Method and apparatus for microphotometering microscope specimens

Abstract

A method of microphotometering individual volume elements of a microscope specimen 10, comprising generating a luminous dot or cursor and progressively illuminating a plurality of part elements in the focal plane 11 of the microscope through the specimen. The mutual position between the specimen and the focal plane is then changed and a plurality of part elements in the focal plane are illuminated. Reflected and/or fluorescent light and transmitted light respectively created by the illumination is collected, detected and stored for generating a three-dimensional image of that part of the specimen composed of the volume elements. Illumination of multiples of part elements is implemented by deflecting the cursor and/or by moving the specimen. The change in the relative mutual position between the specimen and the focal plane of the microscope is effected either by displacing the specimen or the objective. Apparatus for carrying out the method include a specimen table 301, a microscope objective and light source 31-32-33. The table or the objective are arranged for stepwise movement along the main axis of the microscope synchronously with punctilinear light scanning of the specimen. The table is arranged for stepwise movement at right angles to the main axis and/or the light source is arranged for deflection over the focal plane 21 through the specimen.

I claim: 
     
       1. A method for microphotometering and subsequent image combination by generating with the aid of a convergent light beam a luminous dot or cursor in the focal plane (11) of a microscope (30), fitting the cursor to a plurality of part elements in the specimen (10), and collecting light created by the luminous cursor and the specimen (10), detecting the collected light and producing corresponding electric signals, characterized by changing the mutual position between the specimen (10) and the focal plane (11) and re-fitting the luminous cursor to a plurality of part elements in the specimen (10); repeating stepwise changes in the mutual position between the specimen (10) and the focal plane (11) and, subsequent to each such change, again fitting or matching the luminous cursor to a plurality of part elements in the specimen; collecting the light created by the luminous cursor and part elements in the specimen (10) and screening-off any disturbing light created synchronously from adjacent (above, beneath, beside) part elements in the specimen (10); detecting the thus collected light and storing measurement values obtained through said detection, said storage optionally being effected synchronously with the matching of the luminous cursor with part elements in the specimen (10) and with the changes in the mutual position between the specimen (10) and the focal plane (11), said measurement values being representative of locations in various layers through the specimen; and combining the measurement values from locations in a plurality of layers, representative of a given volume of the specimen, in dependence upon a planned/desired analysis of the specimen.[...]. .Iadd.in a manner yielding a projected representation from a desired angle of at least a portion of the specimen. .Iaddend. 
     
     
       2. A method according to claim 1, characterized in that matching of the luminous cursor with a plurality of part elements in the specimen (10) is effected by .[.delinking.]. .Iadd.deflecting .Iaddend.stepwise the convergent light beam in two dimensions (x- and y-directions); and in that the stepwise change in the mutual position between the specimen (10) and the focal plane .[.(10).]. .Iadd.(11) .Iaddend.of the microscope is effected by moving stepwise the microscope object table (301) on which the specimen is placed (z-direction). 
     
     
       3. A method according to claim 1, characterized in that matching of the luminous cursor with a plurality of part elements in the specimen (10) is effected by relative rapid stepwise deflection of the convergent light beam in one dimension (y-direction), and by relatively slow stepwise displacement of the microscope object table (401) on which the specimen (10) is placed in a further dimension (x-direction); and in that the stepwise change in the mutual position between the specimen (10) and the microscope focal plane .[.(10).]. .Iadd.(11).Iaddend.is effected by stepwise displacement of the microscope object table (401) (z-direction). 
     
     
       4. A method according to claim 1, characterized in that matching of the luminous cursor with a plurality of part elements in the specimen (10) is effected by stepwise displacement of the microscope object table (501) along a surface (x-y-plane) perpendicular to the main axis of the microscope; and in that the stepwise change in the mutual position between the specimen (10) and the focal plane of the microscope (50) is effected by stepwise displacement of the object table (501) of the microscope (50) (z-direction). 
     
     
       5. A method according to claim 4, characterized in that collection of light (reflected fluorescent light) created by the luminous cursor and part elements in the specimen (10) is effected on that side of the object table (501) on which the microscope (50) is placed. 
     
     
       6. A method according to claim 5, characterized in that collection of light (transmitted light) created by the luminous cursor and part elements in the specimen (10) is effected on the opposite side of the object table (661) to that on which the microscope (60) is placed. 
     
     
       7. Apparatus for the microphotometering and subsequent image combination of a specimen, comprising a microscope (30) having an object table (301), a light source (31-32-33), a detector (35) and a control and data-collecting assembly (36), characterized in that the object table (301) of the microscope (30) is arranged for stepwise movement in a direction corresponding to the main axis (z-direction) of the microscope (30), said movement being controlled and effected in response to guide pulses from the control and data-collecting assembly (36) in synchronization with the scanning of the light source (31-32-33) of part elements in a microscope specimen (10) placed on the object table (301); and in that the apparatus also includes .[.equipment.]. .Iadd.means .Iaddend.(37, 38,39) for storing, processing and visually displaying data originating from said measurement values.[...]. .Iadd.in a manner yielding a projected representation from a desired angle of at least a portion of the specimen. .Iaddend. 
     
     
       8. Apparatus according to claim 7, characterized in that the object table (401) of the microscope (40) is arranged for stepwise movement in a first direction (x-direction) at right angles to the main axis of the microscope (z-direction); in that the light source (41-42-43) is arranged to scan stepwise part elements in the specimen in a further direction (y-direction) at right angles to the main axis of the microscope (z-direction); and in that movements of the object table (401) and the light source (41-42-43) are co-ordinated for scanning a first plurality of part elements in a first plane through the specimen, and then of a second plurality of part elements in a second plane through said specimen, said second plane extending plane parallel with the first plane, etc. for scanning the whole specimen. 
     
     
       9. Apparatus according to claim 7, characterized in that the object table (501) of the microscope (50) is arranged for relatively slow stepwise movement in a first direction (x-direction) at right angles to the main axis (z-direction) of the microscope (50) and in a relatively rapid stepwise movement in a further direction (y-direction) at right angles to the main axis (z-direction) of the microscope, wherewith movements of the object table (501) in planes at right angles to the main axis of the microscope and parallel with the main axis are co-ordinated through control pulses from the control and data-collecting assembly (56) for scanning part element after part element through the whole of the specimen. .Iadd. 
     
     
       10.  A method for microphotometering a 3-dimensional specimen using a light detection apparatus, the specimen defining a plurality of layers, each layer defining a plurality of part elements, and the detection apparatus defining a focal plane, the method comprising the following steps: (a) generating a cursor of light in the focal plane;   (b) positioning at least one of (1) the focal plane and (2) the specimen such that a desired one of the layers lies in the focal plane;   (c) selecting at least one of the part elements in the selected layer;   (d) positioning at least one of (1) the cursor and (2) the specimen to illuminate by means of the cursor the selected part element;   (e) screening-off unwanted light from part elements adjacent the selected part element;   (f) detecting light from the selected part element;   (g) producing signals indicative of predetermined characteristics of the detected light;   (h) storing representations of the signals;   (i) repeating selected ones of steps (a)-(h) a desired number of times; and   (j) analyzing the representations of the signals to produce measurements of the specimen representative of at least one 3-dimensional characteristic of at least a portion of the specimen. .Iaddend. .Iadd.   
     
     
       11.  The method of claim 10 wherein the measurements produced in step (j) are volumetric measurements. .Iaddend. .Iadd.12. The method of claim 10 wherein the measurements produced in step (j) are surface area measurements. .Iaddend. .Iadd.13. The method of claim 10 wherein the measurements produced in step (j) are light intensity measurements. 
     
     
        .Iaddend. .Iadd.14.  The method of claim 10 wherein the measurements produced in step (j) are distance measurements. .Iaddend. .Iadd.15. The method of claim 10 wherein the measurements produced in step (j) are angular measurements. .Iaddend. .Iadd.16. The method of claim 10 wherein the measurements produced in step (j) are surface parameter measurements. 
     
     
        .Iaddend. .Iadd.17.  A method for microphotometering a 3-dimensional specimen using a light detection apparatus, the specimen defining a plurality of layers, each layer defining a plurality of part elements, and the detection apparatus defining a focal plane, the method comprising the following steps: (a) generating a cursor of light in the focal plane;   (b) positioning at least one of (1) the focal plane and (2) the specimen such that a desired one of the layers lies in the focal plane;   (c) selecting at least one of the part elements in the selected layer;   (d) positioning at least one of (1) the cursor and (2) the specimen to illuminate by means of the cursor the selected part element;   (e) screening-off unwanted light from part elements adjacent the selected part element;   (f) detecting light from the selected part element;   (g) producing signals indicative of predetermined characteristics of the detected light;   (h) storing representations of the signals;   (j) repeating selected ones of steps (a)-(h) a desired number of times; and   (j) analyzing the representations of the signals to produce a projected representation from a desired angle of at least a portion of the specimen.   
     
     
        .Iaddend. .Iadd.18.  The method of claim 17 wherein the projected representation is a 2-dimensional representation of a portion of the specimen bordered by arbitrarily oriented parallel virtual planes. .Iaddend. .Iadd.19. The method of claim 17 wherein the projected representation is a 2-dimensional representation of a portion of the specimen bordered by arbitrarily oriented nonparallel virtual planes. .Iaddend. .Iadd.20. An apparatus for microphotometering a 3-dimensional specimen, the specimen defining a plurality of layers, each layer defining a plurality of part elements, the apparatus comprising: means for detecting light from a focal plane;   means for generating a cursor of light in the focal plane;   means for positioning at least one of (1) the focal plane and (2) the specimen such that a desired one of the layers lies in the focal plane;   means for positioning at least one of (1) the cursor and (2) the specimen to illuminate by means of the cursor a selected part element;   means fore preventing unwanted light from part elements adjacent the selected part element from being detected by the means for detecting light;   means for producing signals indicative of predetermined characteristics of the detected light;   means for storing representations of the signals; and   means for analyzing the representations of the signals to produce measurements of the specimen representative of at least one 3-dimensional   
     
     
        characteristic of at least a portion of the specimen. .Iaddend. .Iadd.21. An apparatus for microphotometering a 3-dimensional specimen, the specimen defining a plurality of layers, each layer defining a plurality of part elements, the apparatus comprising: means for detecting light from a focal plane;   means for generating a cursor of light in the focal plane;   means for positioning at least one of (1) the focal plane and (2) the specimen such that a desired one of the layers lies in the focal plane;   means for positioning at least one of (1) the cursor and (2) the specimen to illuminate by means of the cursor a selected part element;   means for preventing unwanted light from part elements adjacent the selected part element from being detected by the means for detecting light;   means for producing signals indicative of predetermined characteristics of the detected light;   means for storing representations of the signals; and   means for analyzing the representations of the signals to produce a projected representation from a desired angle of at least a portion of the specimen. .Iaddend.