Patent Publication Number: US-2021181493-A1

Title: Confocal microscope with positionable imaging head

Description:
This application claims priority to U.S. Provisional Patent Application No. 62/421,270, filed Nov. 12, 2016, which is incorporated herein by reference. 
     Field of the Invention 
     The present invention relates to a confocal microscope having a positionable imaging head mounted to a platform, and particularly to, a confocal microscope having an imaging head mounted to a platform positionable in one mode to image tissue samples disposed upon the platform, such as an ex-vivo tissue specimen on a movable specimen stage, and in another mode to image tissue samples beside the platform, such as in-vivo skin tissue of large animals or humans. 
     BACKGROUND OF THE INVENTION 
     Confocal microscopes optically section tissue to produce microscopic images of tissue sections without requiring histological preparation of the tissue on slides (i.e., slicing, slide mounting, and staining). Such sectional images produced may be on or under the surface of the tissue. An example of a confocal microscope is the VivaScope® manufactured by Caliber Imaging &amp; Diagnostics, Inc. of Henrietta, New York. Examples of confocal microscopes are described in U.S. Pat. Nos. 5,788,639, 5,880,880, 7,394,592, and 9,055,867. In particular, U.S. Pat. No. 7,394,592 describes an imaging head of a confocal microscope mounted on a multi-positionable arm extending from an upright station having a computer system connected to the imaging head, where the computer system shows on a display confocal images captured by the microscope. While useful for imaging in-vivo tissue, such as a skin lesion without removal from a patient, it is cumbersome when one wishes to image ex-vivo tissue samples as may be mounted on a microscope stage. Other confocal microscopes have been developed for use in imaging ex-vivo tissue samples, such as may be mounted in tissue cassette holders, as described in U.S. Pat. Nos. 6,411,434, 6,330,106, 7,227,630, and 9,229,210. It would be desirable to provide a confocal microscope from a common platform which can be used both for imaging ex-vivo tissue samples mountable upon a stage and in-vivo tissue samples of a patient or animal. 
     SUMMARY OF THE INVENTION 
     Accordingly, it is an object of the present invention to provide a confocal microscope having an imaging head supported from a platform where the imaging head is positionable over the platform or beside the platform as desired for imaging tissue samples, thereby enabling ex-vivo imaging of tissue samples as may be disposed on a stage upon the platform and in-vivo imaging of tissue samples as may be disposed beside the platform. 
     Briefly described, the present invention embodies a microscope for imaging tissue having an imaging head with an optical system for capturing optically formed microscopic sectional images, and a platform upon which is disposed first and second stages. The first stage is coupled to the imaging head for moving the imaging head along a vertical dimension perpendicular to a horizontal dimension along which the platform extends, such as to adjust a height of the imaging head. The second stage rotates the imaging head about the vertical dimension. The imaging head is positionable using at least the first and second stages in a first mode to image at least a first tissue sample disposed between the imaging head and the platform (i.e., upon the platform), and in the second mode to image at least a second tissue sample disposed beside the platform (i.e., not upon the platform). Preferably, the optical system is operative by confocal microscopy, such that the microscope of the present invention is referred to as a confocal microscope, but other modalities for capturing optically formed microscopic sectional images may be used. 
     In the first mode of the microscope operation, the first tissue sample may be an ex-vivo tissue specimen (e.g., excised from a patient/subject) or in-vivo tissue of small animal or subject disposed upon the platform. While in the second mode of microscope operation, the second tissue sample may be in-vivo skin tissue of a human or large animal subject. 
     The optical system of the imaging head comprises optics having at least an objective lens for focusing and collecting illumination from the first and second tissue samples when each face the objective lens. The second stage is preferably a rotary stage between the first stage and the platform for rotating the first stage and the imaging head coupled thereto 360 degrees about the vertical dimension. The first stage may be a linear slide stage having a carriage movable along the vertical dimension, in which such carriage is coupled to the imaging head by a mounting arm. The mounting arm has a first portion fixed to the carriage, and a second portion having a rotary stage for rotating the imaging head about a normal axis perpendicular to the optical axis of the objective lens. The second portion is further tiltable with respect to the first portion to adjust tilt of the imaging head along the normal axis with respect to the horizontal dimension. The rotary stage and tilt adjustment provided by the mounting arm, and the first and second stages, provides the imaging head with multiple (four) degrees of freedom of motion so it can be set by a user to different positions to image ex-vivo or in-vivo tissue samples from a common platform upon which the imaging head is mounted in either first or second modes. 
     A third stage, such as an x-y stage, may be mounted to the platform movable along x and y orthogonal axes along the horizontal dimension along which the platform extends. The first tissue sample, such as ex-vivo tissue, is mounted upon such third stage for moving the tissue sample with respect to the objective lens during first mode operation of the microscope. The optical axis of the objective lens is aligned to extend along a z axis perpendicular to the x and y axes. This may be achieved by one or more of the above described rotation about the normal axis and tilt of the imaging head until the optical axis aligns along the z axis. An optional fourth stage may be mounted to the third stage moveable along such z axis, where the first tissue sample is mounted instead upon the fourth stage to enable the tissue sample to be movable using the third and fourth stages along x, y, and z axes with respect to the objective lens. While the objective lens is movable within the imaging head along its optical axis, the fourth stage can provide additional control for positioning the tissue sample along the z axis. The microscope may further be used in the first mode with the third and fourth stages removed from the platform, if desired. The third stage (and fourth stage if mounted thereto) may be referred to herein as a specimen stage. 
     The microscope has a computer system connected to the imaging head to receive signals representative of the images of the first or second tissue samples when imaged. The computer system shows on a display and/or store in its memory the images captured by the microscope. The computer system controls operation of the imaging head responsive to a user via user interface device(s) provided. Movement of x and y axis motors of the third stage, and z axis motor of the fourth stage (if present), are also preferably controlled by the computer system, but may alternatively be controlled by a joystick if provided. 
     The optical system in the imaging head can utilize multiple discrete laser wavelengths for illumination, and selectable wavelengths for detection. However, a single wavelength of laser illumination and detection may be used. The objective lens may be removably mounted to the microscope, such as by magnetics, so that different objective lens may be mounted thereto, as desired by a user. 
     In the preferred embodiment, the objective lens of the optical system focuses and collects scanned illumination from a tissue sample, where the scanned illumination travels along a first path via the objective lens to the tissue sample, and collected return illumination travels along a second path via the objective lens. The second path has at least a beam splitter that splits the return illumination into first and second beams. The first beam travels to a first detector via a first pinhole and a first selected position of an optical filter or opening (such along of a first filter wheel), and the second beam travels to a second detector via a second pinhole and a second selected position of another optical filter or an opening (such along of a second filter wheel). One or both of the first pinhole and second pinhole are each separately adjustable in position to align their first and second beams, respectively, onto their first detector and second detector, respectively. 
     The optical system may further have a mirror in the second path to reflect the return illumination onto the beam splitter. Such mirror may be adjustable in position to align the first beam when split by the beam splitter onto the first detector via the first pinhole, which may then be non-adjustable in position, and the second pinhole is adjustable in position to align the second beam via the second pinhole onto the second detector. Alternatively, the mirror may be non-adjustable in position, and one (or preferably) both the first and second pinholes are each separately adjusted in position to align their respective first and second beams onto their respective first and second detectors. 
     As the illumination of the tissue sample is of multiple discrete wavelengths, the first and second detectors receive different wavelengths of the collected illumination to enable simultaneous capture of a same one of the images at the different wavelengths or wavelength range on the first and second detectors in accordance with the first selected position and the second selected position having at least one of the optical filter and the another optical filter, respectively. Where one or more of the discrete wavelengths of illumination can activate fluorescent dye(s) that may be applied to tissue sample, the optical filter in the path of one of the first or second beams is selected to enable detection of fluorescent wavelength(s) associated with the dye(s) on their associated detector. Where non-fluorescent imaging is desired, an open position is selected in the path of one of the first or second beams to detect light of a discrete wavelength of illumination that was present along the first path to the tissue sample. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing objects, features, and advantages of the invention will become more apparent from a reading of the following description in connection with the accompanying drawings, in which: 
         FIGS. 1 and 2  are two perspective views taken from different angles of the imaging head of the microscope of the present invention mounted to a platform, where  FIG. 1  shows an example of an ex-vivo tissue sample being imaged and  FIG. 2  shows an example of an in-vivo tissue sample being imaged; 
         FIG. 3  is another view of the imaging head of  FIG. 1  and the platform in which the assembly of a rotary stage and a vertical stage are shown exploded from the platform, and an x-y stage is shown with an exploded optional z-stage mountable upon the x-y stage; 
         FIG. 4  is another view of  FIG. 1  with the z axis stage of  FIG. 3  shown mounted upon the x-y stage, and the imaging head removed; 
         FIG. 5  is a partial cross-sectional view of  FIG. 1  taken along line  5 - 5  in the direction of arrows at the end of the line showing the mounting arm which couples the imaging head to a carriage of the vertical stage; 
         FIG. 6  is another view of  FIG. 1  in which the assembly of the mounting arm of  FIG. 5  is shown exploded between the imaging head and the carriage of the vertical stage, where the z-stage of  FIG. 3  is mounted to the x-y stage; 
         FIG. 7  is another exploded view of the assembly of the mounting arm of  FIG. 5 , but from a different angle of that of  FIG. 6 , and having the rotary stage, vertical stage, and platform removed; 
         FIG. 8  is a block diagram of the microscope of the present invention having an imaging head supported over a platform as shown in  FIG. 1  with a computer system, a display for showing images captured by the microscope, and a multi-wavelength laser light source which may provide additional imaging wavelengths of light, as well as other components for controlling position of the vertical stage, x-y stage, and z-stage; 
         FIG. 9  is an exploded perspective view of the imaging head of  FIG. 1  apart from the rest of the microscope of  FIG. 8 ; 
         FIG. 10  shows the imaging head of  FIG. 9  assembled with the housing of the imaging head removed; and 
         FIG. 11  is an optical diagram of the optical system in the imaging head of  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION ON THE INVENTION 
     Referring to  FIGS. 1, 2, and 3 , an imaging head  12  of a microscope  10  ( FIG. 8 ) is shown in a housing  13  supported over a platform (or base)  14  having an upper surface  14   a  along a horizontal or dimension or plane. A second (or rotary) stage  16  is mounted to platform  14  for rotating a first (or vertical) stage  18  about a vertical dimension, i.e., perpendicular to the horizontal dimension along which upper surface  14   a  of platform  14  extends. Vertical stage  18  is a vertically disposed linear slide stage which carries a movable carriage  20  for translation along such vertical dimension. Carriage  20  is coupled by a mounting arm  22  to a base  15  of housing  13  of imaging head  12 . The mounting arm  22  enables adjustment of tilt and rotation of the imaging head  12  at a desired adjustable rotational position and height position as set by rotary stage  16  and vertical stage  18 , respectively, as will be described later below in more detail. 
     The imaging head  12  has an optical system  11  for capturing optically formed microscopic sectional images of tissue samples. The operation and structure of imaging head  12  may be the same as the confocal microscope of U.S. Pat. No. 9,055,867 which is incorporated herein by reference, but the preferred optical system  11  is shown in  FIGS. 9-11 . The optical system  11  has an objective lens  128  within an extending snout  127  of the imaging head  12  for focusing and collecting illumination from tissue samples facing the objective lens. Objective lens  128  has an optical axis  128   a , and perpendicular to such optical axis  128   a  is a virtual normal axis  128   b . Axes  128   a  and  128   b  are depicted as dashed lines in  FIGS. 1 and 2 . 
     Rotary stage  16  has a base  24 , which is mounted to platform  14  by screws  25  via threaded holes  26  ( FIG. 3 ) in platform  14 , and a turntable  28  which rotates with respect to base  24 , as indicated by arrow  30 , responsive to rotation of a graduated knob (or hand crank)  29  to gearing (not shown) disposed to rotate turntable  28 . A locking pin  27  may slide or be turned to move in and out with respect to a hole in base  24  in order to lock and unlock, respectively, the rotational position of turntable  28  with respect to base  24 . Turntable  28  is rotatable 360 degrees and may have graduations along its outer circumference in rotational degrees with respect to marking(s) along base  24 , which may be utilized by a user when manually turning knob  29  clockwise or counterclockwise to effect desired rotation. The rotary stage  16  is preferably a Velmex rotary table, model no. A4872TS (manufacturer: Velmex, Inc., Bloomfield, N.Y. USA), but other rotary tables may also be used. A circular adapter plate  32  is mounted atop turntable  28  by screws  33  received in threaded holes  34  of the adapter plate  32  and threaded holes  35  of turntable  28 . Adapter plate  32  may be made of stainless steel. 
     Vertical stage  18  has a housing  36  that extends upwards from rotary stage  16  and platform  14 . Carriage  20  is received along an open side  36   a  of housing  36  and mounted for vertical translation between sides  36   b  and  36   c  of housing  36 . As best shown in  FIG. 4 , housing  36  has a bottom wall  40  and a top wall  41  between which is journaled are two ends of a rotatable vertical lead screw  42  that extends through a vertical opening  46  of carriage  20 . Carriage  20  has two vertical slots or grooves  43  that ride along two inwardly protruding rails  44  from opposing sides  36   b  and  36   c  of housing  36 . Lead screw  42  extends along a threaded hole  49  of a nut  48  (shown in dashed lines) fixed in carriage  20 . The threads of lead screw  42  engage the threads of nut  48  along hole  49  so that rotation of lead screws  42  moves nut  48  and thus carriage  20  attached thereto up and down vertically along rails  44 . Rotation of the lead screw  42  in a first direction causes the carriage  20  to move upwards, and in a second direction causes the carriage to move downwards, as indicated by arrow  45 . A stepper motor  50  is mounted along the top of housing  36  and extends through an opening in top wall  41  to engage the top end of lead screw  42 . Motor  50  control the rotation and direction of rotation of lead screw  42  and thus the vertical height of carriage  20  with respect to the horizontal dimension along which platform  14  extends. Vertical stage  18  is preferably a Velmex BiSlide® model number MN-0100-M02-21, but other vertical stages may also be used. Stepper motor  50  may be a Vexta Stepper Motor Model No. PK266-03A-P1 (manufacturer: Oriental Motor Co. Ltd., Japan), but other stepper motors may be used. To mount vertical stage  18  to turntable  28  of rotary stage  16 , bottom wall  40  of housing  36  has holes  54  ( FIGS. 3 and 4 ) through which four screws  55  extend into threaded holes  56  of the adapter plate  32 , where two screws  55  are shown in  FIG. 3 , and the other two screws  55  are shown in  FIG. 4 . 
     Two limit switches  52  are provided each having a switch element  52   a  which actuates when abutted by an extension  53  from carriage  20  to define the uppermost extent and lowermost extent of carriage  20  travel up and down, respectively. When actuated, the limit switch  52  sends a signal to a below discussed controller  135  to turn off motor  50  operation to avoid over travel of carriage  20  in housing  36 . 
     The mounting arm (or assembly)  22  coupling carriage  20  to housing  13  of imaging head  12  will now be described and is best shown by the cross-sectional view of  FIG. 5 , and exploded views of  FIGS. 6 and 7 . An adapter mount plate  58  is attached to a slanted/angled support member  60  by a screw  61  via hole  62  in plate  58  and threaded hole  63  in support member  60 , where pins  64  extend from support member  60  and align with holes  65  of adapter mount plate  58 . A tray member  68  is attached to support member  60  by a screw  70  via a hole  69  of tray member  68  received in a threaded hole  71  of support member  60 , where pins  72  extend from support member  60  and align with holes  74  in the bottom of tray member  68 . 
     Tray member  68  receives a tilt plate  76  which has been attached by screws  77  via holes  78  in tilt plate  76  into threaded holes  79  of a base  80   a  of a rotary stage  80 , which has a turntable  80   b  rotatable with respect to base  80   a . Two ball bearings  66  are disposed in tray member  68  between the inside of tray member  68  and bottom of tilt plate  76 . Such ball bearings  66  are each glued in a semicircular pocket along the interior bottom of tray member  68  prior to receiving tilt plate  76  with attached rotary stage  80  in order to provide two points of contact with tilt plate  76  near the top left and top right of tray member  68 . Four screws  82  extend through holes  83  in tilt plate  76  and holes  84  in tray member  68  and are each captured by one of four nuts  86  via one of four springs  85 . Springs  85  and nuts  86  are also shown in  FIGS. 1-3 . Springs  85  bias tilt plate  76  toward the inside bottom of tray member  68  with two ball bearings  66  being disposed there between. An adapter mount plate  88  is attached by screws  89  via holes  90  in plate  88  to threaded holes  91  along turntable  80   b  of rotary stage  80 . Screws  92  extend through holes  94  in plate  88  into threaded holes  95  ( FIG. 7 ) along base  15  of housing  13  of imaging head  12 . The mounting arm  22  is mounted by four screws  96  which extend via holes  98  of plate  58  into threaded holes  100  ( FIG. 6 ) for receiving such screws  96  along carriage  20 . The slant/angled support plate  60  is set at a desired upward angle or slope to dispose the rest of the mounting arm  22  mounted to plate  60  at a higher height than would be if support plate  60  was horizontally disposed in order to obtain a desired range of vertical travel of imaging head  12  as set by the position of switches  52  along housing  36 . However, other angle or non-angled (i.e., horizontal) support member may be used depending on the desired height of vertical stage  18 , and the range of vertical travel of carriage  20  as set by limit switches  52 . For purposes of illustration, only one of each set of screws, springs, holes, and nuts, are labeled in  FIGS. 6 and 7 . 
     As best shown in  FIG. 5 , a thumb screw  102  with attached handle or knob  102   a  is provided to adjust tilt of plate  76  with respect to tray member  68 . Thumb screw  102  extends through a threaded hole  103  near the center bottom of plate  76  against an opening  104  along the inside of tray member  68  which abuts the end of thumb screw  102 . The thumb screw  102  rotation changes the distance of the bottom of plate  76  with respect to the bottom of tray member  68  under the bias of springs  85 , while the ball bearings  66  provide two points of contact upon which plate  76  tilts, thereby controlling the tilt or yaw (or tilt angle) of the normal axis  128   b  ( FIGS. 1 and 2 ) of the housing  13  of imaging head  12  with respect to the horizontal via the attached rotary stage  80 , as denoted by arrows  105  ( FIG. 1 ). The tilt position of the imaging head  12  may thus be varied from the horizontal as desired by the user, such as for example at or between 0 to 5 degrees. Tray member  68 , plate  76 , adapter plates  58  and  88 , and support member  60  may be made of aluminum. 
     Rotary stage  80  operates to rotate turntable  80   b  with respect to base  80   a  responsive to rotation of a knob or micrometer  81  to gearing (not shown) disposed to rotate turntable  80   b  as indicated by arrow  106  ( FIG. 1 ) about the normal axis  128   b . A locking pin  107  may slide or be turned to move in and out with respect to a hole in base  80   a  in order to lock and unlock, respectively, the rotational position of turntable  80   b  with respect to base  80   a . Turntable  80  is rotatable 360 degrees to rotate imaging head  12  about normal axis  128   b  and may have graduations along its outer circumference in rotational degrees with respect to marking(s) along base  80   a , which may be utilized by a user when manually turning knob  81  clockwise or counterclockwise to effect desired rotation. The rotary stage  80  is preferably a Newport Precision Rotation Stage Model No. UTR80 (manufacturer: Newport Corporation, Irvine, Calif., USA), but other rotary stages may also be used. Thus, plate  58 , support member  60 , and tray member  68  extend along a first portion of mounting arm  22  which is fixed to carriage  20  as described above, and tilt plate  76 , rotary stage  80 , and plate  88  extend along a second portion of mounting arm  22  coupled to the imaging head  12 , as described above, so that the second portion tilts with respect to the first portion by tilting plate  76  in tray member  68  of the first portion to adjust tilt of the imaging head  12  along normal axis  128   b  with respect to the horizontal dimension along which surface  14   a  of platform  14  extends. 
     In summary, the entire mounting arm  22  with imaging head  12  can rotate with vertical stage  18  using rotary stage  16  (arrow  30 ) to a desired rotational position about the vertical dimension (with locking pin  27  temporarily released until new rotational position is reached) using hand crank  29 . The entire mounting arm  22  with imaging head  12  can be set to a desire height or distance along the vertical dimension using vertical stage  18  (arrow  45 ) from the horizontal dimension along which surface  14   a  of platform  14  extends. Thumbscrew  102  of the mounting arm  22  can be turned using handle  102   a  to adjust tilt plate  76  to a desired tilt position with respect to tray member  68  (arrow  105 ), and imaging head  12  can rotate using rotatory stage  80  (arrow  106 ) to a desired rotational position about normal axis  128   b  that extend through of the imaging head  12  and along the rotational axis of rotary stage  80  about which turn table  80   b  rotates (with locking pin  107  temporarily released until new rotational position is reached). This freedom of motion along arrows  30 ,  45 ,  105 , and  106  allows imaging head  12  in a first mode of operation of microscope  10  to be moved to a position, such as shown in  FIG. 1  to image, for example, a first (or ex-vivo) tissue sample  110  between imaging head  12  and platform  14 , and in a second mode of operation of microscope  10  moved to a position such as shown in  FIG. 2  to image, for example, a second (or in-vivo) tissue sample  113  of a patient/subject beside (i.e., at the side of, nearby, but not upon) platform  14 . In-vivo tissue sample  113  has, for example, a skin lesion  113   a . The patient/subject is not shown to scale in  FIG. 2 . 
     As stated earlier, objective lens  128  extends in snout  127  from housing  13 . Snout  127  may have an optional snout cover  127   a  with a material plate window  127   b  of optically transparent material, such as glass or plastic. Preferably, window  127   b  is thick, such as 1 mm. Cover  127   a  is shaped to extend over snout  127  and objective lens  128  with imaging being carried out through window  127   b . It is especially useful so that pressure may be applied by the window  127   b  against a surface of tissue being imaged, such as an in-vivo tissue sample, to assist in stabilization of the optical system  11  of the imaging head  12  to such tissue to improve imaging performance. The snout cover  127   a  and window  127   b  may also be used to image in-vivo samples, such as small animals, that may be placed upon platform  14  with or without specimen stage(s) present. 
     In such first mode of operation, ex-vivo tissue sample  110  may be mounted onto a movable specimen stage provided by a third (or x-y) stage  108  movable along x and y orthogonal axes (depicted as x and y arrows in  FIG. 1 ) which are parallel to the horizontal plane of surface  14   a  of platform  14  upon which stage  108  is mounted. For example, ex-vivo tissue sample  110  may a non-histologically prepared tissue specimen (i.e., without being mechanically sliced thin sections mounted on slides) removed from a patient/subject which is disposed upon a block  111 . Block  111  may represent a substrate, such as of glass or plastic, or a cassette which retains the tissue sample  110  in a desired orientation on stage  108 . Mounting features or inserts  109  receive block  111 , and clips  112  retain block  111  position in stage  108 . However, other mechanisms for retaining block  111  may be used. While stage  108  may be a typical translation stage for moving tissue sample  110  along x and y orthogonal dimensions, preferably stage  108  is a Marzhauser X-Y Stage Scan Plus  Model No. 00-24-579-0000 (manufacturer: Märzhäuser Wetzlar GmbH &amp; Co. KG, Germany). Stage  108  is mounted to platform  14  as typically in mounting stages to bases, such as by screws  109   a  through holes in stage  108  in stage mounts  108   a  attached to platform  14 . Preferably, two stage mounts  108   a  are used as shown in  FIG. 2  between stage  108  and platform  14 . Stage mounts  108   a  may be attached to platform  14  by screws via holes through such mounts  108   a  and platform  14 . Rubber o-rings and washers (such as tooth lock washers) may be disposed along such screws  109   a  in attaching the stage  108  and stage mounts  108   a  ( FIG. 2 ), in which o-rings aid in minimization of vibration to block  111  holding tissue sample  110 . Other mechanisms for coupling stage  108  to platform  14  may also be used. Four rubber feet  115  are attached to the underside of platform  14 , such as by screws through holes in the platform, so that the platform may rest on a tabletop or other surface upon such rubber feet. 
     Optionally, an additional fourth (or z) stage  114 , such as shown in  FIGS. 3, 4, and 6 , may be attached atop stage  108  in which the mount for tissue sample  110  as shown in  FIG. 1  is removed and placed upon stage  114  instead. Stage  114  is movable along a z axis orthogonal to the x and y axes of stage  108 . Stage  114  preferably is a Marzhauser Piezo Z-Stage Model No. 00-55-550-0800, which attaches by clips onto stage  108 , for coupling such stages as set forth by the manufacturer. 
     Referring to  FIG. 8 , an example of microscope  10  in a desktop or table configuration is shown having platform  14  disposed upon a surface  116  with imaging head  12  mounted to platform  14  as described above to enable both first and second modes operation of the microscope. The microscope  10  has a computer system  118 , such as a personal computer or workstation programmed in accordance with software in its memory. Computer system  118  is connected to a display  120  and user interface devices (such as keyboard  121  and mouse  122 ). Display  120  may be a touchscreen display which provides an additional user interface device to graphical user interface software operating on computer system  118 . Computer system  118  is connected by a cable  124  to imaging head  12 , and via such cable the computer system controls operation of imaging head  12  and receive signals therefrom representative of one or more microscopic images of optically formed tissue sections at one or more locations within or upon tissue sample  110  or  113  for output to display  120  and storage in memory of the computer system in the same manner as VivaScope® confocal microscopes manufactured by Caliber Imaging &amp; Diagnostics, Inc. of Henrietta, N.Y., USA. The location of the platform  14  upon surface  116  may be different than shown in  FIG. 8  so that imaging head  12  can be properly positioned with respect to a patient or subject that needs to located beside both surface  116  and platform  14  in order to image in-vivo tissue of such patient or subject in the second mode operation of microscope  10 . 
     In microscope  10  operation, scanned laser illumination is focused and collected by objective lens  128  along its optical axis  128   a  into tissue sample  110  or  113 , where collected light by the lens is representative of a tissue section at a cellular level below the surface of the tissue sample facing objective lens  128 . While  FIG. 8  shows an example of first mode operation for imaging an ex-vivo tissue sample of  FIG. 1 , the housing  13  is movable for imaging an in-vivo tissue sample as described earlier for second mode operation of microscope  10  as shown in  FIG. 2 . As stated earlier, tissue samples may also be positioned on platform  14  for imaging by imaging head  12  without stage(s)  108  or  114  present, such as may be useful for imaging in-vivo tissue samples of small animals. 
     The electronics of imaging head  12 , and the computer system  118  of microscope  10  with display  120 , for viewing microscopic sectional images of tissue samples from light focused and collected via objective lens  128 , may be the same as described in incorporated by reference U.S. Pat. No. 9,055,867. While in  FIG. 8  both stages  108  and  114  are shown when imaging ex-vivo tissue samples, z-stage  114  is optional, since objective lens  128  is movable in housing  13  along its optical axis  128   a  which can be aligned with a z axis orthogonal with the x and y axes of stage  108  so that objective lens  128  is thus movable along such z axis in order to select the depth of focus of a beam scanned at locations upon or within the tissue sample being imaged. However, when z-stage  114  is present upon platform  14 , z-stage  114  may also be used to select the depth of focus a beam scanned at locations upon or within the tissue sample being imaged. Imaging head  12  is temporarily fixed in position with respect with respect to the x and y axes of x-y stage  108  in the first mode of operating microscope  10 . 
     Computer system  118  controls movement of x, y motors of the stage  108  and reading x and y positions thereof, and z axis motor and reading z portion thereof of stage  114  (if present), via one or more cables  130  to ports  129 .  FIG. 3  shows ports  129  with cable  130  removed. Preferably a stage controller card is provided in the computer system  118  to enable such interface with stages  108  (and  114  if present) via cable(s)  130 . In the case of Marzhauser X-Y Stage and Marzhauser Z-Stage as described earlier are utilized, such stage control card may be a Scan Plus  Marzhauser X&amp;Y Stage Controller Card, Part Number 00-76-150-0813, located inside the case of computer system  118 . An optional joystick  132 , may also be used by a user to control movement of x, y axis motors of the stage  108  (and z axis motor of stage  114  if present), via a cable  133  to one of ports  130 . 
     The stepper motor  50  of vertical stage  18  is controlled by a motor controller  134  which drives motor  50  via signals along a cable  136  to rotate lead screw  42  of the vertical stage  18  in first and second directions that enable up and down motion, respectively, of carriage  20  and the imaging head  12  mounted thereto by mounting arm  22 . Limit switches  52  are also connected to motor controller  134  via cable  136  to receive signals therefrom and control motor  50  accordingly as described earlier. Buttons and switches  135  along the motor controller  134  are provided to control motor operation. Preferably, motor controller  134  is a Velmex Stage Controller Model No. VXM-1. An optional joystick  138  may connected by cable  139  to motor controller  134  to facilitate user control of motor  50  to provide desired up and down motion of carriage  20  of vertical stage  18 . Preferably, joystick  138  is a Velmex Digital Joystick Model No. 4-2121. While rotary stage  16  is shown as being manually controlled by knob  29 , optionally stage  16  may have a motor instead of knob  29  for rotating rotary stage  16 , such as manufactured by Velmex. This optional motor may be controlled by signals from motor controller  134  which can additional drive such motor. Although not shown, power is supplied to various components in  FIG. 8  to enable their operation. 
     In operating microscope  10  using stage  108 , the optical axis  128   a  of objective lens  128  is aligned along a z axis perpendicular to the x and y axes of stage  108 , such as shown in  FIG. 1 . This further enables alignment along the z axis of stage  114  if present. Such alignment is enabled by adjusting the tilt of imaging head  12  using handle  102   a  of thumb screw  102  and rotation of imaging head  12  using rotary table  80 , as described earlier. Such may be aided by alignment mark(s) if present along rotary table  80  along turntable  80   b  and base  80   a . Further, a target or features may be placed on the platform  14 , or stage mount (in place of block  111  of  FIG. 1 ), in view of objective lens  128  to assist in electronic calibration of images on screen  125  of display  120  as such calibration alignment is carried out to assure horizontal levelling of imaging head&#39;s normal axis  128   b  with respect to platform  14 , where such normal axis  128   b  lies perpendicular to the z axis. 
     While the operation and structure of imaging head  12  may be the same as described in incorporated U.S. Pat. No. 9,055,867 using the laser illumination source provided therein (such as light source  146  of  FIG. 9 ), the imaging head of the incorporated patent preferably is adapted to that of optical system  11  ( FIGS. 9-11 ) which utilizes multiple discrete laser wavelengths for illumination provided from a multiple wavelength laser light source  140  via a fiber optic cable  142  to imaging head  12 . For example, light source  140  may be a Toptica iChrome MLE-L Multi-Laser Engine (manufacturer: Toptica Photonics AG, Germany) with collimated laser diode assemblies manufactured by Blue Sky Research of Milpitas, Calif. USA. The additional laser illumination provided by light source  140  is combined with the laser illumination produced in imaging head  12  and scanned together via objective lens  128 , and then returned scanned illumination via objective lens  128  is split for detection onto two detectors that sense particular wavelength(s), as described in more detail below in connection with  FIGS. 9, 10 , and  11 . 
     Referring to the optical system  11  of  FIG. 11 , linear polarized light of multiple discrete wavelengths generated by light source  140  (e.g., 405 nm, 488 nm, 561 nm, and 640 nm) passes along optical fiber cable  142  to optics  143  which collimates and expands its size to provide a beam  144 , such as to 4.3 mm in diameter. Optics  143  are preferably contained in a cylindrical tube  143   a  that receives optical fiber cable  142  and extends through an opening  13   a  ( FIG. 1 ) of housing  13 . A laser  146 , preferably a laser diode which is associated with an opto-detector for monitoring laser power as described in the incorporated patent, provides a linearly polarized beam  148  at a single wavelength (e.g., 785 nm). Beam  144  and beam  148  are combined into a beam  150  by a dichroic beamsplitter  149 , and beam  150  then passes through a polarizing beamsplitter  150 . A resonant scanner  152  presents its scanning mirror  152   a  to beam  150 , and the beam from the resonant scanner mirror  152   a  is then incident scanning mirror  154   a  of a galvanometer  154  to provide a scan beam  155 . Mirrors  152   a  and  154   a  oscillate so that mirror  152   a  provides fast or horizontal line scans in a raster being scanned, and slow or vertical scan and retrace are provided by mirror  154   a , as described in more detail in the incorporated patent. The axes of oscillation of these mirrors  152   a  and  154   a  are orthogonal (perpendicular) to each other. The separation distance may be approximately a minimum separation distance to provide clearance between the mirrors  152   a  and  154   a  as they scan. A telescope  156  magnifies the beam (e.g., 2.3×) and relays scanning beam  155  to objective lens  128  via a quarter wave plate shifter  157 , and the objective lens  128  focuses the scanning beam  155  to the sample, such as sample  110  or  113  for example as earlier described. 
     The returned light  158  from the tissue sample  110  or  113  passes through objective  128 , wave plate  157 , telescope  156 , and scanning mirrors  154   a  and  152   a . The return light thus is descanned at mirrors  154   a  and  152   a  into a stationary beam  160  and enters the polarizing beamsplitter  151  which reflects beam  160  via a focusing lens  161 , a reflecting mirror  162 , and a notch filter  163 , to a dichroic beamsplitter  164  which splits the returned light into a first beam  165  and a second beam  169 . Beam  165  is incident a small aperture provided by pinhole  166  onto a detector  168  provided by a photomultiplier tube, via one of selectable open or filter positions along a filter wheel  167 . Beam  165  is incident a small aperture provided by a pinhole  170  onto a detector  172  provided by a photomultiplier tube, via one of selectable open or filter positions along a filter wheel  171 . Lens  161  focuses the light of their respective beams onto pinholes  161  and  170 . Although not shown in  FIG. 11 , a turning mirror  173  ( FIG. 9 ) is provided between beamsplitter  151  and mirror  152   a  to reflect beam  150  onto mirror  152   a , and reflect beam  160  from mirror  152   a  to beamsplitter  151 . 
     Each of filter wheels  167  and  171  has a shaft mounted for rotation by stepper motors  174  and  175 , respectively, to select the desire opening or filter along the wheel. For example, at least four filters are provided along wheel  167  for different wavelength(s) or range of wavelengths onto detector  168 , where filter  167   a  passes light only in range of 405-561 nm wavelength, filter  167   b  passes only 630 nm wavelength light, filter  167   c  passes only 670 nm wavelength light, and filter  167   d  passes both 832 nm and 837 nm wavelength light. At least two filters are provided along wheel  171 , where a filter  171   a  passes only 520 nm wavelength light, and a filter  171   b  passes only 450 nm wavelength light. Spaces on one or both filter wheels are open so that unfiltered light may pass there through, such as for detection of light of the wavelength of laser  146  in the path of light for detection by their respective detectors  168  and  172 . For example, opening  176   e  is provided on filter wheel  176 , and opening  171   c  is provided on filter wheel  171 . Additional openings/filters illustrated on filter wheel  171  may have filters for other wavelengths or wavelength ranges. 
     The wavelengths provided along fiber optic cable  142  can activate fluorescent dyes that may be applied to tissue samples. Thus, one of the filter wheels enables selection of a filter to detect on their associated detector the fluorescent wavelength(s) of the returned light  160 , while the other of the filter wheels is set to an open position to detect light of wavelength of laser  146  in the returned light  60 . Notch filter  163  allows selectable discrete wavelengths or ranges of wavelengths to assist in detecting wavelengths with filters along the filter wheels. Preferably, notch filter  163  allows light of wavelength of laser  146  (e.g.  785 nm), and blocks light of wavelengths received from fiber optic cable  143  which may interfere with imaging at fluorescent wavelengths associated with the filters disposed along filter wheels  167  and  171  in the path of light for detection by their respective detectors  168  and  172 . Further, dichroic beamsplitter  164  may filter light such that beam  165  has wavelengths  405 nm and  408 nm, and a beam  169  has wavelengths 581 nm, 640 nm, and 785 nm. Other wavelengths than set forth above may be used for light sources  140  and  146 , and detected beams  165  and  169 , as well as other wavelength filtering may be used by notch filter  163  and along filter wheels  167  and  171 . 
     Each motor  174  and  175  is driven by electronics on a printed circuit board  185  having a Hall effect sensor which reads a magnet along the wheel to sense the home position of the wheel and rotate the wheel to the desired filter or open location along the wheel by actuation signals received from computer system  118 . The rotational position of each filter or opening along filter wheels  167  and  171  may be stored memory of the computer system  118  so that motors  174  and  175  can be actuated by computer system to arrive at the rotational position associated with the desired filter or opening along the wheels. 
     The optical components and electronics of the imaging head  12  are mounted along a chassis  176  and support plate  177  as shown in  FIGS. 9 and 10  to provide a preferred compact mounting of such components. Two printed circuit boards  190  with electronics for controlling imaging head  12 , responsive to computer system  118 , are attached to chassis  176 , where circuit board  190  are connected to other circuit boards described herein in housing  13 . A first structure or block  178 , such as of aluminum, is mounted to chassis  176  which supports light source  146 , beamsplitter  149 , and cylinder  143   a  with attached fiber optic cable  142 . Structure  178  has a receptacle  179  into which cylinder  143   a  plugs into when additional wavelengths for imaging from fiber optic cable  142  is desired. Detectors  167  and  172  have a circuit board  182  and  183 , respectively, which is mounted to support plate  177 . A second structure or block  180 , which may also be of aluminum, is mounted to chassis  176  to support filter wheels  167  and  171 , pinholes  167  and  170 , beamsplitter  164 , and notch filter  163 , for imaging onto such detectors  167  and  172  as described earlier. The circuit board  185  for driving and controlling motors  174  and  175  may be supported on circuit board  182 . 
     In order to properly align beams  165  and  169  for detection, mirror  162  and pinhole  166  are each adjustable in position. Mirror  162  is mounted upon an adjustable flexure  162   a  attached to a bracket or flange  186  of chassis  176  for steering beam  169  via beamsplitter  164 . Flexure  162   a  may be adjustable by screws, and for example may be a stainless steel flexure mirror mount, such as Flexure Industrial Optical Mount with Allen (or hex) key adjustments, model No. MFM-050 manufactured by Newport Corporation of Irvine, Calif., U.S.A. This adjustability of mirror  162  spatial position is denoted by arrows beside mirror  162  in  FIG. 11 . Pinhole  166  (i.e., provided by a thin substrate with light blocking material having a small aperture) is retained in a cylinder (or cylindrical cell)  188  mounted in structure  180 , where pinhole  166  is spring loaded using two spring steel flexures. Two orthogonally oriented set screws  189  push pinhole  166  into a desired position against the spring force of such flexures, so that turning screws  189  adjusts pinhole  166  position. Optionally, such adjustability may be similar provided in structure  180  in a third orthogonal dimension. This adjustability of pinhole  166  spatial position in two dimensions orthogonal to the incident beam  165 , or in three dimensions, is denoted by arrows beside pinhole  166  in  FIG. 11 . 
     When imaging head  12  is assembled, mirror  162  is adjusted in position to steer beam  169  in alignment with pinhole  170 , which is fixed in position, so that beam  169  detected by detector  172  can be properly imaged. To aid in such alignment, an image is displayed on display  120  by computer system  118  from detector  172 , and adjustable mirror  162  is moved until beam  169  is aligned with the fixed pinhole  170  such that highest signal level from detector  172  is achieved on display  120 . Then an image from detector  168  is displayed on display  120 , and adjustable pinhole  166  is moved until the highest signal from detector  168  is achieved on display  120 . Thus, beam  165  is now aligned with pinhole  166  so that beam  165  detected by detector  168  can be properly imaged. Alternatively, adjustable flexure  162   a  is not used so that mirror  162  is mounted non-adjustable in position when imaging head  12  is assembled, and pinhole  170  is manually adjustable in position in the same way pinhole  166  is disposed. In such case, one or preferably both pinholes  166  and  170  are each separately adjusted in position to align their respective beams  165  and  169  onto their respective detectors  168  and  172  by the highest signal being achieved on display  120  from their respective detectors  168  and  172 . 
     The adjustability in alignment of beams  165  and  169  assures proper operation of dual detection path of optical system  11  of the beams onto detectors  168  and  172 , respectively, so that microscope  10  can simultaneously provide images of microscopic structures of the same tissue sample using two different wavelengths or wavelength range of detected returned light from the sample. Filter wheels  167  and  171  are each set to one of openings or filters accordingly, so that one or both images of the desired wavelengths or wavelength range can appear on display  120  by computer system  118  from received signals of detectors  168  and  172 . Pinholes  166  and  170  may be identical, and they enable confocal imaging on their respective detectors by limiting returned scattered light of their respective beams to a particular section within or on the tissue sample  110  or  113 . 
     Attached to the forward end of chassis  176  is a fixed tube  192  with optics providing telescope  156 . Objective lens  128  is disposed in a generally cylindrical mounting  194  that attaches to a barrel  196  providing a tube or sleeve moving axially (along optical axis  128   a ) over tube  192  by a linear actuator as described in the incorporated patent. A magnetic strip is provided on the side of barrel  196  which is read by a sensor  197  on chassis  176  that linearly encodes position of the barrel  196  to the electronics in the imaging head  12 , thereby enabling computer system  118  to actuate the linear motor to adjust the position of objective lens  128  with respect to telescope  192  and hence the focus of such lens with respect to the tissue sample  110  or  113 . 
     Preferably, cylindrical magnets  198  are attached, such as by adhesive, to holes  199  along the interior annular ring  200  at end of barrel  196 , as shown in  FIG. 9 . A metal ring  201  is attached to the objective lens mounting  194 . Such ring  201  attaches along ring  200  by magnetic attraction to magnets  198 , so that mounting  194  is retained to barrel  196 . The objective lens  128  shown in the figures represents a liquid immersion lens. Such objective lens  128  is useful when a refractive index matching fluid is applied to a tissue sample prior to being imaged (the fluid matches or approximately matches the refractive index of the tissue sample) as the objective lens is brought into contact with the surface of the tissue sample. The index matching fluid reduces undesirable reflections and spherical distortions from the tissue sample&#39;s surface facing the lens that can negatively effect imaging performance. However, different objective lens may be provided in different mountings  194 , each providing a different imaging performance, such as in terms of magnification, or are of non-immersion or different immersion type lenses. When a different objective lens  128  is desired by a user, the user can pull mounting  194  away for magnetics  198 , and replace with a different mounting  194  with the desired objective lens. Alternatively, the mounting  194  may be attached such as by adhesive, to the end of barrel  196 , without metal ring  201  or magnetics  198 . 
     The earlier described snout  127  is provided by barrel  196  with attached mounting  194  having objective lens  128 . As shown in  FIG. 9 , snout cover  127   a  is provided by a cylindrical tube having a window  127   b  mounted in a cap  127   c  that is received in an opening  127   c  at the distal end of such tube which is shaped to receive cap  127   c . To mount snout cover  127   a  to imaging head  12 , a cylindrical mounting  202  is provided having three legs  203  that attach, such as by adhesive, to the front of chassis  176  and extend via an opening  13   b  at the front of housing  13  with barrel  196 . Along the front inner annular rim  204  of mounting  202  are holes  204   a  for cylindrical magnets  205  which are retained in the holes by adhesive. A metal ring  127   d  is attached at the rear of the tube providing snout cover  127   a . Attraction of ring  127   d  to magnets  205  along rim  204  retains snout cover  127   a  in position for imaging through window  127   b  via objective lens  128 . Snout cover  127   a  may be removed from its mounting  202  by pulling cover  127   b  away from magnetics  205  when snout cover  127   a  is not needed for imaging. 
     Housing  13  has a series of ribs  208  extending from base  15  of housing  13  onto which chassis  176  and plate  177  are mounted, such as by screws into holes along such ribs. Left and right housing portions  206  and  207  provide shells that mate with each other and attach to ribs  208  by screws. Two handles  210  are then attached to housing portions  206  and  207  to assist in manually moving housing  13  by a user if desired with respect to platform  14 . If a fan is provided in housing  13 , a fan cover  211  may be used. Other manner of coupling imaging head  12  components within housing  13  may be used than shown in  FIGS. 9 and 10 . 
     Computer system  118  via cable  124  has an I/O interface with electronics in the imaging head  12  to enable their operation, such as to control of operation of resonant scanner  152  and galvanometer  154 , control the linear actuator or motor for positioning objective lens  128  along its optical axis  128   a , and power to light source  146 , as may be described in more detail in the incorporated patent. The signals from detectors  168  and  172  are received along separate channels via cable  124  as raster images in memory of computer system  118  for display on screen  125  as desired by the user. Computer system  118  via cable  124  also sends signals to motor  174  and  175  and reads sensors associated therewith to rotate filter wheels  167  and  171 , respectively, in accordance with the rotational position of the particular filter or opening along such filter wheels as desired by the user. 
     Different locations along tissue samples  110  or  113  are selected to provide optical sectioned microscopic images of the sample at such locations presented to objective lens  128  by one or more of moving imaging head  12  as described earlier, moving stage  108  along its x and/or y axes, changing depth of the scanned beam  115  in and under the surface of the tissue sample by moving objective lens  128  along its optical axis  128   a  in imaging head  12 , such optical axis being co-axial with the z axis if aligned thereto as described earlier, or along z axis to change such depth by using stage  114  if present. The selection of different locations along a tissue sample  110  or  113  may be performed automatically by computer system  118  stepwise movement along x and/or y axes of stage  108  (and z axis of stage  114  if present), and/or stepwise movement of objective lens  128  along its optical axis  128   a . For example, computer system  118  can fix the position of galvanometer mirror  154   a  to be stationary, and instead move stage  108  in a stepwise fashion along the y axis to provide comparable raster scan imaging. Power and ground to electronics and other components, such as laser source  146 , in imaging system  12  is also provided by wires within cable  124 . 
     Further, although imaging head  12  is described herein having an optical system for capturing optically formed microscopic sectional images of tissue sample  110  or  113  operative by confocal microscopy, other modalities for imaging optically sectioned microscopic images of sample may be incorporated in imaging head  12  by optical coherence tomography (OCT) or interferometry, such as described in Schmitt et al., “Optical characterization of disease tissues using low-coherence interferometry,” Proc. of SPIE, Volume 1889 (1993), or by a two-photon laser microscopy, such as described in U.S. Pat. No. 5,034,613. 
     Other positions of imaging head  12  may be provided than shown in the figures. Also, different non-histologically tissue samples than tissue samples  110  and  113  shown in the figures may be imaged by microscope  10 . 
     From the foregoing description, it will be apparent that a confocal microscope having a positionable imaging head has been provided. Variations and modifications in the herein described microscope, and system and method for mounting an imaging head of such microscope in accordance with the invention, will undoubtedly suggest themselves to those skilled in the art. Accordingly, the foregoing description should be taken as illustrative and not in a limiting sense.