Abstract:
A sample carrier for microscopic examination of biological samples includes a base body and a filter membrane. The base body has at least one recess in which the filter membrane is disposed. The filter membrane makes an essentially flush closure with the surface of the sample carrier. The sample carrier has a circular shape.

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S) 
       [0001]    The present application hereby claims priority under 35 U.S.C. §119 to German patent application number DE 10 2011 083 215.7 filed Sep. 22, 2011, the entire contents of which are hereby incorporated herein by reference. 
       BACKGROUND 
       [0002]    1. Field 
         [0003]    Example embodiments relate to sample carriers for microscopic examination of biological samples, and methods for detecting fluorescence-marked objects in a biological sample. 
         [0004]    2. Description of Related Art 
         [0005]    The separation of particles, such as cells, bacteria or the like from biological fluids, such as blood or urine, is increasingly undertaken nowadays using microfiltration. This makes use of the fact that the particles sought (e.g., tumor cells circulating in blood) are significantly larger than most of the other particles or cells contained in the biological fluid. As a result, the particles separated in this way by filtration can be colored with the usual cytological or immunochemical methods and can subsequently be viewed, identified and documented under a microscope. 
         [0006]    Thin polymer films having a porosity of the desired order of magnitude are mostly used for microfiltration. After filtration these microfilters are transferred onto an object carrier so that they are able to be viewed in the microscope. 
         [0007]    This step is time-consuming and also prone to the risk of incorrect transfer or destruction of the membrane. 
         [0008]    A further problem in analysis of particles obtained by microfiltration lies in the large filter surface on which the particles must be searched for in the microscope. In many applications the number of particles sought is so small that the entire filter surface must be evaluated in order to obtain usable and reliable results. Since the particles sought are small relative to the filter surface, a large number of greatly enlarged microscope images of the filter surface must be made in order to cover the entire filter surface, but not overlook any of the particles sought. This too is very time-consuming and labor-intensive. 
         [0009]    A microfilter is known from DE 10 2010 001 322 A1, which is integrated directly into an object carrier for microscopy. This enables the complex step of transferring the filter to the object carrier to be dispensed with. Even with these types of filter devices, however, the coverage of the entire filter surface with microphotographs is still extremely time-consuming and complex. 
       SUMMARY 
       [0010]    Example embodiments provide sample carriers and methods with which more rapid detection of marked particles in biological samples is possible. 
         [0011]    At least one example embodiment provides a sample carrier for microscopic examination of biological samples. The sample carrier comprises a base body with at least one recess in which a filter membrane is disposed. The filter membrane essentially makes a flush closure with a surface of the sample carrier. In this case, the sample carrier may be embodied as a circular carrier. The round shape of the sample carrier makes it possible to carry out an especially rapid microscopic sampling of the filter surface. In contrast with sample carriers having integrated microfilters known from the prior art, such a sample carrier can be accommodated rotating under a fluorescence microscope or the like, so that the sampling of the entire filter surface can be carried out more rapidly than with an object carrier accommodated on a conventional cross table. This merely requires the sample carrier to be set into rotation, wherein a relative movement in a radial direction between sample carrier and microscope is sufficient to capture the entire surface of the sample carrier microscopically. This is also relatively simple mechanically. 
         [0012]    According to at least some example embodiments, the sample carrier has a central, circular through-opening. A spigot of a corresponding drive spindle can engage in this opening in order to make the sample carrier rotate for analysis. 
         [0013]    This through-opening may have a diameter of about 15 mm and an edge height of about 1.2 mm. This corresponds to the compact disk (CD), digital video disc (DVD) or Blu-ray standard, so that apparatuses for handling such a sample carrier can be manufactured simply and at lower cost from components which are produced in large volumes and are thus cheaper. Other dimensions or adaptation to other standards are of course also possible. 
         [0014]    In at least one example embodiment, the at least one recess, as well as the corresponding filter membrane, is circular. This makes possible an especially simple homogeneous application of the fluid to be filtered to the filter membrane, which can for example be done easily by pipetting the fluid into the center of the circular membrane. 
         [0015]    As an alternative, the at least one recess as well as the assigned filter membrane can also form a ring concentric to the sample carrier. On the other hand this simplifies scanning of the membrane surface on the rotating sample carrier since the membrane surface can be captured continuously and without interruption during a single rotation of the sample carrier. 
         [0016]    The at least one recess may have an outlet channel for the filtrate. This makes possible a residue-free observation of the cells or other particles remaining on the filter surface as retentate. 
         [0017]    In at least one other example embodiment, a support element for supporting the filter membrane may be disposed in the at least one recess. This makes it possible to use especially thin and fragile filter membranes which, because of the support element, can easily withstand the mechanical stress when the sample is applied, during the filtration and during the rotational sampling. 
         [0018]    The filter membrane may have a maximum pore size of about 20 μm, but may also have a pore size between about 5 μm and 20 μm, inclusive. Such a filter is especially suitable for separation of suspended tumor cells in blood, since a large part of the leukocytes, erythrocytes and other cellular blood components can easily pass through this type of filter while the considerably larger tumor cells will be held back on the filter surface. 
         [0019]    In order to simplify microscopy, the base body may be embodied from transparent material, such as glass, polycarbonate or the like. 
         [0020]    At least one other example embodiment provides a method for detecting fluorescence-marked objects, such as cells, in a biological sample. For this purpose the sample is initially applied to a circular sample carrier with at least one filter membrane and is filtered through this filter membrane. The pore size of the filter membrane in this case is selected such that the objects to be detected will be held back, while other particles contained in the sample can pass through the filter. To simplify the detection and to be able to distinguish the objects actually sought from other objects also present in the retentate, the retentate subsequently remaining on the filter membrane is treated with at least one fluorescence marker. Immunologically-coupled fluorescence markers are especially useful for this purpose, which for example bind themselves specifically to surface structures of the sought cells. 
         [0021]    After the marking of the sought objects the sample carrier is accommodated rotatably in a holder. The holder and thereby the sample carrier are made to rotate and the sample carrier is sampled with a laser, the frequency of which corresponds to the excitation frequency of an assigned fluorescence marker. At the same time, fluorescence events are detected with a photodetector. High-resolution microscopy is not yet undertaken at this stage. Instead, the coordinates of the recognized fluorescence events are first stored. This may be done by using a polar coordinate system because of the circular shape of the sample carrier. Only after the complete sampling of the sample carrier with the laser is the actual microscopy undertaken. To do this a high resolution microscope is moved to the stored coordinates and microphotographs are taken in each case. 
         [0022]    Thus an especially rapid method is produced overall for detecting fluorescence-marked objects or particles or cells in biological samples. Through the filtration of the sample, which removes a majority of the disruptive cellular or particulate components of the sample, especially low-noise viewing is made possible. Sensitivity and selectivity are further increased by the subsequent fluorescence marking. The method is also compatible to using sampling facilities known per se for circular sample carriers, which are currently used to analyze samples brushed onto their surface. 
         [0023]    A plurality of biological samples may be applied to a plurality of filter membranes of the sample carrier. This makes possible the simultaneous or contemporaneous analysis of a plurality of samples, so that the throughput of the method can be further increased. 
         [0024]    The biological sample may be treated before and/or after the filtration with a lyze agent for lysis of the given, desired or predetermined cell type. For example, an ammonium chloride lysis can be carried out during the examination of blood samples in order to fragment the erythrocytes present in large numbers and facilitate filtering them away. 
         [0025]    The biological sample and/or further fluid media may be applied to the sample carrier by a robotic pipetting system. Such automation allows the method to be carried out in an especially rapid and reliable manner. 
         [0026]    It is expedient in such cases to dispose the sample carrier rotatably in the pipetting system and to rotate the sample carrier to specific positions for applying fluid media. This allows mechanically simple pipetting systems to be used and exploits the advantages of the circular sample carrier. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0027]    Example embodiments will be described in more detail hereinbelow by referring to the accompanying drawing, in which: 
           [0028]    The single FIGURE in this application shows a schematic view of an example embodiment of a sample carrier. 
           [0029]    It should be noted that these Figures are intended to illustrate the general characteristics of methods, structure and/or materials utilized in certain example embodiments and to supplement the written description provided below. These drawings are not, however, to scale and may not precisely reflect the precise structural or performance characteristics of any given embodiment, and should not be interpreted as defining or limiting the range of values or properties encompassed by example embodiments. For example, the relative thicknesses and positioning of molecules, layers, regions and/or structural elements may be reduced or exaggerated for clarity. 
       
    
    
     DETAILED DESCRIPTION 
       [0030]    Various example embodiments will now be described more fully with reference to the accompanying drawings in which only some example embodiments are shown. Specific structural and functional details disclosed herein are merely representative for purposes of describing example embodiments. The present invention, however, may be embodied in many alternate forms and should not be construed as limited to only the example embodiments set forth herein. 
         [0031]    Accordingly, while example embodiments of the invention are capable of various modifications and alternative forms, embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that there is no intent to limit example embodiments of the present invention to the particular forms disclosed. On the contrary, example embodiments are to cover all modifications, equivalents, and alternatives falling within the scope of the invention. Like numbers refer to like elements throughout the description of the figures. 
         [0032]    It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element could be termed a second element, and, similarly, a second element could be termed a first element, without departing from the scope of example embodiments of the present invention. As used herein, the term “and/or,” includes any and all combinations of one or more of the associated listed items. 
         [0033]    It will be understood that when an element is referred to as being “connected,” or “coupled,” to another element, it can be directly connected or coupled to the other element or intervening elements may be present. In contrast, when an element is referred to as being “directly connected,” or “directly coupled,” to another element, there are no intervening elements present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between,” versus “directly between,” “adjacent,” versus “directly adjacent,” etc.). 
         [0034]    The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments of the invention. As used herein, the singular forms “a,” “an,” and “the,” are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the terms “and/or” and “at least one of” include any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises,” “comprising,” “includes,” and/or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. 
         [0035]    Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. 
         [0036]    Spatially relative terms, such as “beneath”, “below”, “lower”, “above”, “upper”, and the like, may be used herein for ease of description to describe one element or feature&#39;s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as “below” or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, term such as “below” can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein are interpreted accordingly. 
         [0037]    Referring to the FIGURE, according to at least one example embodiment, a sample carrier for microscopic examination of biological samples identified overall by the number  10  comprises a circular base body  12  made of a transparent material, such as glass, polycarbonate or the like. The base body  12  has a central through-opening on which it can be rotatably supported by, for example, suitable spindles, spigots or the like. In one example, it is possible to design the central through-opening  18  in accordance with the CD, DVD or Blu-ray standard, so that widely-used components can be employed to drive the sample carrier  10 . 
         [0038]    The sample carrier  10  also has a plurality of recesses  14  let into the base body  12 . Each recess is spanned by a microfiltration membrane  16 . Support elements are also provided in the recesses  14 . The support elements support and stabilize the microfiltration membrane  16 , so that it withstands the mechanical stresses of the application of the sample, filtration and rotational movement. 
         [0039]    The properties of the microfiltration membrane  16  are governed by the actual analysis task for which the sample carrier  10  is to be employed. 
         [0040]    In this example embodiment, the detection of tumor cells in the blood is to be illustrated by the sample carrier  10 . The use of polymer microfiltration membranes  16  with a pore diameter of between about 5 and about 20 μm, inclusive, is suited for this purpose. 
         [0041]    Since tumor cells circulating in the blood are only present in an extremely small number, it is expedient to first separate these from other blood components or to concentrate them for analysis. For this purpose the blood to be investigated is first applied to the microfiltration membranes  16 . If necessary, the separation of the tumor cells from other blood components can also be supported by an erythrocyte lysis, such as and ammonium chloride lysis. After application of the sample prepared in this way to the microfiltration membranes  16 , the pore size of which allows the passage of lysated erythrocyte fragments, leukocytes and other small particulate blood components, while the microfiltration membrane  16  holds back the significantly larger tumor cells as retentate on its surface, the actual filtration follows. The filtrate can in this case run out of the recesses  14  through channels not shown in any greater detail. 
         [0042]    The sought tumor cells now remain on the membrane surface, as well as if necessary other blood components which may not have been filtered away. To facilitate the detection of the tumor cells present in extremely small numbers, a fluorescence coloring is undertaken in the next step. This can likewise be carried out on the surface of the microfiltration membranes  16 . To this end, an immunofluorescence marker, which is specific for surface proteins of the sought tumor cells, is applied to the membrane surfaces by a pipette, where it binds itself specifically to the corresponding targets. Likewise, depending on the cytological or immunohistochemical coloring method used, further treatment steps are necessary. Surplus markers can finally be washed away. 
         [0043]    Like the application of the sample, these coloring steps can also be undertaken by an automatic pipetting system. In such cases it is expedient to support the sample carrier  10  by the recess  18  rotatably in the pipetting system, so that each point of the surface of the sample carrier  10  can be reached by a radial translation movement of the pipetting robot as well as by rotation of the sample carrier  10 , so that the pipetting system is simple to design. 
         [0044]    After successful coloration or marking of the sought tumor cells on the membrane surface, the sample carrier  10  is brought into a corresponding detection device. In this device, the sample carrier  10  is once again supported rotatably on the through-opening  15 . Using, for example, at least one laser, the entire surface of the sample carrier  10  is sampled and simultaneously observed with a photo detector. The wavelength of the at least one laser corresponds to the excitation wavelength of the fluorescence colorant used. If fluorescence events are recognized, the respective coordinates are stored. Naturally in such cases it is possible to also carry out a multiple fluorescence coloration (e.g., for different surface proteins of different tumor types) to carry out specific antibody fluorescence label complexes. Ideally the fluorescence colorants of these complexes have different excitation and emission wavelengths. The sampling is then undertaken in accordance with a plurality of lasers, wherein for each detected fluorescence event, not only the coordinates but also the detected emission wavelength—and thus the type of fluorescence label used—is determined. 
         [0045]    If the entire surface of the sample carrier  10  or the entire surface of the microfiltration membranes  16  has been sampled in this way, then the detected fluorescence events are observed microscopically in greater detail on the basis of the stored coordinates. A high-resolution microscope moves in such cases to the stored coordinates and creates corresponding microphotographs. Because of the transparent nature of the base body  12 , this can initially be done in simple available light. A fluorescence excitation is also possible here in order to recognize the presence of the sought tumor cells in the sample on the basis of the specific and selective fluorescence marking. All known techniques of fluorescence microscopy, such as confocal fluorescence microscopy, can be employed here. 
         [0046]    During the application of the sample and the analysis the sample carrier  10  can be rotated in such cases at considerable speeds of several hundred to several thousand rpm, so that especially rapid scanning is possible. The separation of the detection of fluorescence events from the actual microscopic recording further speeds up the scanning in such cases since the entire membrane surfaces do not have to be recorded microphotographically. 
         [0047]    A further accelerated variant of the method is well suited to the routine laboratory analysis of the large number of samples. In this case blood samples of a number of patients are applied to the individual microfilter surfaces and simultaneously analyzed in the way described. 
         [0048]    An alternative design of the recesses  14  and the assigned microfiltration membrane  16  is also possible. For example, the recesses  14  and membranes  16  can run around the circumference of the sample carrier  10  in the form of concentric rings, so that an interruption-free observation of the membrane surface is made possible during a complete rotation of the sample carrier  10 . Naturally, such ring structures are then to be attached coaxially to the central through opening  18 . 
         [0049]    Overall the method illustrated is to be carried out especially quickly in this way, but also at lower cost and/or in a highly selective and sensitive manner. 
         [0050]    Example embodiments being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.