Abstract:
A flow cytometry system suitable for characterizing multicellular aggregates during culture and before implantation combines a low shear flow channel with a multiphoton laser scanning microscope, the latter permitting the characterization of interior and exterior cells in optical isolation from other cells for a representative sampling of fluorescent activity. Imaging capabilities permit sophisticated statistical measurements reflecting individual cell characteristics.

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0001]    This invention was made with United States government support awarded by the following agency:
       NIH EB000184       
 
         [0003]    The United States government has certain rights in this invention. 
     
    
     CROSS-REFERENCE TO RELATED APPLICATIONS 
     Background of the Invention 
       [0004]    The present invention relates generally to flow cytometers and in particular to a flow cytometer using a multiphoton laser scanning microscope to provide interior fluorescence measurements of multicellular aggregates during flow processing. 
         [0005]    The promise of regenerative medicine, using stem or other cells, requires a practical method of assessing multicellular aggregates as they are cultured to determine the viability, proliferative capacity and/or functional competence of the cultured population before implantation. The monitoring system should lend itself to monitoring large numbers of aggregates in an automated or semi automated fashion. 
         [0006]    Flow cytometry is a known technique for monitoring individual cells in an automated fashion. In a typical cytometer, individual cells are dispersed in a liquid medium and passed through a channel that hydrodynamically focuses the cells in a line to pass an inspection region. In the inspection region, the cells may be electronically monitored, for example by light absorption, scattering, or fluorescence, and cataloged via high-speed computer. The monitored data may be used to operate a sorting mechanism that may sort the cells according to the monitored data into different channels for separation. 
         [0007]    Multicellular aggregates can be disrupted under the sheer forces typically generated in a typical flow cytometer. In addition, conventional flow cytometry monitoring systems are not well adapted for the analysis of three-dimensional cellular structures. 
       SUMMARY OF THE INVENTION 
       [0008]    The present inventors have determined that multicellular aggregates can be processed using flow cytometers with properly designed fluid handling systems that reduce shear forces acting on the multicellular aggregates. In addition, it has been determined that the cells can be sufficiently stabilized at low flow rates to permit analysis of interior structure using a multiphoton laser scanning microscopy. This analysis combines imaging and fluorescence permitting a wide range of characterizations of multicellular aggregates which can be output or used for real time sorting. 
         [0009]    Specifically then, the present invention provides a flow cytometry system having a pump communicating with a reservoir to provide a volume flow of liquid from the reservoir to a channel containing the liquid flow. The channel and volume flow are designed to provide a hydrodynamic focusing of multicellular aggregates within the liquid without disruption of intercellular connections of the multicellular aggregate. A multiphoton laser scanning microscope is positioned to illuminate multicellular aggregates within the liquid flow and to record fluorescence of multiple cells of the multicellular aggregates isolated to a focal plane through the multicellular aggregate as the multicellular aggregates reach an analysis point in the channel. A control system, executing at least one stored program, receives data from the multiphoton laser scanning microscope to provide an output signal assessing the multicellular aggregate based on recorded fluorescence from the multiple cells along the focal plane. 
         [0010]    It is thus a feature of at least one embodiment of the invention to provide a method of rapidly characterizing interior cells in multicellular aggregates without damage to the inter-cell connections. 
         [0011]    The output signal may provide a mapping of fluorescent intensity as a function of two dimensions along the focal plane. 
         [0012]    It is thus a feature of at least one embodiment of the invention to combine imaging and fluorescence measurements to provide for more sophisticated analysis metrics. 
         [0013]    The output signal may indicate, for example, a proportion of cells exhibiting fluorescence. 
         [0014]    It is thus a feature of at least one embodiment of the invention to provide for more sophisticated fluorescence measurements distinguishing fluorescence on a cell-by-cell basis. 
         [0015]    The pump may provide a sample stream and a sheath stream combined within the channel so that the sample stream is hydrodynamically focused by the sheath stream and wherein the volumetric flux of the sample stream is 0.1-1 meters per second and a volumetric flux of the sheath stream is 0.5-3.5 meters per second. Alternatively or in addition, sample stream and sheath stream may provide shear forces between cells at the interface between the sheath stream and the sample stream of less than 0.6 mdynes. 
         [0016]    It is thus a feature of at least one embodiment of the invention to provide a flow cytometer adapted for analysis of multicellular aggregates. 
         [0017]    The pump may be a surface tension pump. 
         [0018]    It is thus a feature of at least one embodiment of the invention to provide a simple and intuitive pump system, readily adaptable to the laboratory environment, that can produce controlled flow rates compatible with processing of multicellular aggregates. 
         [0019]    The pump may include at least one automated metering pipette applying a series of drops to a channel at a rate and size to provide a surface tension pumping action. 
         [0020]    It is thus a feature of at least one embodiment of the invention to provide for accurate control of flow rates of liquids holding multicellular aggregates in a low shear environment. 
         [0021]    The pump may use automated metering pipettes to deliver a different series of drops to ports feeding the sheath stream and sample stream to provide different volumetric flow rates in the sheath stream and sample stream. The sheath and sample stream flow rates may be adjusted to provide a flow stream varying in diameter between 300-1600 μm. 
         [0022]    It is thus a feature of at least one embodiment of the invention to provide for accurate control of the volumetric ratios, for example, to control the sample stream size for different multicellular aggregates, increasing the versatility of the instrument. 
         [0023]    The channel maybe formed of an elastomeric material such as polydimethylsiloxane. 
         [0024]    It is thus a feature of at least one embodiment of the invention to provide a high accuracy versatile channel material. 
         [0025]    The channel may include a downstream branching, providing alternative paths for the multicellular aggregates, and the flow cytometry system may further including a cell sorter driven by the controller according to the output signal for directing the multicellular aggregates preferentially to a downstream branch. 
         [0026]    It is thus a feature of at least one embodiment of the invention to permit sophisticated automated cell sorting. 
         [0027]    The cell sorter may use at least one surface tension pump providing a stream intersecting the channel to redirect the multicellular aggregates. 
         [0028]    It is thus a feature of at least one embodiment of the invention to provide a sorting mechanism compatible with the goal of preventing damage to the multicellular aggregates. 
         [0029]    These particular objects and advantages may apply to only some embodiments falling within the claims, and thus do not define the scope of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0030]      FIG. 1  is an exploded orthogonal view of a flow cytometer according to the present invention showing computer-controlled pipettes providing low shear rate passive pumping in conjunction with a channel assembly positioned for scanning by a multiphoton laser scanning microscope at an inspection point in the channel; 
           [0031]      FIG. 2  is a top plan view of the channel assembly used for conducting multicellular aggregates past the inspection point; 
           [0032]      FIG. 3  is a cross-sectional view along line  3 - 3  of  FIG. 2  showing deposited drops providing flow pressure according to the Young-Laplace relationship; 
           [0033]      FIG. 4  is an expanded cross-sectional view of one droplet of  FIG. 3  showing three stages in its evolution with decreasing drop volume; 
           [0034]      FIG. 5  is a plot of droplet pressure as a function of time for the three stages of  FIG. 4  showing timing of a computer-controlled pipette to refresh the droplet to provide substantially constant pumping pressure; 
           [0035]      FIG. 6  is a detailed cross-sectional view of the channel of  FIG. 1  showing a suspended multicellular aggregate between sample and shield flows and the acquisition of image and fluorescence data as may be used to develop an output signal characterizing the multicellular aggregate; 
           [0036]      FIG. 7  is a flow chart of a program executed by the computer of  FIG. 1  to control the computer-controlled pipettes. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0037]    Referring to  FIG. 1 , a flow cytometer  10  of the present invention generally provides a flow plate  12  receiving liquids from a pump assembly  14  so that liquid flowing through the flow plate  12  may be monitored by a multiphoton laser scanning microscope  16 . 
         [0038]    Referring to  FIG. 2 , the flow plate  12  provides a generally horizontal plate  18  holding an analysis channel  20  extending generally along a horizontal axis  21 . In one embodiment of the invention, the analysis channel  20  may be 0.7 mm deep (measured along the vertical axis perpendicular to the plane of the plate  18 ) and 5 mm wide (measured perpendicularly to the axis  21 ). 
         [0039]    An upstream end of the analysis channel  20  communicates with a downstream end of a sample channel  22 , axially aligned with the analysis channel  20 , and with downstream ends of left and right shield channels  24   a  and  24   b  which intersect sample channel  22  at equal and opposed angles. Flow from the shield channels  24   a  and  24   b  thus flanks the flow from sample channel  22  to hydrodynamically focus flow from the sample channel  22  into a narrow stream through the analysis channel  20 . 
         [0040]    The upstream end of the sample channel  22  communicates with a sample port  26  being a vertical bore passing through material forming an upper wall of the sample channel  22  exiting an upper surface of the horizontal plate  18 . Similarly, upstream ends of the shield channels  24   a  and  24   b  converge on a shield port  28  also being a bore passing upward to the upper surface of the plate  18 . 
         [0041]    Liquid received through the sample port  26  ultimately is received by the analysis channel  20  to pass an inspection region  30  where it may be inspected by the multiphoton laser scanning microscope  16 . The liquid then passes to a sorting junction  32  at a downstream end of the analysis channel  20 . 
         [0042]    Downstream ends of control channels  34   a  and  34   b  communicate with the sorting junction  32  at equal and opposite angles on either side of axis  21 . Upstream ends of control channels  34   a  and  34   b  terminate at inlet ports  36   a  and  36   b  that may receive fluid to provide for corresponding streams in control channels  34   a  and  34   b . These streams from control channels  34   a  and  34   b  serve to direct the flow of liquid from the downstream end of analysis channel  20  either leftward or rightward into corresponding sorting channels  38   a  and  38   b  respectively. 
         [0043]    The sorting channels  38   a  and  38   b  diverge at equal angles from the axis  21  on either side of the axis  21 . These sorting channels  38   a  and  38   b  in turn terminate at upstream ends in a collection port  40   a  or  40   b . A stream of fluid from control channel  34   a  will thus direct the flow of liquid from the analysis channel  20  into sorting channel  38   b  and a flow of liquid from control channel  34   b  will direct the flow from the analysis channel  20  into sorting channel  38   a.    
         [0044]    When liquid is introduced at neither port  36   a  nor  36   b , the flow from the downstream end of the analysis channel  20  proceeds axially into waste channel  38   c  to collection port  40   c.    
         [0045]    Referring to  FIG. 3 , the flow plate  12  may be constructed of a lower glass substrate  42  adhered to an upper layer of elastomer  44  incorporating the channels  20 ,  22 ,  24 ,  34 , and  38 , for example, as molded into the elastomer  44 . In one embodiment, the elastomer  44  may be polydimethylsiloxane molded using a template prepared from patterned photoresist on a substrate, the photoresist providing raised portions that form the cores for the channels  20 ,  22 ,  24 ,  34 , and  38 . In this way, the elastomer  44  may form left, right, and top walls of the channels with the glass substrate  42  forming the bottom walls. The glass substrate  42  may be attached to the elastomer  44  by treating the former with an oxygen plasma. Bores for the ports  26 ,  28 ,  36 , and  40  may be cut through the elastomer  44  using a sharpened hypodermic needle acting as a tube drill. 
         [0046]    Referring again to  FIG. 1 , liquid may be introduced into the ports  26 ,  28 , and  36  by means of multiple computer-controlled pipettes  44   a - 44   d  position above the flow plate  12  to deposit metered drops of liquid on the appropriate ports. Computer-controlled pipettes suitable for this purpose may make use of a nanoliter dispensing valve, for example, the VHS valve commercially available from the Lee Company of Connecticut USA. In this example, computer-controlled pipette  44   a  may deposit drops over port  28 , computer-controlled pipette  44   b  may deposit drops over port  26 , computer-controlled pipette  44   c  may dispense drops onto port  36   a , and computer-controlled pipette  44   d  may dispense drops onto port  36   b.    
         [0047]    Computer-controlled pipette  44   a  will be pre-charged with a shield fluid, for example a saline solution compatible with a multicellular aggregate to be studied. Computer-controlled pipette  44   b  will be pre-charged with a fluid similar to that of the shield fluid but containing the multicellular aggregates to be analyzed. Computer-controlled pipettes  44   c  and  44   d  also will be charged with fluids similar to the shield fluid. 
         [0048]    Each of the computer-controlled pipettes  44   a - d  may be controlled by a central controller  46 , for example a computer, providing the necessary electrical interfaces. The central controller  46  will generally include a processor  48 , a memory  50  holding a stored program to be described, and a connected conventional user input device  52  and graphics display screen  54  as is generally understood in the art. 
         [0049]    Referring now to  FIG. 3 , the present invention may use surface tension pumping in which a drop  56 , for example, is deposited at port  26  by computer-controlled pipette  44   b . The surface tension of the liquid of this drop  56  will produce an internal pressure communicating through the port  26  to the sample channel  22  that will move multicellular aggregates  58  into the analysis channel  20 . This pressure is resisted by a countervailing pressure, for example by a drop  60  at the waste port  40   c , and thus it is the net difference between these pressures that will dictate the flow rate through the analysis channel  20 . Generally drop  56  will be on the order of 0.1 to 5 μL and drop  60  one hundred times larger and, as a result, the surface tension pressure generated by drop  56  will be substantially higher than the surface tension pressure generated by drop  60 . The large size of drop  60  is further selected so that its surface tension pressure will be substantially constant. The flow rate in the analysis channel  20  will be a function of the density of the fluid, the surface energy of the communicating drops, and the channel resistance. 
         [0050]    Referring now also to  FIGS. 4 and 5 , passive pumping harnesses the higher internal pressure of smaller drops of liquid compared to larger drops of liquid  56  and further takes advantage of a relatively constant region of pressure as drop size shrinks with fluid flow. During a first phase  63  of fluid flow from drop  56  when drop  56  has a peak volume  58   a , the internal pressure  61  is substantially constant being bounded by less than a 10% range around an average pressure of 150 Newtons per meter squared. This first phase  63  peaks at droplet contact angles of 90°. At a second phase  64  corresponding generally to smaller peak volume  58   b , the pressure varies substantially until the flow ceases at phase  66 . The present invention, accordingly, provides a refill droplet from a computer-controlled pipette at times  68  sometime in phase  62  causing the pressure to rebound as indicated by dotted line  70  for substantially continuous pumping pressure with extremely low and accurately controlled pressure ranges suitable for multicellular aggregates. Changing the times  68  can change the average pressure and thus the flow rate provided by the pump. 
         [0051]    Generally the refill period may be controlled in an open loop fashion by the controller  46  as will be described below. This passive pumping system provides an extremely simple mechanism that may be readily adapted to a variety of different analysis tasks and compares favorably in accuracy to syringe micro pumps which provide mean pressure values that can change by 5% or more. 
         [0052]    Referring to  FIG. 6 , the multicellular aggregates  58  may be hydrodynamically focused within a stream  90  from the sample channel  22  as flanked by streams  92  from the shield channels  24   a  and  24   b . Although this process is shown in two dimensions, it will be understood that this hydrodynamic focusing may occur in three dimensions to locate the stream  90  and thus the multicellular aggregates  58  in a center of the analysis channel  20  both vertically and horizontally. See generally, “Two simple and rugged designs for creating microfluidic sheath flow” Howell et al, Lab Chip 2008, 8, 1097-1103 thereby incorporated by reference. Generally the size of the stream  90  may be controlled by controlling the relative volume flow rates a being the sum of volumetric flow rates of both shield channels  24   a  and  24   b  over the volumetric flow rate of the sample channel  22 . In this regard, channel widths varying from 300 μm to 1500 μm (at α=2) may be obtained in one embodiment of the invention producing a flow in the outlet channel equal to 600 μL per minute. 
         [0053]    Referring again to  FIGS. 1 and 2 , as multicellular aggregates pass by the inspection region  30 , they may be analyzed by the multiphoton laser scanning microscope  16 . Such microscopes which are generally known in the art may include a laser  71 , for example a Ti:Sapphire laser (for example, as is available from Spectra Physics (Newport Corporation) of Irvine Calif. USA under the tradename Tsunami), providing for wavelength tuning in a range of 700-1100 nm. The beam  76  from the laser  71  may be attenuated by a Pockel&#39;s cell  72  (for example, such as commercially available from Conoptics of Danbury Conn.) or similar device controlled by the controller  46  to provide the desired level of illumination of the multicellular aggregates. This beam  76  is then received by a beam expander  74  and a computer-controlled mirror  78  (for example, a galvanometer scanning head such as is commercially available from Cambridge Technologies of Lexington, Mass. USA). The resulting beam  76  may be deflected in a raster pattern by computer-controlled mirror  78  to be received by a scan lens  80  and transfer lens  82 . The resulting light is then focused by objective lens  84  (for example, as part of an inverted microscope such as the TE2000 commercially available from Nikon of Japan) on the inspection region  30 . Light  91  passing through the inspection region  30  is received by a photodiode  86  whose output is received by the controller  46  to create a coarse bright field image  88  (shown in  FIG. 6 ). Light  93  returned from the multicellular aggregates by two photon or multiphoton induced fluorescence passes back through the objective lens  84  and is received by a dichromatic mirror  83  selecting for the fluorescence frequency which directs the fluorescence to a photomultiplier tube detector  91  (for example, as is commercially available from Hamamatsu of Japan under the tradename  7422 ) that may comparably make a fluorescence image  94  (shown in  FIG. 6 ) of the multicellular aggregates  58 . Generally, the fluorescence image  94  will show fluorescent regions  98  associated with particular cells  99  and non-fluorescent regions  101  that may be distinguished within the resolution of the image  94 . In one embodiment, the objective lens  84  may be maintained at a fixed focal distance so that the data is collected on a single focal plane  87  (shown in  FIG. 6 ). A scan speed of approximately 2.33 frames per second with a resolution of 256×256 pixels may be obtained by this system. Scanning speed increases are possible through reduced data acquisition resolution, line scanning approaches and higher-speed acoustic opticalcomputer-controlled mirrors. Accordingly a given multicellular aggregate may be scanned multiple times while in the inspection region  30  and these images averaged for improved image quality. 
         [0054]    Referring again to  FIG. 6 , the combination of fluorescence data (intensity) and image data (a mapping of direct fluorescence or reflected fluorescence along two dimensions of the focal plane  87 ) may be combined to provide for sophisticated analysis of the multicellular aggregates  58 . In a simple example, the fluorescence may be integrated over the fluorescence image  94  and divided by the total area of the image taken from the fluorescence image  94  or the bright field image  88 . This provides a measure of the proportion of cells providing fluorescence such as may indicate a certain metabolic activity (for inherent fluorescence) or uptake of exogenous florophores providing information about the phenotype of the cell, the ability of the cell to function or the interaction of the cell with its microenvironment. More sophisticated image processing of the fluorescence image  94  can provide for a cell count in the image and can associate individual cells with peaks of fluorescent activity to also provide a proportion of the cells within the sample exhibiting fluorescence. Note that the operation of the multiphoton laser scanning microscope  16  is such as to reject the measurement of cells outside of the focal plane  87  with a high rejection rate caused by the precise focus of the multiple photons necessary for multiphoton fluorescence on the focal plane  87 . 
         [0055]    The fluorescence image  94  and/or bright field image  88  may be output to the display screen  54  (shown in  FIG. 1 ) as well as a quantitative measurement  100  extracted by an image processing engine  103 , for example, providing morphometric filters providing the analysis described above with respect to indicating a proportion of cellular fluorescence. The quantitative measurement  100  may also be received by a range comparator  102  that may be used for sorting purposes to sort cells according to whether they are above or below pre-established ranges (A or B) and provide a sorting signal  105  that may operate a cell sorting device. 
         [0056]    Referring again to  FIG. 1 , the sorting signal  105  (shown in  FIG. 6 ) may be used to activate computer-controlled pipettes  44   c  or  44   d  for sorting of cells when they reach the sorting junction  32  (shown in  FIG. 2 ). 
         [0057]    Referring now to  FIG. 7 , in this regard the controller  46  executing the stored program  104  in memory  50  may at a first process block  106  check a shield fluid delivery schedule and activate computer-controlled pipette  44   a  if a drop is scheduled to be delivered to port  28 . Similarly at succeeding process block  108 , a sample fluid delivery schedule is checked and computer-controlled pipette  44   b  activated if a drop is scheduled to be delivered to port  26 . At decision block  110 , the sorting signal  105  is evaluated to see if a sorting should occur. If so, at process block  112  or process block  114 , depending on which of the ranges A or B has been crossed, after a suitable transport delay time between inspection region  30  and sorting junction  32 , one of computer-controlled pipettes  44   d  or  44   c  is activated as appropriate. Again, this surface tension pumping provides for flow rates consistent with the preservation of the integrity of the multicellular aggregates  58 . 
         [0058]    The present invention may be used for the evaluation of embryonic bodies or pancreatic islets and many other forms of multicellular aggregates. The data collected may be used to analyze these multicellular aggregates not simply with respect to the spatial distribution of fluorescence within the cells or the phenotypes of the cells or other techniques described above, but also with respect to the spectra of the fluorescence and fluorescence lifetime in order to get a read out of chemical interaction between the cells and the microenvironment. Spectra can be obtained by modification of the photomultiplier measuring fluorescence to provide for spectrographic capabilities, for example, by appropriate optics. The analysis of the intrinsic fluorescence may allow a metabolic profiling of stem cell differentiation on the basis of NADH and FAD signals. 
         [0059]    It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims.