Abstract:
In a process for cutting sections from a probe for microscopic analysis, an ultramicrotome device is used having a blade with a cutting edge, the cutting edge extending at least approximately in a first direction. The process includes the steps of: vibrating the blade in the first direction; and moving the blade relative to the probe to be cut in a second direction, the second direction being perpendicular to the first direction. This eliminates, or at least strongly reduces, compression of the cut sections.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
   This application is a continuation of U.S. patent application Ser. No. 10/646,354, filed Aug. 22, 2003, now abandoned which is a continuation-in-part of U.S. patent application Ser. No. 09/718,636, filed Nov. 22, 2000, now abandoned which is a continuation-in-part of U.S. patent application Ser. No. 09/207,284, filed Dec. 8, 1998, now abandoned which claims priority to European Patent No. EP 97 811 004.7, filed Dec. 19, 1997, all of which are incorporated herein by reference in their entirety. 

   BACKGROUND OF THE INVENTION 
   1. Field of the Invention 
   The present invention relates to a process for cutting sections from a probe for microscopic analysis by using an ultramicrotome device. 
   2. Description of Related Art 
   Microtomes and ultramicrotomes are used to cut thin respective ultra-thin sections from a sample for microscopic analyses. The sample is mounted on a cross-slide which can be advanced horizontally in steps according to the desired thickness of the sections and vertically for performing the cutting operation. A cutting blade with a horizontal cutting edge is mounted on a holder. Microtomy is concerned with a range of thickness of 0.5 to 50 μm of the sections and is mainly used for optical microscopy. Ultramicrotomy is concerned with a range of thickness of 10 to 100 nm of the sections. This range of thickness is required for transmission electron microscopy. Ultramicrotomy has proved to be a very fast and efficient technique not only for TEM but also for surfacing samples for STM and AFM. 
   In microtomy mainly steel blades are used for cutting. German Patent No. 913 112 discloses an older type of a microtome in which the cutting blade is horizontal and the sample advances upwardly in steps between the cuts. The blade is fastened between two parallel leaf springs and driven by magnets for oscillating movement parallel to the cutting edge. The cutting edge of a steel blade is relatively rough when viewed under an electron microscope and relatively blunt. With the oscillating motion of the blade, therefore, a sawing action is achieved: the jags of the cutting edge act like saw teeth. 
   This sawing action of the blade in a microtome is also described in the DDR Patent No. 156 199, in which the blade is driven by an electroacoustical transducer at high frequency, and in the Belgian Patent No. 440 928 which uses an ultrasound emitter to oscillate the blade. 
   In ultramicrotomy the sections are so thin that extreme care must be taken to shield the ultramicrotome from all possible external and internal vibrations because they would adversely affect the cutting result. It therefore seemed impossible to transfer the sawing action of the cutting blade known from microtomy to an ultramicrotome. For this reason much sharper and perfectly rectilinear cutting edges are required in ultramicrotomy. This has been achieved by cutting blades of diamond. U.S. Pat. No. 4,697,489 describes a holder with such a diamond cutting blade for ultramicrotomes. With the perfectly rectilinear cutting edge, even when viewed under an electron microscope, of a diamond blade no sawing action can be achieved as with steel blades. 
   For the ultramicrotomy at room temperature, usually the cutting is performed on a knife mounted in a boat which contains water. The water forms a horizontal surface behind the cutting edge of the knife. Due to the surface tension the sections float on the water surface and can be collected. The water acts as a lubricant during sectioning process. 
   However, in ultramicrotomy a different problem arises which does not occur in microtomy: the problem of section compression. This phenomenon occurs at a thickness of the sections below 100 nm. Depending on the mechanical properties of the sample (flexibility) and on the sectioning angle φ of the knife the sections undergo considerable distortion (compression) during cutting ( FIG. 8 ). In  FIG. 8 ,  1  designates the diamond blade or knife with the cutting edge  2 .  3  is the sample. The sample  3  may be one of a great variety of industrial or biological samples. A is the vertical movement of the sample  3 .  4  is the cut section floating on a waterbed  5 .  6  designates the direction of compression in the section  4 .  7  is a region of intense shearing, and  8  is the region of compression in the sample  3 . 
   Water sensitive samples  3  have to be cut dry. Due to the missing lubrication and to the friction on the knife surface the sections  4  are even more compressed as the ones cut on water. In cryo-UM most samples have to be cut dry. The amount of compression depends on different factors:
         The sectioning angle of the knife.   The hardness of the sample.   The triboelectrical properties of the sample.
 
The most critical factor is the sectioning angle φ. The sectioning angle φ is the sum of the wedge angle β of the knife  1  and the clearance angle δ. It was shown that reducing the wedge angle β results in a reduction of compression. However, the wedge angle β may not be reduced ad infinitum. We have found an angle of 30° to be a limit. A further reduction of the wedge angle results in a lower cutting edge  2  quality and in a considerably shorter service time of the knife  1 . In cryo-UM the compression in sections was found almost equal with the sectioning angle φ. Therefore, a knife  1  working with a sectioning angle φ of 40° (wedge angle β 30°, clearance angle δ 10°) would result in a compression in the sections  4  of approximately 40%.
       

   SUMMARY OF THE INVENTION 
   In order to preserve the original ultrastructure and form of matter, it would be desirable to eliminate the distortion (compression) in the sections  4 . 
   The problem to be solved with the present invention is to create a process used in an ultramicrotome which reduces or eliminates the compression of the sections. This problem is solved by the present inventive process for cutting sections from a probe for microscopic analysis, by using an ultramicrotome device having a blade, especially a diamond blade, with a cutting edge, wherein this cutting edge extends in a non-vibrated position at least approximately in a first direction. This process comprises the steps of vibrating said blade in the first direction and moving the blade relative to the probe to be cut in a second direction, the second direction being perpendicular to the first direction. 
   Briefly stated, the device used in such a process comprises a holder and a block attached to the holder by at least one spring. A diamond blade is attached to the block. The cutting edge of the blade is substantially horizontal in operation. A vibrator cooperates with the block to vibrate it substantially parallel to the cutting edge. Preferably, the vibrator comprises a piezoelectric transducer. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a schematic diagram showing the effective sectioning angle α when the blade or knife  1  is moved in the direction of the edge  2  during cutting; 
       FIG. 2  is a side view of a first embodiment according to the present invention; 
       FIG. 3  is a front view, partially in section, of the first embodiment of  FIG. 2 ; 
       FIG. 4  is a side view of a second embodiment according to the present invention; 
       FIG. 5  is a front view, partially in section, of the second embodiment of  FIG. 4 ; 
       FIG. 6  is a side view of a third embodiment according to the present invention; 
       FIG. 7  is a front view of the third embodiment of  FIG. 6 ; and 
       FIG. 8  is a schematic diagram depicting the cutting action of a blade or knife according to the prior art. 
   

   DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
   In the present invention an oscillating movement of the blade or knife  1  parallel to the cutting edge  2  and perpendicular to the cutting direction A is used to eliminate or at least strongly reduce compression of the sections  4 . When the knife  1  moves in the direction of the cutting edge  2  while the probe  3  moves in the direction A, an effective cutting direction B results which forms an acute angle γ with the cutting edge  2  ( FIG. 1 ). If y is the vertical movement of the probe  3  per time unit and z is the effective relative movement between knife  1  and probe  3  in the same time unit, it can be seen from  FIG. 1  that 
                 tan   ⁢           ⁢   α     =     x   z       ;     ⁢                           sin   ⁢           ⁢   γ     =     y   z       ;                 tan   ⁢           ⁢   ϕ     =       x   y     .           
It follows:
 
   
     
       
         
           
             sin 
             ⁢ 
             
                 
             
             ⁢ 
             
               γ 
               · 
               tan 
             
             ⁢ 
             
                 
             
             ⁢ 
             ϕ 
           
           = 
           
             
               
                 y 
                 z 
               
               · 
               
                 x 
                 y 
               
             
             = 
             
               
                 x 
                 z 
               
               = 
               
                 tan 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 
                   α 
                   . 
                 
               
             
           
         
       
     
   
   When the knife  1  vibrates, the effective sectioning angle α varies (maximum effective sectioning angle α equal to φ, minimal effective sectioning angle α close to 0°). The theoretical value of compression reduction is as follows: An assumed mean effective sectioning angle α depends on the amplitude C (mm) and the frequency ν (Hz) of the vibration and on the cutting speed v (mm/sec). Only small effective sectioning angles α are considered. Under this assumption it can be shown that
 
tan α=( v/C ·ν)tan φ.
 
To give an example, the following parameters are assumed:
 
φ=45°; v=0.1 mm/sec; C=1 μm; ν=1 kHz.
 
It follows
 
tan α=(0.1 mm/sec)/(0.001 mm·1000 Hz)·1=0.1
 
resulting in a mean effective sectioning angle α of about 5.7°.
 
   The theoretical assumptions seem to be correct because on a prototype the oscillating knife has shown to significantly reduce the compression of the sections  4 . 
   In ultramicrotomy the persons skilled in the art have taken extreme care to shield the microtome from all possible external and internal vibrations because they adversely affect the cutting result. The inventor has overcome this prejudice and could show that by vibrating the knife  1  substantially parallel to the cutting edge  2 , no adverse effect of the vibration was observed. 
   A first embodiment of the invention is shown in  FIGS. 2 and 3 . The blade  1  is sintered in a bronze holder  16  or vacuum brazed in a tungsten carbide holder. The holder  16  is mounted on an inclined face of a recess  17  in a block  18 . The block  18  is mounted to a holder  19  by means of a leaf spring  20 . The plane of the leaf spring  20  is substantially vertical and perpendicular to the cutting edge  2 . The spring  20  is mounted to the block  18  and the holder  19  by flat plates  21  and screws  22 . Alternatively, the spring  20  may be designed as an integral part of the block  18  and the holder  19 . 
   An arm  23  extends upward from the base  24  of the holder  19 . The arm  23  has a cylindrical horizontal boring  25  and a slot  26  on one side. The cylindrical housing  29  of a vibrator  30  with a piezoelectric transducer  31  and an actuating rod  32  is held in the boring  25  by means of a screw  33 . The spherical face end  34  of the rod  32  is slightly pressed against a plane face  35  of the block  18 . The axis  36  of the vibrator  30  is parallel to the cutting edge  2 . The spring  20  may be slightly bent towards the vibrator  30  in the unloaded state before the vibrator  30  is mounted in position such that with the deflection of the spring  20  required for the preload force of the block  18  against the rod  32  the spring  20  gets plain and vertical. The axis  36  passes through the center of gravity  40  of the block  18 . 
   The vibrator  30  is connected to an oscillator  37  by means of a cable  38 . Two adjustment knobs  39  on the oscillator  37  allow the selection of the amplitude and frequency of the oscillation of the vibrator  30 . Preferably, the frequency is selected in the ultrasound range above 15 kHz. The required amplitude is then only in the range of 10-1000 nm. 
     FIGS. 4 and 5  show a second embodiment. Similar parts are designated with the same reference numerals so that a detailed description of those parts is omitted. The embodiment of  FIGS. 4 and 5  has two parallel leaf springs  20  of equal active length L. The upper and lower ends of the active length L of the two springs  20  lay in horizontal planes which are parallel to the cutting edge  2 . This arrangement has the advantages that the cutting edge  2  moves more parallel to itself than in the first embodiment. In the first embodiment it makes a minute pendulum motion, and that vibrations around a vertical axis are strongly restricted. 
   In this embodiment the piezoelectric thickness transducer  31  is directly attached, e.g., bonded with one of its plane end faces  46  to a vertical face  47  of the block  18 . A counter mass  48  is fastened to the opposite end face  49  of the transducer. Instead of or in addition to directly bonding the faces  46  and  49  to the block  18  and counter mass  48 , a pressing force by springs  64  may be used which may bear against arms  65  attached to the holder  19 . This variant is shown in dash-dotted lines in  FIG. 4 . 
   This arrangement of the vibrator  30  has the advantage that considerably higher accelerations of the block  18  towards the counter mass  48  are possible. This is particularly of advantage when higher frequencies are used, e.g., in the ultrasound range because the accelerations increase with the square of the frequency. 
   The embodiment of  FIG. 5  is shown in the variant for dry ultramicrotomy, e.g., without the water  5  in a trough behind the blade  1 . Instead, the upper, horizontal face  55  of the block  18  has a depression  56  which is filled with a plastic insert  57  with a plane upper surface  58 , the plane of which intersecting the front face  59  of the blade  1  at an angle of 75° to 85°, preferably about 80°. Therefore, when the blade  1  is set at the recommended clearance angle of 10°, the surface  58  is exactly horizontal which greatly facilitates observation of the cut sections  4  with a stereo microscope, e.g., for section pick-up since no refocusing is required when moving the microscope horizontally. A material with good triboelectrical properties for the insert  57  is an epoxy resin. 
   Instead of the piezoelectric transducer  31 , other types of transducers could be used, e.g., magnetic transducers. A suitable transducer would be a moving coil transducer similar to the one used in moving coil loudspeakers. The moving coil would be mounted to the block  18  and connected to the oscillator  37 . The (e.g., permanent) magnet surrounding the coil and acting as counterweight could be elastically suspended (e.g., like the block  18  in  FIG. 4 ) on the holder  19 . The axis of the coil would be coincident with the axis  36 . 
     FIGS. 6 and 7  show a third embodiment. Similar parts are again designated with the same reference numerals. In this embodiment, the holder  19 , the block  18  and the leaf spring  20  are manufactured from a single piece of metal. The spring  20  is a web connecting the holder  19  and the block  18 . The holder  19  is mounted on a base  72 . In operation, the block  18  oscillates with an amplitude a o  and with a frequency in radians ω=2π·ν, wherein ν is the frequency in Hz, in a horizontal first direction x parallel to the cutting edge  2 . The oscillating movement is a o  sin ωt and the oscillating speed v h  is a o  ω cos ωt. 
   A first slide  73  is slidably guided on first guide rails  74  of the base  72  which extend in a horizontal second direction y perpendicular to the first direction x. The movement of the slide  73  is controlled by a first actuator  75  for stepwise advance of the probe  3  towards the cutting edge  2  between successive cuts. Second guide rails  76  are mounted on the slide  73  and extend in the vertical direction z which is perpendicular to the first direction x and the second direction y. A second slide  77  is slidably guided in the rails  76 . The movement of the second slide  77  is controlled by a second actuator  78  which controls the vertical cutting speed v c  of the probe  3  relative to the cutting edge  2 . 
   A base  79  of a chuck  80  is mounted to the slide  77  by means of a second leaf spring  81 . The base  79 , spring  81  and slide  77  are again shown as manufactured from a single metal block. The plane of the spring  81  is horizontal, i.e., parallel to the cutting edge  2  and perpendicular to the plane of the spring  20 . The chuck  80  clamps the probe or sample  3 . A second vibrator  82  is mounted on the chuck  80 . It consists of a piezoelectric transducer  83 , which is bonded with one face end to the chuck  80 , and a counter mass  84  which is bonded to the opposite face end of the transducer  83 . 
   In operation, the chuck  80  and therewith the probe  3  is advanced vertically by the actuator  78  with a constant cutting speed v c  for cutting. A vertical oscillation by the vibrator  82  is superimposed on the cutting speed v c  with an amplitude b o  and a frequency 2ω which is twice the oscillating frequency of the vibrator  30 . The oscillating movement is b o  cos(2ωt−π/2) and the oscillating speed v v  is −2b o ω sin(2ωt−π/2). The total vertical speed v p  of the probe is therefore
 
 v   p   =v   c   +v   v   =v   c −2  b   o ω sin(2ω t −π/2)
 
The vertical amplitude b o  and the frequency ω are now chosen such that
 
2 b   o   ω≧v   c  
 
In this way the actual vertical cutting speed v p  of the probe is zero or negative when the horizontal speed v h  is zero, i.e., when ωt=π/2+n·π where n is an integer number.
 
   In other words, the phase angle, the amplitude b o  of the vertical oscillation and the frequency ω are chosen such that the actual vertical speed v p  of the probe is zero or negative when the horizontal movement of the knife  1  reaches its reversal points. 
   It is also possible to vibrate the probe in a horizontal direction, i.e., at least approximately parallel to the cutting edge of the blade. Preferably, the probe and the blade are vibrated such that when the blade  1  reaches its reversal points, the probe is still moving, preferably at maximum speed and vice versa. Preferably, the probe and the blade are vibrated at the same frequency, but not in the same phase. 
   By vibrating the probe either in a vertical or a horizontal direction, section compression can be completely avoided even in these reversal points. 
   As an example for the vibration in a vertical direction: when the horizontal frequency ω is 2π·16 kHz=10 5 s −1  and the advance speed v c =2 mm·s −1  then the vertical amplitude b o  would have to be at least 10 nm. The horizontal amplitude a o  is again considerably less than 1 μm. In the above example, with the requirement that tan ∝≦0.1 the horizontal amplitude a o  of the knife  1  would have to be at least 200 nm (a o ω≦20 mm/s). With lower cutting speeds v c , the amplitudes a o  and b o  can be reduced accordingly. 
   Of course, the relative movement between the knife  1  and the probe  3  can be achieved in different ways than the one specifically shown in FIG.  6 ., e.g., the slide  73  and/or the slide  77  could be associated with the holder  19  instead of with the chuck  80 , or the horizontal and vertical vibrations could be reversed, i.e., that the knife  1  oscillates vertically and the chuck  80  horizontally, or both vibrations could be imparted on the same elements, knife  1  or chuck  80 .