A femtosecond Raman generator for long wavelength two-photon and third harmonic generation imaging

We demonstrate a femtosecond single pass Raman generator based on an YVO crystal pumped by a high energy fiber laser at a wavelength of 1064 nm and a repetition rate of 1 MHz. The Raman generator shifts the pump wavelength to 1175 nm, in a broad band spectrum, making it suitable for multi-photon microscopy. We use the Raman generator for third harmonic generation imaging of live plant specimens as well as for two-photon fluorescence imaging of red fluorescent protein (RFP) expressing HeLa cells. We demonstrate that the photo-damage to a live specimen is low

Small average differences in attenuation corrected images between men and women in myocardial perfusion scintigraphy: a novel normal stress database

<p>Abstract</p> <p>Background</p> <p>The American Society of Nuclear Cardiology and the Society of Nuclear Medicine state that incorporation of attenuation-corrected (AC) images in myocardial perfusion scintigraphy (MPS) will improve image quality, interpretive certainty, and diagnostic accuracy. However, commonly used software packages for MPS usually include normal stress databases for non-attenuation corrected (NC) images but not for attenuation-corrected (AC) images. The aim of the study was to develop and compare different normal stress databases for MPS in relation to NC vs. AC images, male vs. female gender, and presence vs. absence of obesity. The principal hypothesis was that differences in mean count values between men and women would be smaller with AC than NC images, thereby allowing for construction and use of gender-independent AC stress database.</p> <p>Methods</p> <p>Normal stress perfusion databases were developed with data from 126 male and 205 female patients with normal MPS. The following comparisons were performed for all patients and separately for normal weight vs. obese patients: men vs. women for AC; men vs. women for NC; AC vs. NC for men; and AC vs. NC for women.</p> <p>Results</p> <p>When comparing AC for men vs. women, only minor differences in mean count values were observed, and there were no differences for normal weight vs. obese patients. For all other analyses major differences were found, particularly for the inferior wall.</p> <p>Conclusions</p> <p>The results support the hypothesis that it is possible to use not only gender independent but also weight independent AC stress databases.</p

Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser

We have studied the wavelength-dependence of the two-photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin- and cellulose- staining dye Calcofluor White and Alexa Fluor 350, in the visible and near-infrared wavelength range (540-800 nm). For several of the dyes we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two-photon microscopy. We also find that although the rate of photo-bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens, to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution based measurements

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic-scale knife-edge in the form of a simple transmission electron microscopy (TEM) grid attached to a glass microscope slide, and a light-collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife-edge at different axial positions relative to the beam waist, the divergence and astigmatism of the post-objective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity, and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multi-photon laser scanning microscopy. Our knife-edge method has several advantages over alternative knife-edge methods used in microscopy including that the knifeedge is easy to prepare, that the beam can be characterized also directly under a coverslip, as necessary to reduce spherical aberrations for objectives designed to be used with a coverslip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited