LO-analysis is a means to elucidate the details of sexine pattern on the basis of light microscopic observations of a pollen grain and spore at different adjustments of the microscope.
The ultra thin section (0.1-0.5 micron thick) and optical section of a pollen grain provide details of sexine. The term optical section refers to the view of a pollen grain and spore under light microscope when the focal plane is halfway through the grain.
In optical section thickness, structure, layers and ornamentation etc. of a grain wall are visible. Sometimes optical section does not make the fine structures of exine as clear as is expected. The advantage of LO- analysis is that the details of sexine can be established with full clarity in un-sectioned pollen and spore.
Erdtman (1952) illustrated LO-analysis where ‘L’ is derived from the Latin word lux meaning light and ‘O’ is derived from the Latin word obscuritas meaning darkness. In sexine of angiosperm pollen, three regions are encountered- suprategillar, tegillar and infrategillar (Fig. 4.13). The term tegillum (syn. tectum) denotes the layer of sexine that forms a roof over columella and baculum etc.
For LO-analysis (Fig. 4.14) a pollen grain is focused under light microscope. The objective is raised until the pollen grain is not in the focus. Then the objective is slowly moved down until details are visible. At this high adjustment the suprategillar elements are visible only. The elements appear as light islands. The objective is further racked down and the light islands turn to dark islands.
So the suprategillar elements exhibit LO-pattern, i.e., light islands at first focus become dark when focused at a slightly lower level (Figs. 4.14A & B, 4.16A & B). The light and dark islands reveal the cross sectional view of elements. On analyzing the light and dark islands at different foci the structure of the element can be deduced.
A small light islet in the higher focus when turns to dark islet in the lower focus with greater diameter, it reveals the shape of spinula that has a broad base and pointed apex.
Similarly when a light islet turns dark islet with smaller diameter at lower focus, it reveals the shape of a clava that has a broad apex. If the diameter of light and dark islands remains unchanged at different foci, it reveals the columnar shape of a suprategillar baculum (Fig. 4.15).
It is to note that the same surface pattern in the first focus is produced by different sculpturing types, e.g. gemmate, clavate, baculate and pilate etc. Analysis of observations of second, third and if necessary other lower foci will elucidate the shape of projecting processes. When the objective is at the focusing level of the base of suprategillar elements, it also focuses other structure present on tegillar surface.
The tegillum may be punctate, scrobiculate, reticulate etc. When the objective is focused on the suprategillar side of punctum, scrobiculus, reticulum etc. all appear as dark islands. In lower focus the dark island turns bright. So the hollow structures like punctum, reticulum and scrobiculus etc. exhibit OL-pattern, i.e. dark islands at suprategillar side turn bright on infrategillar side (Figs. 4.16C & D).
The size, shape and diameter etc. of dark and bright islands differentiate between punctum, scrobiculus and reticulum etc. When the objective is moved further down and adjusted at the top level of infrategillar region small white islands appear. These are caused by the bacula standing on nexine. They later become dark at further low adjustments.
In intectate pollen bacula, clava, pila etc. directly stand on nexine. The infrategillar elements exhibit LO-pattern. The Patterns exhibited on the different layers of sexine are referred to as S-, T- and I-pattern where S = suprategillar, T = tegillar and I = infrategillar. It is to note that solid objects like spine, clava and baculum etc. exhibit LO-pattern whereas the hollow structures like punctum, scrobiculus and reticulum etc. reveal OL- pattern.
Transitional forms are observed when the objective is moved to lower focus slowly. Sometimes a supratectal process may be bifurcated at the tip. LO- analysis clearly analyses it (Fig. 4.15). In this case two completely separate light islands are seen at the higher focus.
As the objective is moved slowly towards lower focus it is observed that the two light islands approach to each other and gradually fuse forming a single dark island. Thus any structure on sexine, whether it is a solid process or hollow structure like punctum etc. can be demonstrated with full clarity by LO-analysis.
The S -, T- and I-patterns are depicted in diagrams. This diagram elucidates the sexine pattern of a pollen grain and spore on the basis of LO-analysis and becomes a part of palynogram. A palynogram (Fig. 4.17) is a diagrammatic representation of a pollen grain and spore illustrating the shape, size, aperture, sporoderm stratification, sexine patterns etc.
Pollen grains and spores are acetolysed for LO-analysis. After acetolysis the grains are mounted in glycerine jelly that has refractive index 1.474. This value is lower than the refractive index of sporopollenin in exine. A difference in refractive index eases to detect a solid structure and its surroundings not only under phase contrast microscope but also in light microscope.
For observation in light microscope oil immersion objective greatly aids in the visualization of exine stratification. The observations are clearer in a binocular microscope. A flat or slightly curved surface on apocolpium/mesocolpium/apoporium/mesoporium is convenient for observation (Fig. 4.18).
It is to note that a punctum and a baculum both may appear as dark islands but at different focal planes. So the structure of exine is to be deduced by concentrating attention on a small area of exine and carefully observing the appearance of different images at different focal planes in that area.
Now a days Scan Electron Microscope (SEM) is introduced to analyze the sexine surface pattern and to assemble three-dimensional structures of certain pollen types. Still the light microscopic observations on the basis of LO-analysis are an indispensable tool because SEM images do not provide clearly the structure of endoaperture. The structure can be established with full clarity on the basis of LO-analysis.