Studying biological tissue with fluorescence lifetime imaging: microscopy, endoscopy, and complex decay profiles.

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.

Carotid artery wall motion estimated from B-mode ultrasound using region tracking and block matching.

The motion of the carotid atheromatous plaque relative to the adjacent wall may be related to the risk of cerebral events. A quantitative method for motion estimation was applied to analyse arterial wall movement from sequences of 2-D B-mode ultrasound (US) images. Image speckle patterns were tracked between successive frames using the correlation coefficient as the matching criterion. The size of the selected region-of-interest (ROI) was shown to affect the motion analysis results; an optimal size of 3.2 x 2.5 mm(2) was suggested for tracking a region at the wall-lumen interface and of 6.3 x 2.5 mm(2) for one within the tissue. The results showed expected cyclical motion in the radial direction and some axial movement of the arterial wall. The method can be used to study further the axial motion of the carotid artery wall and plaque and, thus, provide useful insight into the mechanisms of atherosclerosis.

Sulphorhodamine-B-labelled albumin uptake around the ostium of the renal artery in rabbits: changes with age.

Because protein transport between blood and artery wall is important in atherogenesis, we have measured the uptake of fluorescently labelled albumin around the aorto-renal branch, an important site for lesions. Tracer concentrations in artery wall sections were quantified using digital fluorescence microscopy. Short-term experiments indicated endothelial permeability, while longer ones indicated the steady-state distribution of native proteins within the wall. In sexually immature rabbits (65-75 days), the permeability of the aorta was greater downstream of the renal ostium than upstream (p < 0.004). However, in mature rabbits (120-156 days), the permeability was greater, on average, at the upstream site compared to downstream. This change in pattern of uptake around the renal ostium with age appears to be due to increased uptake at the upstream site in mature animals, which was over fourfold greater than that measured at the equivalent site in immature animals (p < 0.01). In the renal artery itself, permeability was low and age independent. In mature animals, endothelial permeability did not correlate with steady-state wall concentrations, suggesting that the latter may be more dependent upon the rate of exit of protein and/or available space in the wall, rather than the rate of entry. The age-dependent changes in the spatial pattern of permeability correspond to the different distributions of spontaneous lipid accumulation in immature and mature vessels of both rabbits and humans, implying that permeability of the wall is important in atherogenesis.

A ratiometric method of autofluorescence correction used for the quantification of Evans blue dye fluorescence in rabbit arterial tissues.

Evans blue dye (EBD) conjugates with albumin in the circulation and is frequently used to measure vascular protein leakage. The fluorescence of the dye from tissue sections can be used to measure its uptake at very specific anatomical locations, but problems arise with dye quantification because tissue components also fluoresce; so-called autofluorescence. We have measured uptake of EBD by blood vessel walls at various points around the aorto-renal branch of rabbits. High resolution, digitised, fluorescence images of histological sections of artery wall allowed detailed microscopic analysis of EBD accumulation; and a ratiometric method was developed to enable autofluorescence to be separated from EBD fluorescence. When EBD-free tissue sections were illuminated with blue light, the ratio of red to green fluorescence was constant throughout the tissue (0.59 +/- 0.03, mean +/- S.D., n = 32). Therefore, at each individual pixel, the level of red autofluorescence could be determined by multiplying the green intensity at that pixel by the calculated red to green ratio. Since EBD fluorescence was detected only in the red region of the spectrum, intensity values of the dye alone were obtained from EBD-exposed tissue by subtracting the red autofluorescence estimated by this ratiometric method. In such cases the red to green fluorescence ratio was measured from adjacent sites known to be free of EDB (0.59 +/- 0.02, mean +/- S.D., n = 56). We were therefore able to increase the sensitivity of tracer quantification by complete elimination of background autofluorescence on a pixel-by-pixel basis. Use of EBD standards allowed calibration of corrected fluorescence intensities and calculation of mass transfer coefficients for albumin into the artery wall. Spatial variations in the permeability of the artery wall around the renal ostium were detected with the present high resolution technique, with an average mass transfer coefficient of (6.8 +/- 0.9) x 10(-8) cm s(-1) for all sites combined (n = 56). The present ratiometric method could potentially be applied to other quantitative fluorescence-based techniques.