Methods of research of structure and functions of biological membranes

To observe structure of a membrane in a usual optical microscope it is impossible. That it to understand, we shall recollect, that is permitting limit of device Z. This minimal distance between two points, which images else can be seen separate. It is natural, that the it is less Z, the more qualitatively the device, as allows seeing smaller structures. For a rough estimate of the permitting ability of a n optical microscope we use parity: .

Instead of let's substitute in the formula the minimal value of length of a wave of visible light( ), we shall receive for a permitting limit Z=200 nanometer. This size approximately in 20 times more thickness of a membrane, therefore about its supervision in an optical microscope cannot be speeches. But use of a microprojection and a microphoto is possible. Formation of the microscopic image occurs to participation of the person and comes to the end with formation of the valid image in an eye. The usual microscope does not create the valid image, however for photographing (microphoto) or projections of the microscopic image to the screen (microprojection) the valid image should be received. For this purpose the image given by objective Ob, it is necessary to arrange further of focal length of eyepiece Ep (fig.1).

Method of a dark field.

T he most widespread reception of optical microscopy is supervision of the fixed and painted preparations in the passing light, named by a method of a light field (see fig. 2 a).

Methods of microscopy of unstable and unpainted objects enter into practice recently. Supervision of such objects in passing light does not give desirable results owing to absence of contrast between elements of structure of object, and also between object and an environment. In these cases the method of supervision in a dark field (see fig. 2, b) is used. For this purpose special set of lenses in a usual biological microscope it is used. Set of lenses a dark field consists of the several lenses of the special form, forming inclined bunches of light which shine a preparation. Light falls on small elements of structure of object, dissipates on them and partially gets in an objective. It does an objective visible on the general dark field of vision of a microscope.

Phase - contrast method.

The method is applied for supervision of non contrast objects; it is based on use of a difference of phases which is formed at passage of light through various structures of investigated object. Intensity of the light wave, which are passing through transparent object, does not change almost, but phases change. These changes depend upon thickness of object and its parameter of refraction. Transparent objects is called out-phasing. Thus to see details of such objects it is impossible. In biophysical researches such objects are necessary for painting, however thus their properties and viability can change. The transparent environment is established on a way of a light bunch. This environment there is a transparent inclusion, for example, a bacterium (fig. 3). The passing bunch of light will be divided on two parts, the first part will pass through transparent object and by a lens will be focused on site Ф of focal plane F (see fig. 4 a, a line 1).

Other part will do diffraction on heterogeneity of object and will gather by lens in a point A of plane I (see fig. 4 b, a line 2). The curve 3 grows out diffraction of light on a bacterium. The difference of phases of curves arises because of different parameters of refraction of environments.

The eye in plane I does not distinguish a wave 1 and 2 as their intensity identical, and eyes does not react on distinction of phases. It is necessary to transform a phase difference in amplitude difference. For this purpose in plane F it is necessary to put the small round phase plate absorbing a wave 1, in this case contrast of a bacterium it will be strengthened.

Phase-contrast devices (a plate, sate of lenses) are additional devices to a microscope.