Aaron Reeves’s research while affiliated with Colorado State University and other places

Achieving the ability to non-destructively, non-invasively examine subsurface features of living multicellular organisms at a microscopic level is currently a challenge for biologists. Optical coherence microscopy (OCM) is a new photonics-based technology that can be used to address this challenge. OCM takes advantage of refractive properties of biological molecules to generate three-dimensional images that can be viewed with a computer. We describe new data processing techniques and a different visualization algorithm that substantially improve OCM images. We have applied OCM imaging, in conjunction with these improvements, to a variety of structures of plants, including leaves, flowers, ovules and germinating seeds, and describe the visualization of cellular and subcellular structures within intact plants. We present evidence, based on detailed examination of our OCM images, comparisons to classical plant anatomy studies, and current knowledge of light scattering by cells and their components, that we can distinguish nuclei, organelles and vacuoles. Detailed examination of vascular tissue, which contains cells with elaborate wall structure, shows that cell walls produce no significant OCM signal. These improvements to the visualization process, together with the powerful non-invasive, non-destructive aspects of the technology, will broaden the application of OCM to questions in studies of plants as well as animals.

The ability to identify individual chromosomes in cytological preparations is an essential component of many investigations. While several computer software applications have been used to facilitate such quantitative karyotype analysis, most of these programs are limited by design for specific types of analyses, or can be used only with specific hardware configurations. MicroMeasure is a new image analysis application that may be used to collect data for a wide variety of chromosomal parameters from electronically captured or scanned images. Unlike similar applications, MicroMeasure may be individually configured by the end user to suit a wide variety of research needs. This program can be used with most common personal computers, and requires no unusual or specific hardware. MicroMeasure is made available to the research community without cost by the Department of Biology at Colorado State University via the World Wide Web at http://www.biology.colostate.edu/MicroMeasure.

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 microm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 x 5 x 10 microm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.

To test assumptions of the autotetraploid chromosome pairing model regarding events during synapsis, whole-mount spreads of synaptonemal complexes (SCs) of Machaeranthera pinnatifida (=Haplopappus spinulosus) (Asteraceae) (2n = 4x = 16) were analyzed by electron microscopy. On the assumption of one synaptic initiation per chromosome arm, each pachytene quadrivalent is expected to have one partner switch (PS), and the frequency of pachytene quadrivalents for each chromosome is predicted to be 2/3 (or 0.67). However, to the contrary, we observed a range of one to four PSs per pachytene quadrivalent with an overall mean of 1.56. This suggests that the number of synaptic initiations is greater than one per chromosome arm (or > two per chromosome), and the predicted frequency of pachytene quadrivalents should be > 8/9 (based on a minimum of three initiations per chromosome). However, in close agreement with the model, the observed pachytene quadrivalent frequency from SCs in this study was 0.69. To explain the apparent discrepancy between the observed frequency of PSs and the observed frequency of quadrivalents, the possibility of nonindependent synaptic initiations and presynaptic alignment are discussed in the context of their potential influence on quadrivalent frequency. Recombination nodules (RNs), which were scored in about half the SC spreads, occurred at a frequency (9.6 per nucleus) comparable with the chiasma frequency at diakinesis (9.3 per nucleus). The frequency of RNs as well as their distribution is consistent with the hypothesis that RNs occur at sites of crossing over and chiasma formation.

We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata.

We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata.