2.5. Image Acquisition and Postprocessing

Image tiles are collected at a lateral spacing of 1 mm in both the x and y directions, which represents an optimal balance between imaging speed and mosaicking accuracy. The vertical step size is 5μm, which satisfies the Nyquist sampling criterion (assuming an axial resolution of ∼12μm). The total z-scanning range is 150μm, which is sufficient to accommodate a majority of the surface irregularities seen at the tissue surface. The integration time for each frame is 50 ms (UV radiant exposure per frame of ∼77.5Jm−2), which represents a balance between speed and signal-to-noise ratio (SNR). Large-area image acquisitions are automatically controlled by a custom-developed LabVIEW program.

Data postprocessing consists of the following steps [Fig. 2(c)]: (1) 3-D deconvolution is performed for contrast and resolution enhancement. Ten iterations (optimized for speed and image quality) of a Lucy–Richardson algorithm (MATLAB) are applied for each vertical image stack (z stack). (2) Surface extraction is performed for surface-irregularity mitigation. For each z stack of deconvolved images, we used an open source ImageJ plugin to perform a complex wavelet transform³⁵ that takes a vertical stack of images and extracts the best focus (tissue surface) for each lateral subregion (the parameters for the algorithm are tunable and provide a trade-off between extraction quality and speed). (3) The images from the two channels at each lateral position are co-registered (aligned) with a normalized cross-correlation algorithm. (4) The aligned images for each channel are mosaicked using the grid/collection stitching plugin in ImageJ.³⁶ (5) The large-area (mosaicked) images are false-colored (by combining the two image channels) to mimic the appearance of gold-standard H&E histology. Here we have optimized an H&E false-coloring algorithm that was previously published.³⁷ In short, localized histogram equalization is applied to each channel, prior to H&E false coloring, to enhance the contrast and consistency of the false coloring across a large-area image.