A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.

Germline stem cells and follicular renewal in the postnatal mammalian ovary

We show that standard silicon nitride cantilevers can be used for tapping mode atomic force microscopy (AFM) in air, provided that the energy of the oscillating cantilever is sufficiently high to overcome the adhesion of the water layer. The same cantilevers are successfully used for tapping mode AFM in liquid. Acoustic modes in the liquid excite the cantilever. o­n soft samples, e.g., biological material, this tapping mode AFM is much more gentle than the regular contact mode AFM. Not o­nly is the destructive influence of the lateral forces minimized, but more important, the intrinsic viscoelastic properties of the sample itself are effectively used to ''harden'' the soft sample.

/pdf/tapping-mode-atomic-force-microscopy-in-liquid-58inpzczst.pdf

Tapping mode atomic force microscopy in liquid

In photosynthesis, the harvesting of solar energy and its subsequent conversion into a stable charge separation are dependent upon an interconnected macromolecular network of membraneassociated chlorophyll–protein complexes. Although the detailed structure of each complex has been determined1–4, the size and organization of this network are unknown. Here we show the use of atomic force microscopy to directly reveal a native bacterial photosynthetic membrane. This first view of any multi-component membrane shows the relative positions and associations of the photosynthetic complexes and reveals crucial new features of the organization of the network: we found that the membrane is divided into specialized domains each with a different network organization and in which one type of complex predominates. Two types of organization were found
for the peripheral light-harvesting LH2 complex. In the first, groups of 10–20 molecules of LH2 form light-capture domains that interconnect linear arrays of dimers of core reaction centre (RC)–light-harvesting 1 (RC–LH1–PufX) complexes; in the second they were found outside these arrays in larger clusters. The LH1 complex is ideally positioned to function as an energy collection hub, temporarily storing it before transfer to the RC where photochemistry occurs: the elegant economy of the photosynthetic membrane is demonstrated by the close packing of these linear arrays, which are often only separated by narrow ‘energy conduits’ of LH2 just two or three complexes wide.

/pdf/the-native-architecture-of-a-photosynthetic-membrane-52xgq8kwck.pdf

The native architecture of a photosynthetic membrane.

A novel method based on AFM was used to attach individual collagen fibrils between a glass surface and the AFM tip, to allow force spectroscopy studies of these. The fibrils were deposited on glass substrates that are partly coated with Teflon AF®. A modified AFM tip was used to accurately deposit epoxy glue droplets on either end of the collagen fibril that cross the glass-Teflon AF® interface, as to such attach it with one end to the glass and the other end to the AFM tip. Single collagen fibrils have been mechanically tested in ambient conditions and were found to behave reversibly up to stresses of 90 MPa. Within this regime a Young's modulus of 2-7 GPa was obtained. In aqueous media, the collagen fibrils could be tested reversibly up to about 15 MPa, revealing Young's moduli ranging from 0.2 to at most 0.8 GPa.

Micromechanical Testing of Individual Collagen Fibrils

Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy.

A stand-alone atomic force microscope (AFM) featuring large scan, friction measurement, atomic resolution, and liquid operation, has been developed. Cantilever displacements are measured using the optical beam deflection method. The laser diode and focusing lens are positioned inside the piezo tube and the cantilever at the end of the piezo tube. Because the laser beam stays o­n the cantilever during scanning, the scan range is solely determined by the characteristics of the piezo tube. In our case 30 x 30 x 9.5 mum3 (xyz). The optical beam deflection detection method allows simultaneous measurement of height displacements and torsion (induced by lateral forces) of the cantilever. AFM images of dried lymphocytes reveal features in the torsion images, which are o­nly faintly visible in the normal height images. A new way of detecting the nonlinear behavior of the piezo tube is described. With this information the piezo scan is linearized. The nonlinearity in a 30-mum scan is reduced from 40% to about 1%, as is illustrated with images of a compact disk. The stand-alone AFM can be combined with a (confocal) inverted microscope, yielding a versatile setup for biological applications.

/pdf/compact-stand-alone-atomic-force-microscope-1efplocrf9.pdf

Compact stand‐alone atomic force microscope

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Compact stand‐alone atomic force microscope