Method for automatically matching serial cross-sections observed with a microscope

Three holes in known relative positions are made in a sample to be examined. After the sample has been serially cross-sectioned, the sections are imaged. Movement and deformation of each section is determined by comparing the positions of the holes in the sections to that of the original known relative positions. Based on the changes, an inverse transformation is determined and applied to the section image. The transformed images then provide serial cross sectional images of the sample without the degradation produced by the sectioning and imaging process.

BACKGROUND OF THE INVENTION 
This invention concerns a process that makes it possible to automate the 
calculation of deformations that make possible the matching of successive 
sections of microscopy, electronic transmission microscopy and optical 
microscopy. 
The matching of successive sections is traditionally done by moving images 
according to the operator's judgment. This method does not make it 
possible to correct the deformations that the mechanical action of the 
cutting introduces to the thin sections. 
Some deformations are related to the techniques used to make it possible to 
visualize successive sections in microscopy. These include 
Translation and rotation related to: 
placement of the sections one after the other within the grids or 
supporting blades which cannot be reproduced exactly, and 
deformations related to the mechanical actions of the microtome or the 
ultramicrotome. 
Also included is 
Systematic rotation related to different powers of enlargement (in the case 
of an electron microscope in transmission mode). 
SUMMARY OF THE INVENTION 
The process of the invention makes it possible to correct all these 
deformations by calculating an inverse transformation function and 
establishing a fixed marker derived from markings previously obtained 
mechanically or by firing a pulsed laser. 
The process according to the invention takes place after the histological 
preparation stage, prior to observation with the optical microscope or the 
electron transmission microscope.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The process according to the invention includes several steps: (See FIG. 1) 
1. "Marking" 
The process according to the invention includes a sample preparation step 
before a series of sections is cut (see FIG. 2). The microscopic 
preparation 1 is pierced by at least three holes 2 not in a straight line 
near area to be studied and made either mechanically or by means of a 
pulsed laser. The barycenters of these holes 2 will be used as marking 
points to establish a fixed marker and for final calculation of the 
inverse transformation function. 
2. "Section in Series" 
The process according to the invention takes the classic step of making 
successive sections 4 of the preparation 1 using a microtome for the 
sections to be viewed by optical microscopy and an ultramicrotome for 
sections to be viewed with an electron microscope (see FIG. 3). 
3. Digitation 
The process according to the invention includes observation steps with the 
optical microscope or with the electron transmission microscope, as well 
as the acquisition and digitation of images by a computer system. This 
computerized system includes a camera and an image-digitation system 
connected to a computer. Three images are acquired for an image to be 
studied (see FIG. 4): 
Image A: The image that is going to be studied. 
Image B: An image obtained at a weaker magnification, without moving the 
slide 3 making it possible to see at least one of the marking holes 2. 
Image C: An image making it possible to visualize all the marking holes 2, 
at the same magnification as for image B. 
4. "Normalization" 
A normalization step for digital processing of all the images A of the 
sections to be studied, making it possible to obtain homogeneous 
information in terms of contrast and distribution of grey levels. 
The distributions of the grey levels of the images are normalized in terms 
of their average and their variance. 
Enhancement is done by dispersion of the diagram distributions. 
Specific mathematical functions are used for the work on the distributions 
(improvement and stabilization). 
5. "Calculation of the Inverse Transformation" 
The process according to the invention includes a step for calculating the 
inverse transformation function making it possible to re-establish more 
closely from an initial section considered a reference a set marker that 
is fixed for all the sections. This method is as follows: 
5.1 Automatic calculation of the translation between images B and C, which 
includes: 
5.1.1 Automatic detection of holes 2 by image-analysis methods 
(thresholding) on images B and C. 
5.1.2 Calculation of the barycenters of the holes 2 of image C. 
5.1.3 Calculation of the barycenters of the hole or holes 2 of image B. 
5.1.4 Superposition of the corresponding barycenters. 
5.1.5 Calculation of the translation necessary between the two images by 
calculating the distance of the two barycenters on the respective images. 
5.2 Calculation of Rotation and Deformation (See FIG. 5) 
These two functions are to be applied to the image A in relation to the 
corresponding image of the preceding specimen section. They make possible 
virtual automatic recording of the series of images A. 
5.2.1 Calculation of Rotation 
The rotation is calculated by the angular sum of rotation related to the 
functioning of the specific apparatus at each magnification and of the 
rotation related to the random orientation of the section on the slide 3 
of the microscope. The function related to the apparatus is given in 
advance; that of the position of the section is calculated on the basis of 
the orientation of the holes 2 in the image C. 
5.2.2 Calculation of the Deformation 
Knowing the position of the holes 2 before the section and their respective 
distances, calculation of the inverse deformation to be applied to the 
successive images in order to record the holes 2 and consequently the 
images that are coordinated with them uses a matrix model already tested 
in prior phases which up to now remains sufficient, namely: 
an affine transformation of the first order with 6 parameters. 
6. "Recording" 
An image-recording step is obtained by digital processing applying to the 
images A the series of transformation functions as well as the rotations 
and translations calculated by the process in the invention described in 
the preceding step 5. 
The process according to the invention is particularly adapted to the 
reconstruction of the information in three dimensions from successive 
sections 4 observed with the electron transmission microscope or an 
optical microscope. The information can then be used by employing digital 
image-processing techniques. 
The process according to the invention makes it possible to automate the 
search for areas of one section on another within successive sections 
observed with the help of electron transmission microscopes whose 
movements of the microscope stage and control parameters are 
computer-controlled. 
Moreover, the process according to the invention makes is possible to 
automate the search for areas of one section on another within successive 
sections observed with the aid of optical microscopes whose movements of 
microscope stage and control parameters are computer-controlled.