ABSTRACT
The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally-distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely-overlapped fluorophores in samples containing ten different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin with up to eight different cell types or tissue structures distinguished.
ABSTRACT
BACKGROUND: To determine the effect of hydration as well as prone versus supine positioning on the pelvic veins during cardiovascular magnetic resonance (CMR) venography. METHODS: Under institutional review board approval, 8 healthy subjects were imaged with balanced steady state free precession, non-contrast CMR venography to measure common and external iliac vein volumes and common femoral vein cross-sectional area in the supine, prone and decubitus positions after dehydration and again following re-hydration. CMR venography from 23 patients imaged both supine and prone were retrospectively reviewed and measurements of common femoral and iliac veins areas were compared using Wilcoxon test. RESULTS: Common femoral vein area on CMR venography increased with prone positioning (83 ± 35 mm2) compared to supine positioning (59 ± 21 mm2) (p = 0.02) and further increased with hydration to 123 ± 44 mm2 (p < 0.01). With right and left side down decubitus positioning, the common femoral vein area on dehydration increased from 29 ± 17 mm2 in the ante-dependent position to 134 ± 36 mm2 in the dependent position (p < 0. 001). Similarly, common and external iliac veins increased in volume with prone, 5.4 ± 1.9 cm3 and 5.8 ± 1.9 cm3 compared to supine positioning 4.6 ± 1.8 cm3 and 4.5 ± 1.9 cm3 (p = 0.01) and further increase with hydration to 6.7 ± 2.1 cm3 and 6.3 ± 1.9 cm3 (p = 0.01). CMR venography on patients also demonstrated an increase in mean common femoral vein luminal area from 103 ± 44 mm2 in supine position to 151 ± 52 mm2 with prone positioning (p < 0.001) as well as increases in common and external iliac vein volumes from 6.5 ± 2.6 cm3 and 8.0 ± 3.4 cm3 in the supine position to 7.5 ± 2.5 cm3 and 9.3 ± 3.6 cm3 with prone positioning (p < 0.01). CONCLUSIONS: Common femoral and common/external iliac vein size on CMR venography may be affected by position and hydration status. Routine clinical CMR venography of the pelvis could include prone positioning and avoiding dehydration to maximize pelvic vein distension.
