Confocal macroscope

Abstract

A confocal macroscope is disclosed. According to one embodiment of the present invention an imaging system includes a specimen stage, a source of a collimated excitation beam centered on a beam axis, and a scan-head movably positioned to focus the collimated excitation beam on a focal point in an object plane above the specimen stage and to receive light emitted or reflected from the object plane. According to another embodiment of the present invention the imaging system has three orthogonal motion axes that harness the quality of a collimated infinity space to stretch in scanning a specimen.

What is claimed is:  
     
         1 . An imaging system comprising: 
 an x stage movable on an x-axis;    a source of a collimated excitation beam centered on a beam axis and defining infinity space; and    a scan-head comprising a deflector mirror to bounce the excitation beam normal to the respective x stage and an objective lens, the objective lens having a central axis aligned coaxial with the beam axis of the collimated excitation beam, the scan-head being movably positioned to stretch the infinity space and to focus the collimated excitation beam on a focal point in an object plane over the x stage and to receive light emitted or reflected from the object plane, the objective lens being movable along a z-axis by a focus mechanism and the scan-head being movable upon a y-axis orthogonal to the x and z-axes and relative to the x stage and relative to the source while the central axis of the objective lens remains coaxial with the beam axis of the collimated excitation beam;    a detector pinhole to receive light emanating from the focal point in the object plane; and    a photodetector behind the detector pinhole to receive the light and to generate a signal based on the light.    
     
     
         2 . The imaging system of  claim 1  wherein: 
 the x stage includes a propulsion source to move the x stage on the x-axis;  
 the scan-head includes a propulsion source to move the scan-head on the y-axis;  
 the source of the collimated excitation beam comprises a multicolor laser and a source pinhole; and  
 the photodetector comprises a PMT or a photodiode.  
 
     
     
         3 . The imaging system of  claim 1 , further comprising: 
 an integrated automatic wide view laser illuminated scrambler/spreader motorized to move in and out of an excitation path;    a beam expander on the beam axis to expand and collimate the excitation beam;    a polychroic beamsplitter having multiple passbands and corresponding reflective bands to direct the collimated excitation beam from an illumination axis parallel to the x-axis of the x-stage toward the y-axis;    a linear motor in the scan-head to move the scan-head along the y-axis;    a linear motor in the x stage to move the x stage along the x-axis; and    a control system to control the focus mechanism to move the objective lens to obtain a focal point in the object plane, to move the scan-head and the x stage alternately or simultaneously to trace out a serpentine, raster, or other pattern with the focal point, and to collect and store data automatically and unattended.    
     
     
         4 . An imaging system comprising: 
 a multicolor laser to generate a collimated excitation beam centered on a beam axis and occupying infinity space;    a beamsplitter to reflect the collimated excitation beam;    a scan-head comprising a flat mirror movably positioned to reflect the collimated excitation beam from the beamsplitter toward an objective lens having a central axis coaxial with the beam axis of the collimated excitation beam, the objective lens being positioned along a z-axis to focus the collimated excitation beam on a focal point in an object plane in a specimen on a specimen stage; and    a detector pinhole to receive light emanating from the object plane.    
     
     
         5 . The imaging system of  claim 4  wherein: 
 the beamsplitter comprises a polychroic beamsplitter;  
 the source of a collimated light beam comprises a multicolor laser;  
 the scan-head comprises the objective lens movable along the z-axis by a piezoelectric actuated lead-screw focus motor, the scan-head being movable by a linear motor along a y-axis orthogonal to the z-axis relative to the source while the central axis of the objective lens remains coaxial with the beam axis of the collimated excitation beam in infinity space, the scan-head being movable along the y-axis to stretch the infinity space; and  
 the specimen stage comprising a linear motor to move the specimen stage along an x-axis orthogonal to the y and z-axes.  
 
     
     
         6 . The imaging system of  claim 5 , further comprising a control system to control a focus mechanism to move the objective lens to obtain a focal point in the object plane, to move the scan-head and the specimen stage alternately or simultaneously to trace out a serpentine, raster, or other pattern with the focal point, and to collect and store data automatically and unattended.  
     
     
         7 . An imaging system comprising: 
 a specimen stage;    a source of a collimated excitation beam centered on a beam axis; and    a scan-head comprising an objective lens having a central axis coaxial with the beam axis of the collimated excitation beam, the scan-head being movably positioned to focus the collimated excitation beam on a focal point in an object plane over the specimen stage and to receive light emitted or reflected from the object plane, the scan-head being movable along a first axis and along a second axis relative to the specimen stage and relative to the source while the central axis of the objective lens remains coaxial with the beam axis of the collimated excitation beam.    
     
     
         8 . The imaging system of  claim 7 , further comprising a flat mirror in the scan-head to reflect the collimated excitation beam from the source to the objective lens and to maintain the central axis of the objective lens coaxial with the beam axis of the collimated excitation beam.  
     
     
         9 . The imaging system of  claim 7  wherein the first axis is normal to the specimen stage.  
     
     
         10 . The imaging system of  claim 7  wherein the second axis is parallel with the object plane.  
     
     
         11 . The imaging system of  claim 9  wherein the specimen stage is movable along a third axis that is parallel with the object plane to cause the scan-head to scan over the object plane in a pattern as the scan-head and the specimen stage are moved.  
     
     
         12 . The imaging system of  claim 11 , further comprising: 
 a first linear motor in the scan-head to move the scan-head along the second axis; and    a second linear motor in the specimen stage to move the specimen stage along the third axis.    
     
     
         13 . The imaging system of  claim 7  wherein the source of the collimated excitation beam comprises: 
 a light source to generate a excitation beam centered on the beam axis;  
 a beam expander on the beam axis to expand and collimate the excitation beam; and  
 a beamsplitter to direct the collimated excitation beam toward the scan-head.  
 
     
     
         14 . The imaging system of  claim 13 , further comprising: 
 an iris on the beam axis to block edges of the expanded and collimated excitation beam and to transmit a central portion of the collimated excitation beam;    a neutral density filter on the beam axis to modulate an intensity of the collimated excitation beam; and    a bandpass filter on the beam axis to transmit a single color in the collimated excitation beam and to block other colors in the collimated excitation beam.    
     
     
         15 . The imaging system of  claim 13 , further comprising: 
 an image pinhole;    a tube lens positioned to receive the emitted or reflected light transmitted through the objective lens and the polychroic beamsplitter and to focus the emitted or reflected light on the image pinhole; and    a photodetector coupled to the image pinhole to detect the emitted or reflected light passing through the image pinhole.    
     
     
         16 . The imaging system of  claim 11  wherein: 
 the second axis is orthogonal to the first axis; and  
 the third axis is orthogonal to the first axis and the second axis.  
 
     
     
         17 . The imaging system of  claim 7 , further comprising a specimen mounted on the specimen stage.  
     
     
         18 . The imaging system of  claim 15  wherein: 
 the light source comprises a krypton-argon ion laser;  
 the beamsplitter comprises a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands; and  
 the photodetector comprises a PMT.  
 
     
     
         19 . An imaging system comprising: 
 a specimen stage;    a source of a collimated excitation beam centered on a beam axis; and    a scan-head movably positioned to focus the collimated excitation beam on a focal point in an object plane above the specimen stage and to receive light emitted or reflected from the object plane.    
     
     
         20 . The imaging system of  claim 19  wherein the scan-head comprises an objective lens having a central axis coaxial with the beam axis of the collimated excitation beam, the scan-head being movable along a first axis and along a second axis relative to the specimen stage and relative to the source while the central axis of the objective lens remains coaxial with the beam axis of the collimated excitation beam.  
     
     
         21 . The imaging system of  claim 19  wherein the first axis is normal to the specimen stage and the second axis is parallel with the object plane.  
     
     
         22 . The imaging system of  claim 21  wherein the specimen stage is movable along a third axis that is parallel with the object plane to cause the scan-head to scan over the object plane in a pattern as the scan-head and the specimen stage are moved.  
     
     
         23 . The imaging system of  claim 22 , further comprising: 
 a first linear motor in the scan-head to move the scan-head along the second axis; and    a second linear motor in the specimen stage to move the specimen stage along the third axis.    
     
     
         24 . The imaging system of  claim 20 , further comprising a flat mirror in the scan-head to reflect the collimated excitation beam from the source to the objective lens and to maintain the central axis of the objective lens coaxial with the beam axis of the collimated excitation beam as the scan-head is moved along the first axis and along the second axis.  
     
     
         25 . The imaging system of  claim 20  wherein the source of the collimated excitation beam comprises: 
 a light source to generate a excitation beam centered on the beam axis;  
 a beam expander on the beam axis to expand and collimate the excitation beam; and  
 a beamsplitter to direct the collimated excitation beam toward the scan-head.  
 
     
     
         26 . The imaging system of  claim 25 , further comprising: 
 an iris on the beam axis to block edges of the expanded and collimated excitation beam and to transmit a central portion of the collimated excitation beam;    a neutral density filter on the beam axis to modulate an intensity of the collimated excitation beam; and    a bandpass filter on the beam axis to pass a single color of the collimated excitation beam and to block other colors in the collimated excitation beam.    
     
     
         27 . The imaging system of  claim 26  wherein: 
 the light source comprises a krypton-argon ion laser; and  
 the beamsplitter comprises a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands.  
 
     
     
         28 . The imaging system of  claim 26 , further comprising: 
 an image pinhole;    a tube lens positioned to receive the emitted or reflected light transmitted through the objective lens and the polychroic beamsplitter and to focus the emitted or reflected light on the image pinhole; and    a PMT coupled to the image pinhole to detect the light passing through the image pinhole.    
     
     
         29 . The imaging system of  claim 22  wherein: 
 the second axis is orthogonal to the first axis; and  
 the third axis is orthogonal to the first axis and the second axis.  
 
     
     
         30 . The imaging system of  claim 19 , further comprising a specimen mounted to the specimen stage.  
     
     
         31 . An imaging system comprising: 
 a specimen stage;    a light source to generate a excitation beam centered on the beam axis;    a beam expander on the beam axis to expand and collimate the excitation beam; and    a beamsplitter to direct the collimated excitation beam;    a scan-head comprising an objective lens having a central axis coaxial with the beam axis of the collimated excitation beam directed by the beamsplitter, the scan-head being movably positioned to focus the collimated excitation beam on a focal point in an object plane over the specimen stage and to receive light emitted or reflected from the object plane, the scan-head being movable along a first axis and along a second axis relative to the specimen stage and relative to the beamsplitter while the central axis of the objective lens remains coaxial with the beam axis of the collimated excitation beam;    an image pinhole;    a lens positioned to receive the emitted or reflected light transmitted through the objective lens and the beamsplitter and to focus the emitted or reflected light on the image pinhole; and    a photodetector coupled to the image pinhole to detect the emitted or reflected light passing through the image pinhole.    
     
     
         32 . The imaging system of  claim 31 , further comprising: 
 an iris on the beam axis to block edges of the expanded and collimated excitation beam and to transmit a central portion of the collimated excitation beam;    a neutral density filter on the beam axis to modulate an intensity of the collimated excitation beam; and    a bandpass filter on the beam axis to pass a single color of the collimated excitation beam and to block other colors in the collimated excitation beam.    
     
     
         33 . The imaging system of  claim 31 , further comprising: 
 a beam scrambler on the beam axis to scramble the collimated excitation beam into a less-coherent, less-speckle inducing excitation beam; and    an ocular head between the image pinhole and the polychroic beamsplitter to view the emitted or reflected light when the collimated excitation beam has been scrambled.    
     
     
         34 . The imaging system of  claim 31  wherein: 
 the beamsplitter comprises a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands;  
 the lens comprises a tube lens;  
 the photodetector comprises a PMT; and  
 the light source comprises a krypton-argon ion laser.  
 
     
     
         35 . The imaging system of  claim 31  wherein the first axis is normal to the specimen stage and the second axis is parallel with the object plane.  
     
     
         36 . The imaging system of  claim 35  wherein the specimen stage is movable along a third axis that is parallel with the object plane to cause the scan-head to scan over the object plane in a pattern as the scan-head and the specimen stage are moved.  
     
     
         37 . The imaging system of  claim 36 , further comprising: 
 a first linear motor in the scan-head to move the scan-head along the second axis; and    a second linear motor in the specimen stage to move the specimen stage along the third axis.    
     
     
         38 . The imaging system of  claim 31 , further comprising a flat mirror in the scan-head to reflect the collimated excitation beam from the polychroic beamsplitter to the objective lens and to maintain the central axis of the objective lens coaxial with the beam axis of the collimated excitation beam as the scan-head is moved along the first axis and along the second axis.  
     
     
         39 . The imaging system of  claim 36  wherein: 
 the second axis is orthogonal to the first axis; and  
 the third axis is orthogonal to the first axis and the second axis.  
 
     
     
         40 . The imaging system of  claim 31 , further comprising a specimen mounted to the specimen stage.  
     
     
         41 . A method comprising: 
 generating a collimated excitation beam centered on a beam axis and defining infinity space;    maintaining the beam axis of the collimated excitation beam coaxial with a central axis of an objective lens in a scan-head in the infinity space;    focusing the collimated excitation beam on a focal point in an object plane in a specimen on a specimen stage with the objective lens;    detecting light emanating from the specimen at the focal point with a photodetector to generate a signal based on the light to indicate image data;    collecting the image data to generate an image of the specimen with an automated and unattended control system; and    storing the data in a mass storage device.    
     
     
         42 . The method of  claim 41 , further comprising: 
 aligning elements generating and attenuating the collimated excitation beam with an excitation path optical alignment fixture;    aligning the beamsplitter with a beamsplitter beam target;    viewing the specimen in an integrated automatic wide view using a laser illuminated scrambler/spreader;    selecting a programmable region of interest in the specimen;    selecting a programmable velocity and acceleration and data sampling rate for an arbitrary scan path through the specimen;    moving the objective lens along a z-axis normal to the specimen stage to move the focal point of the objective lens to a different object plane in the specimen;    moving the objective lens along a y-axis orthogonal to the z-axis and parallel with the object plane in the specimen to scan the specimen in an arbitrary scan path inside the region of interest along the y-axis in a stretching infinity space; and    moving the specimen stage along an x-axis orthogonal to the y and z-axes to scan the specimen along the x-axis in the arbitrary scan path inside the region of interest.    
     
     
         43 . The method of  claim 41  wherein collecting the image data to generate an image of the specimen further comprises: 
 integrating the signal from a PMT indicating the instantaneous light passing through the pinhole to generate time-averaged rolling window values in a sampling period;  
 triggering a sampling of the integrated signal on a movement in the position of the scan-head or the specimen stage or the objective lens detected with a position encoder; and  
 generating an image of the specimen in the focal plane with the samples of the signal.  
 
     
     
         44 . A method comprising: 
 generating a collimated excitation beam centered on a beam axis;    focusing the collimated excitation beam with an objective lens on a focal point in an object plane in a specimen on a specimen stage;    maintaining the beam axis of the collimated excitation beam coaxial with a central axis of the objective lens;    moving the objective lens along a first axis relative to the specimen;    moving the objective lens along a second axis relative to the specimen; and    detecting light reflected or emitted from the specimen at the focal point to generate image data.    
     
     
         45 . The method of  claim 44  wherein moving the objective lens along a first axis comprises moving the objective lens along a first axis normal to the specimen stage to move the focal point of the objective lens to a different object plane in the specimen.  
     
     
         46 . The method of  claim 44  wherein moving the objective lens along a second axis comprises moving the objective lens along a second axis parallel with the object plane in the specimen to cause the focal point to traverse the specimen in the object plane.  
     
     
         47 . The method of  claim 46 , further comprising: 
 moving the specimen stage along a third axis parallel with the object plane to cause the focal point to traverse the specimen in a pattern;    detecting light reflected or emitted from the specimen at the focal point to generate image data for the pattern; and    generating an image of the object plane in the specimen traversed by the focal point.    
     
     
         48 . The method of  claim 44  wherein focusing the collimated excitation beam further comprises reflecting the collimated excitation beam toward the objective lens with a flat mirror in a scan-head fixed to the objective lens.  
     
     
         49 . The method of  claim 44  wherein generating a collimated excitation beam comprises: 
 generating a excitation beam centered on the beam axis from a laser;  
 expanding and collimating the excitation beam in a beam expander; and  
 directing the collimated excitation beam toward the objective lens with a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands.  
 
     
     
         50 . The method of  claim 47  wherein detecting light comprises: 
 transmitting light reflected or emitted from the specimen through the objective lens and the polychroic beamsplitter having a plurality of passbands;  
 focusing the reflected or emitted light on to a conjugate image plane with a tube lens;  
 filtering the reflected or emitted light through a pinhole in the conjugate image plane; and  
 detecting the light passing through the pinhole with a PMT.  
 
     
     
         51 . The method of  claim 47  wherein: 
 moving the objective lens along a second axis further comprises moving the objective lens along the second axis that is orthogonal to the first axis; and  
 moving the specimen stage further comprises moving the specimen stage along a third axis that is orthogonal to both the first axis and the second axis.  
 
     
     
         52 . A method comprising: 
 generating a collimated excitation beam centered on a beam axis;    focusing the collimated excitation beam on a focal point in an object plane in a specimen on a specimen stage with an objective lens; and    detecting light reflected or emitted from the specimen at the focal point to generate image data.    
     
     
         53 . The method of  claim 52 , further comprising maintaining the beam axis of the collimated excitation beam coaxial with a central axis of the objective lens.  
     
     
         54 . The method of  claim 53 , further comprising: 
 moving the objective lens along a first axis normal to the specimen stage to move the focal point of the objective lens to a different object plane in the specimen; and    moving the objective lens along a second axis parallel with the object plane in the specimen.    
     
     
         55 . The method of  claim 54 , further comprising: 
 moving the specimen stage along a third axis parallel with the object plane to cause the focal point to traverse the specimen in a pattern;    detecting light reflected or emitted from the specimen at the focal point to generate image data for the pattern; and    generating an image of the object plane in the specimen traversed by the focal point according to the pattern.    
     
     
         56 . The method of  claim 52  wherein focusing the collimated excitation beam further comprises reflecting the collimated excitation beam toward the objective lens with a flat mirror in a scan-head fixed to the objective lens.  
     
     
         57 . The method of  claim 55  wherein detecting light comprises: 
 transmitting light reflected or emitted from the specimen through the objective lens and the polychroic beamsplitter;  
 focusing the reflected or emitted light on to a conjugate image plane with a tube lens;  
 filtering the reflected or emitted light through a pinhole in the conjugate image plane; and  
 detecting the light passing through the pinhole with a PMT to generate the image data.  
 
     
     
         58 . The method of  claim 55  wherein: 
 moving the objective lens along a second axis further comprises moving the objective lens along the second axis that is orthogonal to the first axis; and  
 moving the specimen stage further comprises moving the specimen stage along a third axis that is orthogonal to both the first axis and the second axis.  
 
     
     
         59 . A method comprising: 
 generating a excitation beam centered on the beam axis from a krypton-argon ion laser;    expanding and collimating the excitation beam in a beam expander;    directing the collimated excitation beam toward an objective lens with a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands;    reflecting the collimated excitation beam toward the objective lens with a flat mirror in a scan-head fixed to the objective lens;    focusing the collimated excitation beam on a focal point in an object plane in a specimen on a specimen stage with the objective lens;    maintaining the beam axis of the collimated excitation beam coaxial with a central axis of the objective lens;    moving the objective lens along a first axis normal to the specimen stage to move the focal point of the objective lens to a different object plane in the specimen;    moving the objective lens along a second axis that is orthogonal to the first axis and parallel with the object plane in the specimen to cause the focal point to traverse the specimen in the object plane;    moving the specimen stage along a third axis that is orthogonal to both the first axis and the second axis and parallel with the object plane to cause the focal point to traverse the specimen in a pattern;    detecting light reflected or emitted from the specimen at the focal point to generate image data for the pattern; and    generating an image of the object plane in the specimen traversed by the focal point from the detected light.    
     
     
         60 . The method of  claim 59 , further comprising: 
 blocking edges of the collimated excitation beam with an iris and transmitting a central portion of the collimated excitation beam through the iris;    modulating the intensity of the collimated excitation beam with a neutral density filter; and    transmitting a selected color and blocking all other colors in the collimated excitation beam with a bandpass filter.    
     
     
         61 . The method of  claim 59  wherein detecting light comprises: 
 transmitting light reflected or emitted from the specimen through the objective lens and the polychroic beamsplitter;  
 focusing the light reflected or emitted from the specimen on to a conjugate image plane with a tube lens;  
 filtering the light reflected or emitted from the specimen through a pinhole in the conjugate image plane; and  
 detecting the light passing through the pinhole with a PMT to generate the image data.  
 
     
     
         62 . The imaging system of  claim 6  wherein: 
 the multicolor laser comprises a krypton-argon ion laser; and  
 the imaging system further comprises: 
 a beam expander on the beam axis to expand and collimate the collimated excitation beam;  
 a tube lens positioned to receive the light from the object plane, the light to be transmitted through the objective lens and the polychroic beamsplitter, the tube lens to focus the light on the detector pinhole;  
 a PMT coupled to the detector pinhole to detect the light received by the detector pinhole; and  
 a mass storage device to store the data collected by the control system from the light detected by the PMT.  
 
 
     
     
         63 . The imaging system of  claim 12  wherein: 
 the source of the collimated excitation beam comprises: 
 a krypton-argon ion laser to generate an excitation beam centered on the beam axis;  
 a beam expander on the beam axis to expand and collimate the excitation beam; and  
 a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands to direct the collimated excitation beam toward the scan-head;  
 
 the second axis is parallel with the object plane; and  
 the imaging system further comprises: 
 a piezoelectric focus motor in the scan-head to move the objective lens along the first axis;  
 a specimen mounted on the specimen stage;  
 a flat mirror in the scan-head to reflect the collimated excitation beam from the source to the objective lens;  
 an image pinhole;  
 a tube lens positioned to receive the emitted or reflected light transmitted through the objective lens and the polychroic beamsplitter and to focus the emitted or reflected light on the image pinhole;  
 a PMT coupled to the image pinhole to detect the emitted or reflected light passing through the image pinhole;  
 a control system to collect image data from light detected by the PMT and to control the piezoelectric focus motor, the first linear motor, and the second linear motor; and  
 a mass storage device to store the image data.  
 
 
     
     
         64 . The imaging system of  claim 18  wherein: 
 the first axis is normal to the specimen stage; and  
 the imaging system further comprises: 
 a piezoelectric focus motor in the scan-head to move the objective lens along the first axis;  
 a first linear motor in the scan-head to move the scan-head along the second axis parallel with the object plane;  
 a second linear motor in the specimen stage to move the specimen stage along a third axis parallel with the object plane, the second axis being orthogonal to the first axis and the third axis being orthogonal to the first axis and the second axis;  
 a specimen mounted on the specimen stage;  
 a flat mirror in the scan-head to reflect the collimated excitation beam from the source to the objective lens;  
 a control system to collect image data from light detected by the PMT and to control the piezoelectric focus motor, the first linear motor, and the second linear motor; and  
 a mass storage device to store the image data.  
 
 
     
     
         65 . The imaging system of  claim 22 , further comprising: 
 a piezoelectric focus motor in the scan-head to move the objective lens along the first axis;    a control system to collect image data from the received light and to control the movement of the scan-head, the objective lens, and the specimen stage; and    a mass storage device to store the image data.    
     
     
         66 . The method of  claim 57  wherein: 
 generating a collimated excitation beam further comprises: 
 generating an excitation beam centered on the beam axis from a krypton-argon ion laser;  
 expanding and collimating the excitation beam in a beam expander;  
 directing the collimated excitation beam toward the objective lens with a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands;  
 blocking edges of the collimated excitation beam with an iris and transmitting a central portion of the collimated excitation beam through the iris;  
 modulating the intensity of the collimated excitation beam with a neutral density filter;  
 transmitting a selected color and blocking all other colors in the collimated excitation beam with a bandpass filter; and  
 reflecting the collimated excitation beam toward the objective lens with a flat mirror in a scan-head including the objective lens; and  
 
 the method further comprises: 
 moving the objective lens with a piezoelectric focus motor along the first axis;  
 moving the objective lens with a first linear motor along the second axis;  
 moving the specimen stage with a second linear motor along the third axis;  
 collecting the image data and controlling the piezoelectric focus motor, the first linear motor, and the second linear motor with a control system; and  
 storing the image data in a mass storage device.  
 
 
     
     
         67 . The method of  claim 56  wherein: 
 generating a collimated excitation beam further comprises: 
 generating an excitation beam centered on the beam axis from a krypton-argon ion laser;  
 expanding and collimating the excitation beam in a beam expander;  
 directing the collimated excitation beam toward the objective lens with a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands;  
 blocking edges of the collimated excitation beam with an iris and transmitting a central portion of the collimated excitation beam through the iris;  
 modulating the intensity of the collimated excitation beam with a neutral density filter; and  
 transmitting a selected color and blocking all other colors in the collimated excitation beam with a bandpass filter;  
 
 detecting light further comprises: 
 transmitting light reflected or emitted from the specimen through the objective lens and the polychroic beamsplitter;  
 focusing the light reflected or emitted from the specimen on to a conjugate image plane with a tube lens;  
 filtering the light reflected or emitted from the specimen through a pinhole in the conjugate image plane; and  
 detecting the light passing through the pinhole with a PMT to generate the image data; and  
 
 the method further comprises: 
 maintaining the beam axis of the collimated excitation beam coaxial with a central axis of the objective lens;  
 moving the objective lens with a piezoelectric focus motor along a first axis normal to the specimen stage to move the focal point of the objective lens to a different object plane in the specimen;  
 moving the objective lens with a first linear motor along a second axis that is orthogonal to the first axis and parallel with the different object plane in the specimen to cause the focal point to traverse the specimen in the different object plane;  
 
 moving the specimen stage with a second linear motor along a third axis that is orthogonal to both the first axis and the second axis and parallel with the different object plane to cause the focal point to traverse the specimen in a pattern;  
 collecting the image data and controlling the piezoelectric focus motor, the first linear motor, and the second linear motor with a control system;  
 storing the image data in a mass storage device; and  
 generating an image of the different object plane in the specimen from the image data.  
 
     
     
         68 . The method of  claim 42  wherein: 
 generating the collimated excitation beam further comprises: 
 generating an excitation beam from a krypton-argon ion laser;  
 expanding and collimating the excitation beam in a beam expander;  
 blocking edges of the collimated excitation beam with an iris and transmitting a central portion of the collimated excitation beam through the iris;  
 modulating the intensity of the collimated excitation beam with a neutral density filter;  
 transmitting a selected color and blocking all other colors in the collimated excitation beam with a bandpass filter; and  
 reflecting the collimated excitation beam toward the objective lens with a flat mirror in the scan-head;  
 
 aligning a beamsplitter further comprises aligning a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands with the beamsplitter beam target to direct the collimated excitation beam toward the objective lens with the polychroic beamsplitter;  
 detecting light further comprises: 
 transmitting light reflected or emitted from the specimen through the objective lens and the polychroic beamsplitter;  
 focusing the light reflected or emitted from the specimen on to a conjugate image plane with a tube lens;  
 filtering the light reflected or emitted from the specimen through a pinhole in the conjugate image plane; and  
 detecting the light passing through the pinhole with a PMT to generate the image data; and  
 
 moving the objective lens along a z-axis further comprises moving the objective lens with a piezoelectric focus motor along the z-axis;  
 moving the objective lens along a y-axis further comprises moving the objective lens with a first linear motor along the y-axis;  
 moving the specimen stage along an x-axis further comprises moving the specimen stage with a second linear motor along the x-axis; and  
 collecting the image data further comprises collecting the image data and controlling the piezoelectric focus motor, the first linear motor, and the second linear motor with the control system.  
 
     
     
         69 . The method of  claim 43  wherein: 
 generating a collimated excitation beam further comprises: 
 generating an excitation beam centered on the beam axis from a krypton-argon ion laser;  
 expanding and collimating the excitation beam in a beam expander;  
 directing the collimated excitation beam toward the objective lens with a polychroic beamsplitter having a plurality of passbands and a plurality of reflective bands;  
 blocking edges of the collimated excitation beam with an iris and transmitting a central portion of the collimated excitation beam through the iris;  
 modulating the intensity of the collimated excitation beam with a neutral density filter;  
 reflecting the collimated excitation beam toward the objective lens with a flat mirror in the scan-head; and  
 transmitting a selected color and blocking all other colors in the collimated excitation beam with a bandpass filter;  
 
 detecting light further comprises: 
 transmitting light reflected or emitted from the specimen through the objective lens and the polychroic beamsplitter;  
 focusing the light reflected or emitted from the specimen on to a conjugate image plane with a tube lens;  
 filtering the light reflected or emitted from the specimen through the pinhole in the conjugate image plane; and  
 detecting the light passing through the pinhole with the PMT to generate the image data; and  
 
 the method further comprises: 
 moving the objective lens with a piezoelectric focus motor along a first axis normal to the specimen stage to move the focal point of the objective lens to a different object plane in the specimen;  
 moving the objective lens with a first linear motor along a second axis that is orthogonal to the first axis and parallel with the different object plane in the specimen to cause the focal point to traverse the specimen in the different object plane;  
 moving the specimen stage with a second linear motor along a third axis that is orthogonal to both the first axis and the second axis and parallel with the different object plane to cause the focal point to traverse the specimen in a pattern; and  
 controlling the piezoelectric focus motor, the first linear motor, and the second linear motor with the control system.  
 
 
     
     
         70 . An imaging system comprising: 
 a specimen stage;    a source of a collimated excitation beam centered on a beam axis;    means for focusing the collimated excitation beam on a focal point in an object plane above the specimen stage; and    means for receiving light emitted or reflected from the object plane.