Design of an aberration corrected low-voltage SEM.

The low-voltage foil corrector is a novel type of foil aberration corrector that can correct for both the spherical and chromatic aberration simultaneously. In order to give a realistic example of the capabilities of this corrector, a design for a low-voltage scanning electron microscope with the low-voltage foil corrector is presented. A fully electrostatic column has been designed and characterised by using aberration integrals and ray tracing calculations. The amount of aberration correction can be adjusted relatively easy. The third order spherical and the first order chromatic aberration can be completely cancelled. In the zero current limit, a FW50 probe size of 1.0 nm at 1 kV can be obtained. This probe size is mainly limited by diffraction and by the fifth order spherical aberration.

Low-energy foil aberration corrector.

A spherical and chromatic aberration corrector for electron microscopes is proposed, consisting of a thin foil sandwiched between two apertures. The electrons are retarded at the foil to almost zero energy, so that they can travel ballistically through the foil. It is shown that such a low-voltage corrector has a negative spherical aberration for not too large distances between aperture and foil, as well as a negative chromatic aberration. For various distances the third- and fifth-order spherical aberration coefficients and the first- and second-order chromatic aberration coefficients are calculated using ray tracing. Provided that the foils have sufficient electron transmission the corrector is able to correct the third-order spherical aberration and the first-order chromatic aberration of a typical low-voltage scanning electron microscope. Preliminary results show that the fifth-order spherical aberration and the second-order chromatic aberration can be kept sufficiently low.

VH gene usage in humans: biased usage of the VH6 gene in immature B lymphoid cells.

Preferential usage of JH-proximal VH genes has been demonstrated in immature murine B cell repertoires. To determine whether this phenomenon is also evident in human repertoires, we studied utilization of VH6, the most JH-proximal human VH gene. Examination of VH gene usage in a panel of precursor B cell acute lymphoblastic leukemia samples indicated that 15% of the IgH rearrangements utilized VH6. VH6 is a single-member family in a total repertoire of 100-200 VH genes; thus, if usage were purely random, one would expect VH6 rearrangement frequency to be less than 1%. Analysis of VH gene usage in normal lymphoid tissues also revealed biased usage of VH6. VH6 was preferentially utilized in 16- to 24-week-old fetal liver as compared to adult peripheral blood mononuclear cells or spleen. Possible implications of the conservation of preferential usage of JH-proximal genes in both immature murine and human repertoires are discussed.

Biased expression of JH-proximal VH genes occurs in the newly generated repertoire of neonatal and adult mice.

We have previously demonstrated a dramatic preference for utilization of the most JH-proximal VH gene segments in the newborn liver versus adult spleen. We now examine in detail the relative expression of different VH gene families throughout ontogeny and in immunodeficient mice to gain insight into factors that cause the shift in VH usage. We find that the relative expression of VH gene families remains constant and biased throughout fetal and neonatal liver development. In addition, the primary VH repertoire expressed in neonatal spleen displays a similarly biased, position-dependent VH repertoire. The pattern of VH gene expression begins to change at 5-7 d postnatally and reaches the adult randomized pattern at approximately 2 wk of age. We also find biased expression of JH-proximal VH gene families in adult bone marrow and in spleens of adult leaky scid mice, suggesting that the spontaneously generated repertoire of adult mice is similar to that observed in neonates. Together, these data suggest that a position-dependent repertoire is generated in differentiating pre-B cells at all stages of ontogeny, at least in part, as a result of preferential rearrangement of proximal VH gene segments. Therefore, mechanisms subsequent to V gene rearrangement, such as regulatory interactions and antigen selection, must play a major role in normalizing the repertoire.