INTERFERENCE MICROSCOPY 



For this application low powers are desir- object, such as a cell, by measurement of its 



able for two reasons. First, it is sometimes area and optical thickness. 



not easy to make the film of constant thick- Assume that the cell is a parallel-sided 



ness right up to its edge, so the distance of disc of thickness t and area A , its refractive 



shear is required to be larger than the width index being Mc , immersed in water of re- 



of the imperfection ; the required large shears fractive index /x„ . Its optical thickness is 



are more easily obtained in low-power in- then 



struments. Secondly, parasitic effects, such _ , _ . 

 as aberrations of the rear focal planes of 



objectives, are usually less pronounced in The cjuantity Mc — Atir is equal to 100 ac, 

 low-powered optics so these give mterference where c is the concentration of dry substance 

 effects of higher contrast. in cell and a is the quantity known as spe- 

 Polarizing instruments such as the shear- cific refraction. The total weight M of dry 

 ing microscopes of Smith and Frangon de- material in the cell is equal to cAt, and from 

 scribed above can be modified in an obvious equation (3) can be given as 

 manner for opaque specimens and are par- m = 4 /inn 

 ticularly suitable for this type of measure- 

 ment. By suppressing the image altogether The value of a is remarkably constant for 

 and virtually converting the microscope into the types of substance which are found in 

 a polarimeter, Dyson (13) has shown that cells, and so from equation (4) a good ap- 

 it is possible to make settings with an error proximation to the dry weight can be ob- 

 not greater than one Angstrom unit of tained in spite of the fact that the cell con- 

 thickness, tents constitute a heterogeneous mixture. 



It is evident from this review that the As the cell has not in fact the elementary 

 situation is less satisfactory for opaque ob- shape suggested above it is necessary to re- 

 jects than for transparent objects. No in- place Ap in equation (4) by an integral of 

 strument is available for opaque objects path difference over the cell surface. This 

 which allows the use of a full range of powers integral can be evaluated by a number of 

 onany type of object and also makes possible methods. The path difference can be meas- 

 the precise measurement of path difference, ured at a number of equally spaced points, 



using a graticule in the eyepiece, or a photo- 

 Applications of Interference Microscopy graph can be taken. By setting the back- 

 Biological Applications. The inter- ^^'^^"^^ P^^^ difference approximately mid- 

 ference method is useful for qualitative ob- '^^^ between the values for maximum and 

 servations in biology because of the freedom "i^^i^^^"^^ brightness, the mtensity can be 

 from optical artefacts which it gives. Thus, a TJ^^ very nearly a hnear function of path 



oi^^r • +• £ 4.- 1 i.1,- 1 dmerence for values of the latter small 



slow variation of optical thickness across a . , , ,, , , , ,•«. 



1, 1, ,1, .• 1 , against a wavelength; hence the path difier- 



cell would probably pass unnoticed when u r i i_ i -x ^^ xi 



, 111 , , ■. . ^n(?6 can be found by densitometry on the 



observed by phase contrast methods, but is . • •,, • , j.- r 



,. , . , ' negative, with appropriate corrections for 



immediately evident by interference con- ^^^ ^^^^^^^^^ characteristics. 



Methods are also available, due to Davies 



A great impetus to the use of interference and Deeley (16) and Mitchison et al. (17), 



microscopy was given, however, by Davies whereby the dry mass can be evaluated im- 



and Wilkins (14) and Barer (15), who dis- mediately by photo-electric means at the 



covered independently the possibility of microscope without the necessity of taking 



measuring the dry weight of a biological photographs. 



418 



