Use of atomic force microscopy to reveal sperm ultrastructure in HIV-patients on highly active antiretroviral therapy.

Atomic force microscopy (AFM) has been employed to examine morphological and topographical changes caused by human immunodeficiency virus (HIV) and the effects of highly active antiretroviral therapy (HAART) on spermatozoon of HIV infected patients. This powerful technique has allowed us to visualize morphological alterations present in the spermatozoa of patients either with or without treatment. In addition to this, even the minute details, such as viral particles, located on the membrane of the spermatozoa, and the merging of such particles on the surface of the spermatozoa were detected with precision. The most important aspect is that AFM, unlike electron microscopy, permits to image virions in their nearly natural environment. Excess of damage of spermatozoon is due to the chemicals involved in HAART rather than the damage made by virus.

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope.

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 microm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique.

Ultrastructural pathology of varicocele spermatozoa by using atomic force microscopy (AFM).

Ultrastructural investigation of pathological spermatozoa of patients having varicocele (grades I and III) in its natural environment (without fixation, dehydration, embedding, sectioning, etc.) was carried out by using Atomic Force Microscope (AFM) in tapping mode. This is a unique technique to examine the topographical structure with precision and to obtain 3-D images. It revealed structural alterations caused by the presence of varicocele. Not only neckpieces and flagella were defective, but also the head; particularly, dimensions and heights are altered.

Effect of an electric field on the motility of Entamoeba histolytica examined by multiple-beam interference microscopy.

A recently developed multiple-beam interference microscopic technique has been used to visualize submicroscopic structures of Entamoeba histolytica and their movements in applied external electric fields. The movements were videorecorded and it was found that at low current (120 microA) pseudopods are filled with hyaline ectoplasm. At slightly higher current (about 150 microA), the amoeba stops extending the pseudopods and loosens its attachment to the surface. At higher currents (200 microA), it forms a cyst and remains immobile for a time. Before this stage is reached a narrow ring is formed around the nucleus due to alterations in the proteins to protect it.

Ultrastructural investigation of human sperm using atomic force microscopy.

Ultrastructural investigation of human sperm in its natural environment (without fixation, dehydration, embedding, sectioning, etc.) was carried out by using atomic force microscope (AFM) in its tapping mode. This technique permits the examination of fine structural details of undamaged sperm and its topography with precision. Moreover, it allows 3D reconstruction of images and enhances the contrast to resolve details such as mitochondria that surround the axoneme at the sperm middle piece. An organized structure has been found in the flageller axoneme region. Ultrastructure also reveals folding and details of the depression of the membrane that cannot be examined with conventional techniques.