The visibility of particles and fibres when illuminated by means of a ring illuminator, is similar to that obtained with dark-ground illumination, except in one respect. With dark-ground illumina tion the light passes up through the lower layers of the specimen and, except when the specimen is very thin, much light may be sent up into the object-glass from these layers. As a result of this, the visibility of any particles or fibres, or of structure generally, may be impaired since, to some extent, the upper layers of the specimen will be illuminated by transmitted light sent up through them from the underlying portions of the object. With the ring illuminator, much of this diffuse transmitted light is avoided and the visibility of particles or fibres is often better than that obtained with ordinary dark-ground illumination.
Minute particles are often much better revealed when intensely illuminated by ordinary dark-ground methods, or by means of a ring illuminator, than when viewed by transmitted light, just as dust particles in a beam of light are visible to an eye looking side ways into the beam but cannot be seen by looking along the beam towards the source of light. Thin objects, such as bacteria or other minute living organisms, in water, are of ten seen better by means of a lens of moderate numerical aperture, when ordinary dark ground illumination is used, than by an immersion lens of much higher aperture used with transmitted light. It is worth while emphasising how much work lies within the scope of dry-front lenses of numerical apertures of 0.35 to 0.85 if advantage is taken of the high visibility of small particles when intensely illuminated, either by ordinary dark-ground methods or by means of the ring illuminator.
For dark-ground work with object-glasses of numerical aper tures between 0.85 and 1.o, immersion object-glasses give more satisfactory visibility of faint objects than do dry object-glasses of corresponding apertures, as stray light from the cover-glass and front surface of the object-glass is largely avoided. For the purpose of rendering very minute particles visible, specially in tense dark-ground illumination is used in conjunction with an ordinary microscope. This method of studying minute particles is called ultra-microscopy.
of small size have the power of scattering light which falls on them, the intensity of the scattered light being greatest in direc tions at right-angles to the path of the illuminating beam. Ad vantage is taken of this in the ultra-microscope, in which the par ticles to be rendered visible are illuminated by an intense beam of light which is directed through or across the object, in a direc tion at right-angles to the axis of an ordinary microscope. Each particle scatters some of this light, and the scattered light which enters the object-glass from any one particle is brought to focus as a diffraction disk in the image plane. If the dimensions of the particle are smaller than half the wave-length of the light used, no deductions as to the form of the particle can be made from the appearance of the diffraction disk seen in the image plane. Some estimate of the relative sizes of the different particles can be made from the relative brightnesses of the corresponding dif fraction disks, but the diffraction disks will all be of substantially the same size. The size of the diffraction disks becomes smaller and smaller as the numerical aperture of the object-glass used for viewing the particles is increased. Each diffraction disk represents a particle ; the number and positions of the particles can thus be determined provided no two particles are so close together that their diffraction disks are practically coincident in the image plane.
The illumination used is of such intensity that an appreciable amount of light is sent into the microscope from a plain slide of glass, either due to lack of homogeneity in the glass, to imperfec tions of its surface polish, or to fluorescence of the glass. For this reason any specimens which require mounting are usually mounted on slides of polished quartz when they are to be examined by the ultra-microscope. For the examination of fluids the Siedentopf Zgismondy ultra-microscope is largely used, the fluid being con tained in a small chamber fitted with quartz windows, through which a very intense beam of light is passed. The larger the particle and the higher its refractive index relative to that of the surrounding medium, the more readily is the particle seen. With modern methods of illumination, particles of colloidal gold having diameters as small as 1.7 j (17 Angstrom units, or about of the wave-length of blue light) have been seen.