Microtome Having Electro-mechanical Knife Controlling Means

Abstract

An apparatus for cutting microtome specimen sections employs electro-mechanical transducer means connected to the specimen holder, or the knife, to generate an electrical signal during the cutting which indicates the uniformity, or any variations in the uniformity of the thickness of the section.

Description

The present invention refers to a method for indicating the thickness and its variations of sections cut in a microtome from a specimen block. The invention also refers to a method for generating variations of the thickness of a section cut from a specimen in a microtome.

When cutting ultrathin sections (<1.000 A) to be used for studies in an electron microscope it sometimes turns out from these studies that the sections are provided with disturbing parallel lines formed by variations of the thickness of the section. These lines might be obtained even if external vibration sources are eliminated, and the variations are thus a product of the sectioning itself. The lines, which are perpendicular to the cutting direction are usually called "chatter." The distance between the lines is as a rule in the order of 5.000 A and they are thus not possible to detect in a light microscope but are discovered only when the section is studied in the electron microscope. The process of eliminating these lines, for instance by varying the angle between the knife edge and the specimen is thus very time consuming.

It is an object of the present invention to provide a method by means of which the generation of chatter could be indicated at the sectioning whereby it will be possible to carry out the necessary adjustments to eliminate the variations of thickness without studying the sections in an electron microscope. The characteristics of the method will appear from the characterizing part of claim 1.

Another problem within the ultramicrotomy consists in determining the distance between different elements in a section when studied in the electron microscope. This determination of distances within the section have hitherto generally been made by distributing small latex balls of well defined diameter over the section so as to obtain a surface scale reference. The drawback of this method consists therein that the structure of the section is often compressed in the direction of cutting and thus the surface scale reference will refer to the compressed section and no exact information will be obtained as to the original distances in the structure of the specimen. It is another object of the present invention to provide a method for performing a distance determination referring to the original specimen. The characteristics of this method will appear from the characterizing part of claim 3.

The invention will now be explained in detail, reference being made to the enclosed drawing in which:

FIG. 1 shows an embodiment of an apparatus for carrying out the method according to the invention and

FIGS. 2 and 3 illustrate the indicating signals obtained from cutting with and without chatter respectively.

Referring now to FIG. 1 reference SH denotes the specimen arm in a microtome, the specimen being provided with a specimen block S. When cutting the specimen block, which for instance might consist of an organic specimen embedded in plastics, the specimen arm is brought downwards in the direction of the arrow towards a knife K, which cuts a section from the block. The means used for feeding the specimen arm and for handling the sections are well known per se and are not shown in FIG. 1. The specimen block arm is further provided with two piezoelectrical sensors P1 and P2 respectively which form part of the specimen arm. When the specimen arm at sectioning is brought downwards towards the knife, the vertical forces from the knife will then give rise to a tractive force on the lower sensor and a compressive force on the upper sensor. The sum of the tractive force on the lower sensor and the compressive force on the upper sensor will then constitute a measure of the transversal force which effects the specimen. The outputs of the electrical sensors are then connected in opposition so as to obtain a voltage between the inputs I1 and I2 respectively of an operational amplifier A corresponding to this force. The amplifier A is an operational amplifier having a high input impedance and a high negative internal amplification. The amplifier is further provided with a feed back path including a capacitor C connected between one output U1 of the amplifier and the input I1. The remaining input and output terminals I2 and U2 respectively are connected to ground.

The hitherto described apparatus works as follows, when the sensors are subject to a tractive or compressive force, a charge Q will be generated in the sensor and transferred to the capacitor C. The voltage across the capacitor will then be =Q/C and this voltage will also be obtained between the output connections U1 and U2 since the feed back path of an amplifier having the above defined properties will imply that the two inputs will be kept at substantially the same potential.

The piezoelectric material of the sensors is choosen so as to make the charge Q as big as possible for a given force. The advantage of measuring the charge generated from the sensors instead of measuring the generated voltage is that the capacitor C will act as a memory element, i.e., if the sensors are subject to a constant force for a certain period of time, the output signal will be constant during this time. If the output voltages between the terminals of the sensors is measured, the measuring signal will at a constant force decrease due to leak currents. It is further possible to prove that when measuring the charge generated the sensitivity will be independent of the length and cross sectional area of the sensors. The sensors could thus be made thin and have a large cross sectional area which is essential for not effecting the elastic properties of the microtome.

In FIG. 2 there is shown to the left a section S1 which has been cut in the direction of the arrow from the specimen block S in FIG. 1. The thickness of the section is denoted .delta.. In the right of the figure there is shown the output signal V obtained from the output terminals U1 and U2 during the cutting. This output signal will thus be lineary decreasing during the cutting, due to the decreasing width of the section. Experiment further indicates that the cutting force and thus the amplitude of this output signal will within certain limits be lineary related to the thickness of the section. The apparatus according to FIG. 1 could thus after calibration be used for determining the thickness of the sections.

In FIG. 3 there is shown to the left another section S2 cut from the specimen block, this section being provided with the parallel lines discussed above, i.e., the thickness of the section varies along the direction of cutting as indicated on the section surface. To the right in FIG. 3 there is shown the corresponding output signal from the apparatus according to FIG. 1. As appears from this diagram the output signal will vary as the thickness of the section varies and one could thus detect if the section is provided with chatter. Adjustments to eliminate the chatter, for instance variation of the knife angle .alpha. could thus be made without studying the section in an electron microscope.

By using the method according to the invention one could thus by measuring the cutting forces determine the thickness of the section as well as variations of this thickness along the cutting circuit. Provisions could then be made immediately to eliminate possible defects which means that time could be saved and the risk of consuming valuable specimens without obtaining any sections useable for electron microscopy studies is eliminated.

The apparatus in FIG. 1 could also be used for generating variations of the thickness of the sections to be used for the scale reference determination as discussed above. If namely both sensors P1 and P2 are connected in series and an alternating voltage of determined frequency is supplied to the terminals I1 and I2 one will obtain variations of the thickness of the sections for instance as shown in FIG. 3. If the frequency of the alternating voltage and the vertical velocity of the arm SH are known one will obtain parallel lines having a well defined distance. Such a section could then be used as a scale reference for subsequent sections in the electron microscope. One will thus obtain an automatic compensation for the compression in the cutting direction which is normally obtained in the sections, as the distance between the lines will be compressed to a corresponding extent. The scale reference will thus be related to the specimen before cutting. It should also be noted that the method is simpler and less expensive than the scale determination using latex balls described above.

It should also be noted that the cutting force determining apparatus according to FIG. 1 also could be used for measuring such variations of the cutting forces which derive from other defects of the cutting process, e.g., due to defects in the embedding of the specimen in the specimen block.

Claims

We claim:

1. In a microtome means of the type comprising two assemblies, and means for moving one of said assemblies with respect to the other of said two assemblies, one of said assemblies including support means for a specimen to be sectioned, the other of the assemblies including support means for a knife for sectioning said specimen, one of said support means including two piezoelectric crystals, one of the crystals being mounted to absorb compressive force during said relative movement, the other of said crystals being mounted to absorb tractive force during said relative movement, electrical current integrating means and circuit means connecting said piezoelectric crystals with said integrating means in electrical opposition to each other, said integrating means generating a signal indicating the total charge generated by said crystals resulting from said relative movement while sectioning a specimen.

2. The invention defined in claim 1, wherein said support means including said piezoelectric crystals includes a specimen arm for supporting a specimen block.

3. The invention defined in claim 1, wherein said electrical current integrating means comprises amplifier means having a high negative internal amplification.

4. The invention defined in claim 3, wherein said electrical current integrating means also includes capacitor means connected between the input of said amplifier means.