Demonstration of flat-top beam illumination in widefield multiphoton microscopy.

Multiphoton microscopy provides a suitable technique for imaging biological tissues with submicrometer resolution. Usually a Gaussian beam (GB) is used for illumination, leading to a reduced power efficiency in the multiphoton response and vignetting for a square-shaped imaging area. A flat-top beam (FTB) provides a uniform spatial intensity distribution that equalizes the probability of a multiphoton effect across the imaging area. We employ a customized widefield multiphoton microscope to compare the performance of a square-shaped FTB illumination with that based on using a GB, for both two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The variation in signal-to-noise ratio across TPF images of fluorescent dyes spans ∼5.6 dB for the GB and ∼1.2 dB for the FTB illumination, respectively. For the GB modality, TPF images of mouse colon and Convallaria root, and SHG images of chicken tendon and human breast biopsy tissue showcase ∼20 % area that are not imaged due to either insufficient or lack of illumination. For quantitative analysis that depends on the illuminated area, this effect can potentially lead to inaccuracies. This work emphasizes the applicability of FTB illumination to multiphoton applications.

Relationships between intra-specific variation in seed size and recruitment in four species in two contrasting habitats.

Large seeds contain more stored resources, and seedlings germinating from large seeds generally cope better with environmental stresses such as shading, competition and thick litter layers, than seedlings germinating from small seeds. A pattern with small-seeded species being associated with open habitats and large-seeded species being associated with closed (shaded) habitats has been suggested and supported by comparative studies. However, few studies have assessed the intra-specific relationship between seed size and recruitment, comparing plant communities differing in canopy cover. Here, seeds from four plant species commonly occurring in ecotones between open and closed habitats (Convallaria majalis, Frangula alnus, Prunus padus and Prunus spinosa) were weighed and sown individually (3200 seeds per species) in open and closed-canopy sites, and seedling emergence and survival recorded over 3 years. Our results show a generally positive, albeit weak, relationship between seed size and recruitment. In only one of the species, C. majalis, was there an association between closed canopy habitat and a positive seed size effect on recruitment. We conclude that there is a weak selection gradient favouring larger seeds, but that this selection gradient is not clearly related to habitat.

Confocal laser scanning microscopy using a frequency doubled vertical external cavity surface emitting laser.

We report on a frequency doubled 980 nm vertical external cavity surface emitting laser for applications in confocal laser scanning microscopy. The beam quality, wavelength flexibility, and low noise characteristics of this compact source make this prolific imaging technique an exemplary tool. Single pass frequency doubling via KNbO(3) was demonstrated, yielding 1.8 mW at 490 nm with a near diffraction limited beam quality. Detailed analysis and comparison of the laser performance with the current standard argon ion laser revealed clear advantages of the solid-state source for confocal imaging. Imaging of fluorescein and eGFP labeled biological samples using the attenuated solid-state source provided high-resolution images at lower cost and with improved reliability.