Abstract:
The method and the system simplify moving interactions by means of virtual reference subjects and flux-based coordinate transformations in order to generate a changeable frame of reference.

Description:
RELATED APPLICATIONS  
         [0001]    This application claims priority of the German patent application 103 15 592.9 which is incorporated by reference herein.  
         FIELD OF THE INVENTION  
         [0002]    The invention concerns a method for performing interactions on microscopic subjects that have changed in space and time. “Interaction” here means on the one hand the performance of manipulations on the microscopic subjects. “Interaction” further means the acquisition, storage, and processing of data recovered from the selected microscopic subjects.  
           [0003]    A second aspect of the invention concerns a system for observing and manipulating microscopic subjects that have changed in space and time. The system encompasses in particular a confocal scanning microscope that guides an illuminating light beam over a subject; several detectors that identify, from the light proceeding from the subject, intensities from different spectral regions; a processing unit; a PC; an input unit; and a display.  
         BACKGROUND OF THE INVENTION  
         [0004]    Numerous methods exist for performing interactions on biological subjects. It must be noted in this context, however, that these biological subjects are stationary or change little in time and space. An evaluation of data from microscopic subjects that change in time and space has not hitherto been performed satisfactorily.  
         SUMMARY OF THE INVENTION  
         [0005]    The object underlying the invention is to create a method for performing interactions on microscopic subjects that change in space and time, and in that context to be able to guarantee, regardless of the motion of the microscopic subjects, a controlled interaction at the selected regions or positions within the microscopic subject.  
           [0006]    The stated object is achieved by way of a method for performing interactions, using a microscope, on microscopic subjects that change in space and time, comprises the following steps:  
           [0007]    acquiring at least one image of a sample that encompasses at least one microscopic subject;  
           [0008]    defining by the user virtual reference subjects on a discrete grid of the acquired image or images, in order to define regions;  
           [0009]    automatic acquiring of a sequence of image data or volume data;  
           [0010]    successive identifying an optical flux based on the sequence of acquired images;  
           [0011]    applying the identified optical flux to the defined reference subjects; and  
           [0012]    performing interactions on the reference subject modified by the optical flux.  
           [0013]    A further object of the invention is to create a system for performing interactions on microscopic subjects that change in space and time, and in that context to be able to guarantee, regardless of the motion of the microscopic subjects, a controlled interaction at the selected regions or positions within the microscopic subject.  
           [0014]    The stated object is achieved by way of a system for interactions on microscopic subjects that change in space and time comprising:  
           [0015]    a confocal scanning microscope that guides an illuminating light beam over a subject;  
           [0016]    several detectors that identify, from the light proceeding from the subject, intensities from different spectral regions;  
           [0017]    a processing unit;  
           [0018]    a PC;  
           [0019]    an input unit;  
           [0020]    a display on which an individual image is presented to the user; the user interactively defines virtual reference subjects on the image shown on the display, using the input unit for position definition;  
           [0021]    a means for determining the optical flux based on the intensities from different spectral regions identified by the detectors is housed in the processing unit; and  
           [0022]    a means for applying the optical flux to the virtual reference subjects is present in the processing unit and the processing unit controls interactions on the basis of the changed reference subjects.  
           [0023]    The invention has the advantage that firstly an image of a sample is generated with a microscope, the image encompassing at least one microscopic subject. On the basis of the image, at least one virtual reference subject is defined interactively by the user. This reference subject defines a discrete point set of interaction locations, continuous regions of interaction locations, or local coordinate systems that are placed in the center points of the imaged subjects. At least one region, or several positions, in the subject are determined by this step. A sequence of images is acquired with the microscope, and an optical flux is calculated from those images. This optical flux describes the motion of the grayscale values from one image to the next. The optical flux calculated from the sequence of images is applied to the set of predefined virtual reference subjects, such as the Cartesian coordinate system, region, or positions in the subject. Lastly, the interactions are performed on the virtual reference subjects modified by the optical flux, and therefore in a modified coordinate system, a modified region, or the modified positions in the microscopic subject that is changing in time and space. This ensures that the interaction is performed at the location desired by the user, despite independent motion or deformation of the microscopic subject.  
           [0024]    The interaction is defined, in the context of this document, as the recovery of measured data from the subject, or also as the controlled manipulation of the subject at at least one position of the subject. Examples including the recovery of intensity parameters by imaging or by calculations such as mean, maximum, variance, or any elements of the intensity distribution within a region. The recovery of measured data can also be the recovery of geometrical data—for example the center point, area, periphery, or volume of a marked region, or the motion of the center point of the marked region—that change under the influence of the flux field. Further classes of measured values result from the detection of collisions of marked subjects under the influence of the flux field. These measured data are extracted in suitable form by means of the method, and are subsequently available for visualization and computer-controlled data analysis. Further examples of interactions is the controlled illumination of positions or areas within the subject with electromagnetic radiation. Manipulation of the subject may involve, for example, controlled bleaching of dyes, controlled photo activation of dyes, or cage-compound release in a specific region of the microscopic subject (e.g. a cell nucleus). Manipulation of the microscopic subject can also encompass the cutting or excision of the specific region or positions of the subject. It is particularly important in this context that the motions of and the change over time in the subject also be sensed, in order to cut or manipulate the microscopic subject at the correct and desired point. The interaction can thus encompass the recovery of all conceivable measured data and also the controlled manipulation of the subject in a selected region or at selected positions of the microscopic specimen.  
           [0025]    The invention will be described below using the example of a confocal microscope, it being clear to one skilled in the art that this same technology can also be applied to other types of microscope. Further advantageous embodiments of the invention are evident from the dependent claims.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0026]    The subject matter of the invention is depicted schematically in the drawings and will be described below with reference to the Figures, in which:  
         [0027]    [0027]FIG. 1 schematically depicts an exemplary embodiment of a scanning microscope, an SP module being placed downstream from the detectors;  
         [0028]    [0028]FIG. 2 shows an embodiment of the selection of a microscopic subject;  
         [0029]    [0029]FIG. 3 shows a further embodiment of the selection of a microscopic subject;  
         [0030]    [0030]FIG. 4 graphically depicts the motion of the subject in the Cartesian coordinate system;  
         [0031]    [0031]FIG. 5 graphically depicts the motion of a subject, a Cartesian coordinate system being defined at the center point of the subject and being co-moved as the subject moves;  
         [0032]    [0032]FIG. 6 schematically depicts the identification of the displacement vector field; and  
         [0033]    [0033]FIG. 7 schematically depicts a subregion of a further embodiment of the microscope system, various positioning elements being provided. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0034]    [0034]FIG. 1 schematically shows a exemplary embodiment of a confocal scanning microscope  100 . Although a confocal scanning microscope  100  is depicted here, this is not to be construed as a limitation of the invention. It is also equally conceivable to implement the subject matter of the invention using a conventional microscope. Illuminating light beam  3  coming from at least one illumination system  1  is directed by a beam splitter or a suitable deflection means  5  to a scanning module  7 . Before illuminating light beam  3  strikes deflection means  5 , it passes through an illumination pinhole  6 . Scanning module  7  encompasses a gimbal-mounted scanning mirror  9  that guides illuminating light beam  3 , through a scanning optical system  12  and a microscope objective  13 , over or through a sample  15 . In the case of non-transparent samples  15 , illuminating light beam  3  is guided over the sample surface. With biological samples  15  or transparent samples, illuminating light beam  3  can also be guided through the sample. For these purposes, non-luminous specimens are prepared, as applicable, using a suitable dye (not depicted, since it is established existing art). The dyes present in the sample are excited by illuminating light beam  3  and emit light in a region of the spectrum characteristic of them. This light proceeding from sample  15  defines a detected light beam  17 . The latter travels through microscope objective  13  and scanning optical system  12 , and via scanning module  7  to deflection means  5 , passes through the latter, and travels through a detection pinhole  18  onto at least one detector unit  19  that is embodied, for example, as a photomultiplier. In the system with scanning microscope  100  depicted here, an SP module  20  is placed before detector  19 . With SP module  20 , the user can select the appropriate region for detection and optionally direct it onto corresponding detectors. It is clear to one skilled in the art that other detectors, for example diodes, diode arrays, APDs, photomultiplier arrays, CCD chips, or CMOS image sensors, can also be used. Detected light beam  17  proceeding from or defined by sample  15  is depicted in FIG. 1 as a dashed line. In detector  19 , electrical detection signals are generated in proportion to the power level of the light proceeding from sample  15 . Because, as already mentioned above, light of more than one wavelength is emitted from the sample, it is advisable to provide SP module  20  in front of the at least one detector unit  19 . The data generated by detector  19  or the detectors are forwarded to a computer system  23 . Associated with computer system  23  is at least one peripheral unit  27 , which is embodied e.g. as a display. The data of sample  15 , acquired by the detectors and supplied to computer system  23 , are displayed graphically to the user with display  27 . The graphical depiction of sample  15  encompasses at least one microscopic subject  30 . Also associated with computer system  23  is an input means that comprises, for example, a keyboard  28  of an adjusting apparatus  29  for the components of the microscope system, and a mouse  25 . With mouse  25  the user can, for example, select microscopic subject  30  so that appropriate manipulations can be performed on the selected subject  30 . In addition to selecting the entire microscopic subject  30 , the user can also select individual positions  63   0  or pixels in the image of sample  15  so that controlled manipulations or interactions can be performed on the basis of those selected positions. The microscopic subject can also be selected by the user, without direct marking, by means of automatic image processing algorithms. These image processing algorithms are sufficiently known from the existing art.  
         [0035]    [0035]FIG. 2 depicts one embodiment for selection of a microscopic subject within sample  15 . The data of sample  15  acquired by scanning microscope  100  are depicted on display  27  in visual or graphical form. The image of sample  15  encompasses several microscope subjects  30 . At least one microscopic subject  30  from among microscopic subjects  30  can be selected by the user. Selection of microscopic subject  30  is accomplished, for example, by means of mouse  25 . To do so the user can, for example, align a crosshairs  35  on the microscopic subject using mouse  25 . That microscopic subject  30  is selected with a mouse click. On the basis of the selection, computer system  23  identifies the center point of microscopic subject  30 . As depicted in FIG. 3, for example, the user can likewise define within a selected microscopic subject  30 , by means of a mouse pointer  37 , a virtual reference subject which stipulates a region  36  within which certain manipulations or interactions can be performed. The user can likewise define, in or on the microscopic subject, multiple positions  38  at which the selected measurements or interactions will be performed.  
         [0036]    [0036]FIG. 4 graphically depicts the motion of a microscopic subject  30  in a static coordinate system. Static coordinate system  40  encompasses an X axis and a Y axis, both arranged perpendicular to one another. A microscopic subject  30  is plotted in coordinate system  40  at time T=0. Microscopic subject  30  is also plotted in FIG. 4 at time T=N. The transition from microscopic subject  30  at time T=0 to microscopic subject  30  at time T=N is depicted by means of a first vector  41  and a second vector  42 . Second vector  42  is longer than first vector  41 , indicating a motion of microscopic subject  30  that results from a translation in combination with a rotation. With this embodiment, it is difficult to define and retrieve the interaction positions and interaction surfaces that were determined and selected. The reason for this is principally that a static coordinate system is used to track the interaction positions and interaction surfaces. It is always difficult to calculate from the coordinate origin of static coordinate system  40  to the new position or location of the regions or locations within microscopic subject  30  at time T=N.  
         [0037]    [0037]FIG. 5 graphically depicts the motion of a microscopic subject, a coordinate system being placed, as a virtual reference subject, at the center point of the microscopic subject and following the microscopic subject as it moves. As already mentioned above, the user selects a microscopic subject  30  in the image of the sample, and computer system  23  identifies center point  50  of microscopic subject  30 . FIG. 5 depicts microscopic subject  30  at time T=0. Center point  50   0  is located inside microscopic subject  30  at time T=0. Center point  50   0  constitutes the origin of coordinate system  52   0  at time T=0. Microscopic subject  30  at time T=0 further encompasses a region  51  at which manipulations or interactions may need to be performed. Microscopic subject  30  at time T=N is derived, by rotation and translation, from microscopic subject  30  at time T=0. In addition, microscopic subject  30  may also have been subjected to deformations. Deformations often occur in biological subjects. There is thus also a change in the location of center point  50  at time T=N within microscopic subject  30  at time T=N. Region  51  at which manipulations or interactions are to be performed has, in the present case, migrated partially out of microscopic subject  30 . The subject motions are followed by tracking the coordinate transformation, by allowing the optical flux field to act as a force on the coordinate system. This method depicted in FIG. 5 sometimes functions adequately, but fails in certain situations. Examples thereof are the deformations of the microscopic subjects already mentioned, which are common in biology.  
         [0038]    [0038]FIG. 6 schematically depicts the identification of a displacement vector field  60  that represents the rotation and translation between a microscopic subject  30  at time T=0 and a microscopic subject  30  at time T=N. Microscopic subject  30  is depicted by a region  61  that is enclosed by a line  62 . Line  62  can also enclose the selected region, and can thus be at least a portion of microscopic subject  30 . Computer system  23  then compares the defined positions  63   0  on microscopic subject  30  at time T=0 with the locations of several positions  63   N  on microscopic subject  30  at time T=N, by calculating a flux field that varies over time. Human eyes can perceive this by tracking individual features, as illustrated in the drawing by several positions  63   0  along line  62 . The eye cannot perceive this transformation if reference features are absent, whereas the mathematical tool supplies a solution. Displacement vector field  60  results from the association of mutually corresponding pixel positions at time T=0 and at time T=1, at the subsequent time T=2, and so on until time T=N. Displacement vector field  60  is determined by the fact that computer system  23  searches successively for correspondences between all pixel positions  63   0  at time T=0 and pixel positions  63   N  at time T=N. The limitation to pixels  63   0 ,  63   N  is made only for illustrative purposes; mathematically, the solution to the flux problem is performed using all the pixels. Although only two successive microscopic subjects are depicted in FIGS. 4 and 5, it is self-evident to one skilled in the art that the method of the invention can be applied to an image sequence of N images. Generalization to more than one subject is also readily possible. Based on the sequence of images or volumes, the deformation and motion are determined by solving the flux problem; these are then applied to the initially defined regions or positions, which can thus be tracked. This method does not require binarization of subjects, but instead operates on intensities and thus on a quasi-continuous model. The displacement vector field is identified, for example, by way of equations 1 through 3, which are also referred to as “optical flux” equations:  
               λ          ∇   2          v   1         =       (           ∂   I       ∂   x            v   1       +         ∂   I       ∂   y            v   2       +       ∂   I       ∂   t         )            ∂   I       ∂   x                 (     equation                 1     )                 λ          ∇   2          v   2         =       (           ∂   I       ∂   x            v   1       +         ∂   I       ∂   y            v   2       +       ∂   I       ∂   t         )            ∂   I       ∂   x                 (     equation                 2     )                 ∇   2          =         ∂   2         ∂   2        x       +       ∂   2         ∂   2        y                   (     equation                 3     )                               
 
         [0039]    where I is the intensity. It is well known to one skilled in the art, however, that there are further flux equations which can replace these. The vector fields deriving from these equations are the modification instructions for adapting, at each time step, the locations defined by the virtual reference subjects.  
         [0040]    The modifications or interactions are always performed on the modified coordinate system, and consequently at the modified position of microscopic subject  30 . This means in practical terms that region  61  or positions  63  marked in microscopic subject  30  are subjected to the coordinate system modification defined by the displacement vector field that was identified. The modifications or interactions are applied, on the basis of the coordinate system modifications, to the virtual reference subjects, i.e. for example, to region  61  or to positions  63 . The geometrical data, e.g. the center point, area, periphery, or volume of the changing microscopic subject  30 , are thus, for example, ascertained as a function of time. The motion of the center point or of similar features over time can likewise be evaluated and graphically depicted. Derived geometrical data, such as velocity, acceleration, or deceleration, can be ascertained from the geometrical data. Collision, combination, and separation statistics of predefined microscopic subjects in the image of sample  15  can likewise be acquired over time, these statistics being generalized in the context of the aforementioned definition as “measurement.” Intensities within a region of interest can likewise be acquired over time. This makes it possible to identify, for example, organelles or other structured subjects identifiable by biologists within a biological mass, and to measure processes in the interior of the microscopic subjects or organelles even though they are vigorously moving or wriggling. In addition, manipulations of the marked pixel regions by means of electromagnetic radiation can be performed with the method according to the present invention. When a scanning microscope is used, the manipulation by radiation at present preferably involves laser radiation for the purpose of bleaching, cutting, cage-compound release, or photoactivation, it being sufficiently clear to one skilled in the art that any additional interaction with electromagnetic radiation that arises in the future is also covered. This manipulation is applied exclusively to the region of microscopic subject  30  defined by the virtual reference subject, regardless of how the region of microscopic subject  30  changes and moves over time. When a conventional microscope or a scanning microscope is used, it is possible, for example, to mark regions or positions of a microscopic subject  30 , track them over time, and cut or manipulate them at a specific point in time. The method according to the present invention ensures in this context that regardless of the motion of microscopic subject  30 , the location which was selected by the user at a time T=0 is cut or manipulated.  
         [0041]    [0041]FIG. 7 schematically depicts a subregion of the microscope system, disclosing the connection of computer system  23  to the various positioning elements of scanning microscope  100 . In a particular embodiment, calculation of the motion of microscopic subject  30  can be performed using a FPGA/DSP  63 . In addition, FPGA/DSP  63  can also be employed to readjust scanning microscope  100  by way of correspondingly controllable positioning elements, if the selected microscopic subject  30 , or the region in microscopic subject  30 , leaves the image field defined by the microscope. FPGA/DSP  63  can also be embodied as a plug-in module in computer system  23  itself. It is likewise conceivable for a software program to implement the calculation for motion tracking and for identifying the displacement vector field. In this context, both the software and/or FPGA/DSP  63  can coact in appropriate fashion. The changes in microscopic subject  30  identified by the software module and/or by FPGA/DSP  63  are employed for correcting the optical systems of the scanning microscope as applicable. For example, if it is necessary to correct the image field of the scanning microscope because microscopic subject  30  has migrated out, actuator signals to corresponding positioning elements are thus supplied to the software program or to FPGA/DSP  63 . Scanning microscope  100  is equipped with an XYZ stage  65  that is oriented adjustably in all three spatial dimensions. For each axis, a positioning motor  66  is provided with which a suitable adjustment of XYZ stage  65  can be made. The signals for the adjustment are generated, on the basis of the tracking, by the software or by FPGA/DSP  63 . The software or FPGA/DSP  63  likewise generates signals to adjust an objective turret  67  of scanning microscope  100  and thus to allow a modification of the image field to be brought about. Objective turret  67  encompasses a first positioning motor  68  for rotating the objective turret, so that one of the several objectives  70  can be brought into the working position. In addition, a second positioning motor or actuator  69  (piezoelement) is provided which generates a relative motion between objective turret  67  and XYZ stage  65 . Selection of a different objective  70  is suggested, for example, when a new image window needs to be selected because of the motions of microscopic subject  30  or of the selected region. Corresponding control signals are likewise supplied by the software or the FPGA/DSP  63 , on the basis of the identified displacement vector field, to the galvanometers of the scanning module, so that they can follow the motion of microscopic subject  30  in appropriate fashion and thus apply the desired interaction or modification exclusively to the selected region of the microscopic subject. The number of positioning possibilities in the microscope system substantially depends principally on the particular equipment desired for the microscope system. A standard configuration of a scanning microscope  100  often has, for example, alongside an XYZ galvanometer control system for controlling the scan point, additionally an XY stage and a coarse Z actuator, resulting in two sets of actuators for each of X, Y and Z that can be used for control purposes.  
         [0042]    The invention has been described with reference to a particular embodiment. It is self-evident, however, that changes and modifications can be made without thereby leaving the range of protection of the claims below.