2.4 Discussion
3.1.2 Measurement of Phyllotaxis
right, what is the nature of the connection between the mechanical and biochemical? Do biochem- ical signals lead to mechanical buckling or is it the other way around? Some progress has been made towards resolving this conflict. Marcus Heisler and Olivier Hamant showed that mechanical perturbation (laser ablation of cells in the meristem) led to the repositioning of auxin transporters in nearby cells[29]. Additionally, through a combination of experiments, image processing and me- chanical computer modeling Richard Smith and colleagues were able to determine that mechanical forces can act as a feedback signal on meristem patterning affecting the biochemistry[30]. But while connections between the biochemical and mechanical have been made, critical pieces of the puzzle remain at large, particularly mechanisms for sensing strain and the pathway leading from auxin accumulation to strain.
In this work I seek to push our understanding of phyllotaxis patterning further by exploring the relationship of the meristem size to the divergence angles produced — a relationship suggested by the models in Chapter 2. This ties in closely with Douady and Couder’s assertion that phyllotaxis patterns are a result of simple geometric ratios and more recently the work by Oliver Hamant and colleagues, who have also made careful measurements of meristem sizes[31]. By using advanced, modern measurement techniques I seek to quantify relevant geometric parameters in the meristem and compare those with the macroscopically observed phyllotaxis patterns.
but larger gains in accuracy were made with the development of the scanning electron microscope (SEM).
Beginning in the 1920’s physicists experimenting with electron beams found they could steer the beams and detect the reflection, providing a method that, in theory, could create much higher resolution images than optical microscopy[33]. For many decades the technique was limited to specialized labs and was an experiment in itself. In 1965 the first commercial scanning electron microscope, the Cambridge Scientific Mark I Stereoscan, was produced and allowed non-physicists to make high magnification, high resolution, high contrast images of the surfaces of their samples[34].
One of the biggest innovations with this device was the development of long-life CRT phosphors that allowed images to be transferred from the CRT screen (which would typically only allow for temporary viewing of the sample) to photographic film. This meant images could be developed into photographs and features could be measured on paper with good accuracy and repeatability, since the magnification ratios of sample to CRT and CRT to film were known.
The first SEM images of plants, published in 1973, were taken by John Owens and Marje Molder who were looking at rates of mitosis in the meristems of conifers[35]. Shortly after, Paul Green also began using this technique to study the regeneration of stems from leaf tissue[36]. Other plant scientists picked up on this[37, 38, 39, 40] and by the mid 1908s SEM had become a routine proce- dure for making precise measurements of microscopic plant morphology, including divergence angles between successive primordia[41]. But while this tool provided very detailed images of meristem morphology it had its drawbacks — primarily that the samples have to be fixed under harsh con- ditions, dehydrated, and placed in a hard vacuum, which can distort some of the morphological features. Additionally, the specimen can only be viewed at one point in time since the process is destructive.
Another technique was developed in Paul Green’s lab that preserved the plant tissue but also pro- vided the high resolution of SEM[42]. In this technique a mold is made of the meristem using the same quick-setting polymers used in dental molds. From the negative mold a positive cast is made from a material that can be sputtered with gold and placed in the vacuum for SEM imaging.
This technique preserves very fine details of the meristem, even small meristems like those of Ara- bidopsis, and is non-destructive to the plant. A series of dental molds can be made from the same specimen to track morphology over time. This method is still used today in situations where the non-destructive nature of the technique is critical and high resolution images of the tissue surface are needed[30].
Even with the benefits that SEM and dental molds have provided for measuring microscopic mor- phological features, there is still room for improvement. While SEM images appear to be three- dimensional since shadows are visible, the appearance of depth is an illusion. In machines with backscatter detectors, the electrons are returned to the detector after being reflected and the return- ing electrons are blocked in the same way that light would be, leading to shadows. But the images produced by the detector are still only two-dimensional. Measurements taken from these images can not take the z-axis into account.
Advances in light microscopy allow for high resolution fluorescent images of the meristem in three dimensions. In confocal microscopy, out of focus light (light not emitted from the focal plane) is blocked by a pin hole, allowing for samples to be optically sectioned by moving the sample through the focal plane and taking a two-dimensional image at each height. The only limit to resolution is the wavelength of the emitted light which, while not as great as SEM, is sufficiently good enough for measuring distances between organs in three dimensions in the meristem. It also provides a view inside the tissue rather than just the surface. Advances to the imaging techniques have actually allowed for resolutions below the diffraction limit[43, 44] but it has been the advances in 3D image processing that have made the largest impact on our ability to quantify the morphology of the shoot apical meristem[45].
In addition to measuring phyllotaxis at the microscopic level of the meristem, it can also be measured by observations of the whole plant. One technique that has been commonly used throughout the history of research on phyllotaxis is to take a picture from the top of the plant. Divergence angles can then either be measured on a printout (or developed photo) or on a computer using a digital image and basic microscopy software like Fiji[46]. This method provides the benefit of being quick and simple, although it is limited to the apical most leaves or flowers as they will obscure the view of those below. Another approach is to use a divergometer, essentially a protractor that can move up and down the vertical axis of the plant to measure the divergence angles visually. A third approach to this problem is to use tomographic techniques for constructing a 3D model of the entire plant. This would provide the benefit of producing divergence angles for the entire plant all at once instead of just a few as is the case with meristem microscopy and could potentially be faster than a divergometer.