Autofluorescence imaging captures heterogeneous drug response differences between 2D and 3D breast cancer cultures.

Two-photon microscopy of cellular autofluorescence intensity and lifetime (optical metabolic imaging, or OMI) is a promising tool for preclinical drug development. OMI, which exploits the endogenous fluorescence from the metabolic coenzymes NAD(P)H and FAD, is sensitive to changes in cell metabolism produced by drug treatment. Previous studies have shown that drug response, genetic expression, cell-cell communication, and cell signaling in 3D culture match those of the original in vivo tumor, but not those of 2D culture. The goal of this study is to use OMI to quantify dynamic cell-level metabolic differences in drug response in 2D cell lines vs. 3D organoids generated from xenograft tumors of the same cell origin. BT474 cells and Herceptin-resistant BT474 (HR6) cells were tested. Cells were treated with vehicle control, Herceptin, XL147 (PI3K inhibitor), and the combination. The OMI index was used to quantify response, and is a linear combination of the redox ratio (intensity of NAD(P)H divided by FAD), mean NADH lifetime, and mean FAD lifetime. The results confirm that the OMI index resolves significant differences (p<0.05) in drug response for 2D vs. 3D cultures, specifically for BT474 cells 24 hours after Herceptin treatment, for HR6 cells 24 and 72 hours after combination treatment, and for HR6 cells 72 hours after XL147 treatment. Cell-level analysis of the OMI index also reveals differences in the number of cell sub-populations in 2D vs. 3D culture at 24, 48, and 72 hours post-treatment in control and treated groups. Finally, significant increases (p<0.05) in the mean lifetime of NADH and FAD were measured in 2D vs. 3D for both cell lines at 72 hours post-treatment in control and all treatment groups. These whole-population differences in the mean NADH and FAD lifetimes are supported by differences in the number of cell sub-populations in 2D vs. 3D. Overall, these studies confirm that OMI is sensitive to differences in drug response in 2D vs. 3D, and provides further information on dynamic changes in the relative abundance of metabolic cell sub-populations that contribute to this difference.

A program for computer-assisted scoring of Southern blots.

SCORE, a program for computer-assisted scoring of Southern blots of clone DNA, retains the use of expert human judgment while taking over much of the drudgery of the scoring task. The primary functions of the program are to help make an aligned overlay of the fluorescence gel image and the autoradiogram blot image, to keep track of band and lane locations and to store the resulting data directly into a database. Use of SCORE has resulted in greatly increased efficiency and accuracy.

Uniformly redundant arrays: digital reconstruction methods.

Several new digital reconstruction techniques for coded aperture imaging are developed which are especially applicable to uniformly redundant arrays (URAs). The techniques provide improved resolution without upsetting the artifact-free nature of URAs. Two new techniques are described; one which allows self-supporting URAs and one which avoids (or at least mitigates) a blur which has been associated with previous correlation analyses. Each of the methods and their resolution improvements are demonstrated with reconstructions of a laser-driven compression. Particular emphasis has been placed on the special sampling required of the encoded picture and the decoding function if artifacts are to be avoided. For large URAs, it is shown that another new digital technique, periodic decoding, is much faster. Periodic decoding does produce artifacts, but they usually are negligible.

Tomographical imaging using uniformly redundant arrays.

Recent work in coded aperture imaging has shown that the uniformly redundant array (URA) can image distant planar radioactive sources with no artifacts. This paper investigates the performance of two URA apertures when used in a close-up tomographic imaging system. It is shown that a URA based on m sequences is superior to one based on quadratic residues. The m-sequence array not only produces less noticeable defocus artifacts in tomographic imaging but is also more resilient to some described detrimental effects of close-up imaging. It is shown that, in spite of these close-up effects, the URA system retains tomographic depth resolution even as the source is moved close to the detector. The URAs based on m sequences provide better images than those obtained using random arrays. This compliments previous studies that have shown random arrays to have better tomographical properties than Fresnel zone plates and nonredundant arrays.

Coded aperture imaging with uniformly redundant arrays.

Uniformly redundant arrays (URA) have autocorrelation functions with perfectly flat sidelobes. The URA combines the high-transmission characteristics of the random array with the flat sidelobe advantage of the nonredundant pinhole arrays. This gives the URA the capability to image low-intensity, low-contrast sources. Furthermore, whereas the inherent noise in random array imaging puts a limit on the obtainable SNR, the URA has no such limit. Computer simulations show that the URA with significant shot and background noise is vastly superior to random array techniques without noise. Implementation permits a detector which is smaller than its random array counterpart.

Comparison of image restoration methods.

Three image restoration methods are compared in a variety of blur and noise conditions. Both numerical and subjective data are evaluated. It is demonstrated that, in certain conditions, one restoration method is preferable to others.