The objectives of this project are to design, construct, and test a computed- tomography imaging spectrometer (CTIS) microscope to be used in studies of physiological changes in function from populations of cells which respond in a heterogeneous manner. The proposed CTIS microscope will also be an important tool to study the effects of drugs and toxic agents on the physiology of the liver. The resultant spectral image data will provide detailed information regarding changes in ion movements (H+, Ca2+, K+) and metabolism (O2, NADH) from cells throughout a functioning hepatic unit. Finally, the CTIS will be used in combination with a confocal endoscope to provide spectral as well as 3D spatial information. The resultant capability to utilize multiple fluorescent dyes and 4D (x,y,z, wavelength) data will be a significant advance, which can lead to improved diagnostic accuracy. To demonstrate the feasibility of this type of imaging, the investigators have carried out experiments involving a combination of the CTIS with an inverted microscope and a combination of thje CTIS with a confocal endoscope developed for in vivo imaging. Preliminary results obtained with both systems have demonstrated: (1) the premise of spectral imaging with both a CTIS-inverted-microscope and a CTIS-confocal- endoscope combination, (2) accurate reconstruction of spectra with sampling intervals of 10 nm, (3) minimum spatial sampling distance of 1.7 microns, (4) (x,y, wavelength) data recorded at frame rates of 15 frames/second and 30 frames/second. Imaging experiments involving cell culture specimens have shown that dynamic changes in pH can be followed using the CTIS microscope. Building on these demonstrations, we will: (a) work to improve the signal-to-noise ratio of the CTIS microscope, (b) increase the instrument's spatial resolution, (c) characterize the spatial and spectral resolution using microspheres, (d) demonstrate the acquisition of 4D (x,y,z, wavelength) image data with a combination of the CTIS, the confocal microscope, and depth of field synthesis algorithms. The proposed development of the CTIS microscope will result in significant imaging capability that will support follow-on biomedical research and will enable improvements in diagnostic accuracy when used in vivo in conjunction with an endoscope.
|Beverage, J L; Shack, R V; Descour, M R (2002) Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor. J Microsc 205:61-75|
|Ford, B K; Volin, C E; Murphy, S M et al. (2001) Computed tomography-based spectral imaging for fluorescence microscopy. Biophys J 80:986-93|