CHAPTER I 



Physical Aspects of Image Formation 



in Microscopy 



1. INTRODUCTION 



Microscopic investigation of image formation and interpretation 

 cannot be based solely on considerations of geometrical optics. In 

 order to ascertain the distribution of light-intensities in the images 

 exhibited by a microscope, the laws of physical optics have to be 

 brought into play. 



The first and simplest case to consider is that of an object sufficiently 

 small as to be likened to a pin-point of light. In the image, the light 

 is distributed according to a law determined by diffraction. This image 

 is the elementary diffraction pattern known as Airy's disk: it is the 

 perfect image that would be exhibited by an aberration-free and 

 perfectly focused microscope. 



Images of large objects are all derived from the more or less complex 

 process of such an elementary picture since any object may be split 

 into elements small enough to be considered as discrete pin-point 

 sources. Therefore, under such conditions, it can be anticipated that 

 diffraction effects shall be the more prominent as the dimensions of 

 the object are closer to those of Airy's disk. The finer details of 

 a specimen are those which are the more markedly altered by diffraction. 

 Since, naturally, it is such details that are the most valuable to micro- 

 scopists, it is essential to determine the results given by the diffraction 

 theory in order to reduce interpretation errors to a bare minimum. 



However, in microscopy, the problem is rather the converse: it is 

 not a given image which needs ascertaining: it is the actual structure 

 of the object corresponding to it. No difficulty is involved when the 

 size of the object is large in relation to the diffraction pattern as the 

 geometrical aspect of the image prevails. Such is not the case when 

 the size of the object approximates that of Airy's disk. Then a com- 



