Calif. and SlowFade® Life Technologies Co., Grand Island, N.Y.).
As shown in
In certain embodiments, the slide carrier may be used with a robotically coupled stainer and imager. The slide carrier may, for example, fit in a standard micro-well plate footprint wherein the micro-well plate contains a biological sample. It may then be moved by robotics such, as a PAA Kinetix® robot (Rockwell Automation, Milwaukee Wis.) or similar device, between a staining station and an imaging station. As such, in certain embodiments, the robotic device configured to engage with the slide carrier and control the position of the slide carrier relative to the one or more multiplexing staining station.
In certain embodiments, a lifter slip may also be used be used for incubation of the mounted biological sample on the slide. The lifter slip provides a raised edge design such that separation occurs between the sample and the lifter slip itself. The lifter slip is designed to be removed prior to imaging.
In certain operations the staging of two or more slide carriers would allow for an operation of staggered staining and imaging of multiple samples. This is shown in
A series of non-cover slip staining and imaging was performed using the steps outlined above to access quality of staining an imaging. A DAPI stain was applied to the fluid chamber and background image obtained using glycerol and an immersion lens. The sample ((Pantomics, Inc. TMA MTU 541 (Richmond, Calif.)) was then prepared for direct conjugation in an incubation chamber and reimaged using glycerol and an immersion lens.
Step 1: Deparaffinization: A standard deparaffinization process was used involving slide clearing and dehydration protocol using xylene, ethanol, and water. After dewaxing, no paraffin or visual residue was observed on the slide and the sample was used for the subsequent multiplexing steps.
Step 2: DAPI stain and Background Image: DAPI was applied for 15 minutes on a biological sample mounted in the slide holder, washed with PBS (2 ml) for 5 minutes, and a back ground image obtained on IN Cell® device (GE Healthcare, Grandview Blvd. Waukesha, Wis.) (DAPI, Cy3, Cy5, FITC) @ 10× using 90% glycerol mounting media (volume 2 mL). The 10× images are shown in
Step 3: Stain and Image on IN Cell with two mounting media types (round 1 multiplexing; Staining was performed using direct Conjugates: Cy 3 (PCK 26); Cy 5 (PCAD) and the sample incubate 1 hour (volume 500 uL). Images where obtained on IN Cell (DAPI, Cy3, Cy5) @ 10× with 2 types of mounting media-90% glycerol mounting media and PBS as mounting media. The 10× images are shown in
Step 4: Bleach process and Image on IN Cell; Deactivation of dyes through bleaching process: 15 min incubation followed by a wash with PBS (volume 2 mL). The sample was re-stained with DAPI (5 min incubation). Images were obtained on IN Cell (DAPI, Cy3, Cy5) @ 10× with 50% glycerol mounting media. The images are shown in
Step 5: Stain and Image on IN Cell with 50% glycerol mm (round 2 multiplexing); the sample was re-stained using direct conjugates Cy 3 (Na,K-Atpase) and incubated for 1 hour (volume 500 uL). Images were obtained on IN Cell at 10× with 50% glycerol mounting media. The images are shown in
Step 5: Stain while using an optional LifterSlip™ (Erie Scientific Company. Portsmouth, N.H.) for Incubation step (round 2 multiplexing) Similar to step 4 above, after staining a lifter slip was added prior to incubation of the sample, the lifter slip did not come into contact with the sample and was removed prior to imaging. Images obtained on an Olympus® microscope @ 20× (Olympus America Inc., Center Valley, Pa.) with 50% glycerol mounting media. Images are shown in
The present invention has been described in terms of the preferred embodiment, and it is recognized that equivalents, alternatives, and modifications, aside from those expressly stated, are possible and within the scope of the appending claims.
