Whole-mount sections displaying microvascular and glandular structures in human uterus using multiphoton excitation microscopy

There has been considerable interest over many years in the precise structural relationships between microvessels and secretory glands in human endometrium. However, microcirculatory networks have rarely been studied in three-dimensions (3D) using modern computerised technologies, this has been partly due to the late arrival of suitable endothelial cell markers. This study was designed to develop a technique to visualize and to reveal the relationships between microvessels, their glandular environment and epithelial boundaries in 3D, using endometrium from human hysterectomy biopsies. Specimens were carefully selected from women with conditions unlikely to affect the microvascular networks. Monoclonal antibodies (mouse anti-human CD 34 and goat anti-mouse fluorescein (FITC)) were used to visualize the microvessels, and polyclonal antibodies (rabbit anti-human keratin and goat anti-rabbit tetramethylrhodamine (TRITC)) were used to visualize the glandular structures. The samples were studied with a Leica multiphoton system using a titanium–sapphire laser (excitation 800 nm with pulses in the 200 fs range) to obtain a stack of two-dimensional (2D) images to a minimal focus depth of 120 μm. The initial data sets acquired were volume rendered using the integrated software of the Leica system to produce 3D images. This software allowed for the acquisition of data sets from the microscope and for an observational morphological assessment to be made, but was limited in preparing the data for any quantitative analysis. The additional use of ImarisBasic 3.1 visualization software allowed for an observational morphological assessment but also included numerous tools for data manipulation.     

Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams

The confocal fluorescence microscope is the instrument of choice for biologists. However, compared to other instruments, its resolution is still limited. We propose a simple technique, based on laterally interfering beams, to improve the resolution. One technique consists in using a halve phase plate to modify the illumination, combined with a laterally offset detection. A 90 nm lateral resolution is obtained for properly prepared specimens using readily available dyes. Another approach is to use several excitation beams, slightly shifted and properly dephased, to decrease the lateral extension of the PSF. With this approach, a lateral resolution of 75 nm is predicted with the advantage of a regular confocal detection. Finally, we show how using these techniques in combination with a two-color two-photon excitation could permit to further improve the resolution to 60 nm.     

A novel excitation scheme to enhance image resolution in dynamic atomic force microscopy

We propose a novel multifrequency excitation technique for the non-contact atomic force microscopy (AFM). The probe is excited at two frequencies that are far from resonances while their subtraction is close to the fundamental frequency. Due to combination resonance occurring in nonlinear systems, the response includes a term with frequency equal to subtraction of excitation frequencies. We suggest to employ this term as the main signal for imaging. It is found that the present excitation improves signal sensitivity to sample topography and increases resolution. This technique is especially convenient for highly-damped environments where non-contact AFMs are very difficult to use.     

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca²⁺; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca²⁺ in biological samples, we demonstrate thatintensity-based measurements of Ca²⁺ with single-wavelength Ca²⁺ probes such as Calcium Crimson may falsely report the actual Ca²⁺ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.