Onion Skin Cells Research Articles

Open Source 3D-printed focussing mechanism for cellphone-based cellular microscopy.

The need to improve access to microscopes in low-resource and educational settings coupled with the global proliferation of camera-enabled cellphones has recently led to an explosion in new developments in portable, low-cost microscopy. The availability of accurate ball lenses has resulted in many variants of van Leeuwenhoek-like microscopes. Combined with cellphones, they have the potential for use as portable microscopes in education and clinics. The need for reproducibility in such applications implies that control over focus is critical. Here, we describe a 3D-printed focussing mechanism based on a rack and pinion mechanism, coupled to a ball lens- based microscope. We quantify the time-stability of the focussing mechanism through an edge-based contrast measure used in autofocus cameras and apply it to 'thin smear' blood sample infected with Plasmodium as well as onion skin cells. We show that stability of the z-focus is in the micrometre range. This development could, we believe, serve to further enhance the utility of a low-cost and robust microscope and encourage further developments in field microscopes based on the Open Source principle. LAY DESCRIPTION: The wide spread of cellphones with cameras makes them an attractive platform for digital microscopy. Such microscopes could help improve microscope access in clinics and classrooms in the form of 'field microscopes', if they could be adapted for imaging cells. We integrate a 3D printed focussing mechanism made with recyclable plastic with ball-lens microscope of the Leeuwenhoek type. We demonstrate how the device can help stabilise to a focal plane for acquiring movies of a thin-smear of blood infected with Plasmodium and onion skin cells using a cellphone. The stability of focus is expectedly less precise as compared to research-grade microscopes, but is of the range of a few micrometers. We believe, the focussing device demonstrates it is possible to obtain reliable and reproducible images of typical samples used in clinics and classrooms. By making the design files of this device open-source we believe it could serve as a small step in improved, affordable and accurate 'field microscopes'.

High-resolution full-field optical coherence microscopy using a Mirau interferometer for the quantitative imaging of biological cells

In this paper quantitative imaging of biological cells using high-resolution full-field optical coherence microscopy (FF-OCM) is reported. The FF-OCM was realized using a swept-source system, a Mirau interferometer, and a CCD camera (a two-dimensional detection unit). A Mirau-interferometric objective lens was used to generate the interferometric signal. The signal was analyzed by a Fourier analysis technique. Optically sectioned amplitude images and a quantitative phase map of biological cells such as onion skin and red blood cells (RBCs) are demonstrated. Further, the refractive index profile of the RBCs is also presented. For the 50× Mirau objective, the experimentally achieved axial and transverse resolution of the present system are 3.8 and 1.2 μm, respectively. The CCD provides parallel detection and measures enface images without X, Y, Z mechanical scanning.

Quantitative cell imaging using single beam phase retrieval method

Quantitative three-dimensional imaging of cells can provide important information about their morphology as well as their dynamics, which will be useful in studying their behavior under various conditions. There are several microscopic techniques to image unstained, semi-transparent specimens, by converting the phase information into intensity information. But most of the quantitative phase contrast imaging techniques is realized either by using interference of the object wavefront with a known reference beam or using phase shifting interferometry. A two-beam interferometric method is challenging to implement especially with low coherent sources and it also requires a fine adjustment of beams to achieve high contrast fringes. In this letter, the development of a single beam phase retrieval microscopy technique for quantitative phase contrast imaging of cells using multiple intensity samplings of a volume speckle field in the axial direction is described. Single beam illumination with multiple intensity samplings provides fast convergence and a unique solution of the object wavefront. Three-dimensional thickness profiles of different cells such as red blood cells and onion skin cells were reconstructed using this technique with an axial resolution of the order of several nanometers.

Ultrabroadband coherence-domain imaging using parametric downconversion and superconducting single-photon detectors at 1064 nm

Coherence-domain imaging systems can be operated in a single-photon-counting mode, offering low detector noise; this in turn leads to increased sensitivity for weak light sources and weakly reflecting samples. We have demonstrated that excellent axial resolution can be obtained in a photon-counting coherence-domain imaging (CDI) system that uses light generated via spontaneous parametric downconversion (SPDC) in a chirped periodically poled stoichiometric lithium tantalate (chirped-PPSLT) structure, in conjunction with a niobium nitride superconducting single-photon detector (SSPD). The bandwidth of the light generated via SPDC, as well as the bandwidth over which the SSPD is sensitive, can extend over a wavelength region that stretches from 700 to 1500 nm. This ultrabroad wavelength band offers a near-ideal combination of deep penetration and ultrahigh axial resolution for the imaging of biological tissue. The generation of SPDC light of adjustable bandwidth in the vicinity of 1064 nm, via the use of chirped-PPSLT structures, had not been previously achieved. To demonstrate the usefulness of this technique, we construct images for a hierarchy of samples of increasing complexity: a mirror, a nitrocellulose membrane, and a biological sample comprising onion-skin cells.

Experimental evidence for eukaryotic fossil preservation: Onion skin cells in silica solution

Research on the origin of eukaryotes often focuses on the exceptional preservation found in silicified Precambrian fossils. Nuclei like subcellular structures in well-preserved fossil becomes confusion and arguments, which focus on whether the partial degradation of prokaryotes produces artifacts that resemble a ‘nucleus’, or fossilized nuclei of eukaryotes. In order to understand the mechanisms of silicification and identify the fossilized subcellular and microstructures in rocks, a series of laboratory controlled experiments were performed for simulating the silicification process. The effects of different silica solutions in eukaryote fossilization were studied in our experiments by exposing onion skin cells (epidermis) to silica solutions. Onion skin provides a good experimental model because of its well characterized cellular structures which are easily observed. The designed experiments revealed that the possibility of onion cell preserved as a “fossil” with nuclear structures, the first week fossilization, or mineralization as rapid as 1 week is important. And the experiment also revealed interactions between silica and the onion skin cell wall surface functional groups were weak. The preservation of nuclei in the onion skin model was due to precipitation in highly supersaturated silica solutions rather than simply the high silica concentration. When the silica gel precipitates slowly at low supersaturation states, the nuclei were not well preserved, but the rapid precipitation at high supersaturated silica conditions preserved nuclear structures. A better understanding of the processes involved in onion skin fossilization will further contribute to issues concerning the silicification of other eukaryotic materials.

Harmonically matched grating-based full-field quantitative high-resolution phase microscope for observing dynamics of transparent biological samples

We have developed a full-field high resolution quantitative phase imaging technique for observing dynamics of transparent biological samples. By using a harmonically matched diffraction grating pair (600 and 1200 lines/mm), we were able to obtain non-trivial phase difference (other than 0 degrees or 180 degrees) between the output ports of the gratings. Improving upon our previous design, our current system mitigates astigmatism artifacts and is capable of high resolution imaging. This system also employs an improved phase extraction algorithm. The system has a lateral resolution of 1.6 mum and a phase sensitivity of 62 mrad. We employed the system to acquire high resolution phase images of onion skin cells and a phase movie of amoeba proteus in motion.

In-Situ atomic force microscopic observation of ion beam bombarded plant cell envelopes

A program in ion beam bioengineering has been established at Chiang Mai University (CMU), Thailand, and ion beam induced transfer of plasmid DNA molecules into bacterial cells (Escherichia coli) has been demonstrated. However, a good understanding of the fundamental physical processes involved is lacking. In parallel work, onion skin cells have been bombarded with Ar+ ions at energy 25keV and fluence1–2×1015ions/cm2, revealing the formation of microcrater-like structures on the cell wall that could serve as channels for the transfer of large macromolecules into the cell interior. An in-situ atomic force microscope (AFM) system has been designed and installed in the CMU bio-implantation facility as a tool for the observation of these microcraters during ion beam bombardment. Here we describe some of the features of the in-situ AFM and outline some of the related work.

Introducing the Cell Concept with both Animal and Plant Cells: A Historical and Didactic Approach

In France, as well as in several other countries, the cell concept is introduced at school by two juxtaposed drawings, a plant cell and an animal cell. After indicating the didactic obstacles associated with this presentation, this paper focuses on the reasons underlying the persistence of these two prototypes, through three complementary interpretations: A pedagogical one: it was, and it is still today, easy to use onion skin and mouth epithelial cells at school, hence both of these kinds of cells became cell prototypes. A historical one: Schleiden worked on plant cells, and Schwann on animal cells; together they established common features between plant and animal cells (1838). A sociological one: since the emergence of the cell concept (19th century) and until recently, Zoology and Botany were clearly separated research fields. Today, besides these traditional animal and plant cell prototypes, a general “egg-cell model” is used increasingly to introduce and develop the cell concept.

Ion bombardment induced formation of micro-craters in plant cell envelopes

Ion beam bombardment of biological material has been recently applied for gene transfer in both plant and bacterial cells. A consistent physical mechanism for this significant result has not yet been developed. A fundamental question about the mechanism is the possible formation of pathways due to ion bombardment that are responsible for the gene transfer. We have carried out investigations of the effects of low-energy bombardment by both gaseous and metallic ion species of onion skin cells on their surface microstructure. Our experimental results reveal evidence demonstrating that the formation of micro-crater-like structures on the plant cell envelope surface is a general phenomenon consequent to ion bombardment, no matter what ion species, under certain ion beam conditions. The micro-craters are about 0.1–1μm in size (diameter) and a few tens of nanometers in depth. The micro-crater formation process seems to be unrelated to the chemical composition of and rapid water evaporation from the cell envelope, but is associated with the special microstructure of the cell wall.

On the origin of microcraters on the surface of ion beam bombarded plant cell walls

Ion bombardment of plant and bacterial cellular material has recently been used as a tool for the transfer of exogenous DNA macromolecules into the cell interior region. The precise mechanism that leads to the transfer of macromolecules through the cell envelope is not yet clear, however it has been observed that the ion bombardment is accompanied by the formation of “microcraters” on the cell wall, and it is possible that these features provide channels for the macromolecule transfer. Thus the nature and origin of the microcraters is of importance to understanding the DNA transfer phenomenon as well as being of fundamental interest. We report here on some scanning electron microscope observations we have made of onion skin cells that have been subjected to electron beam bombardment of sufficiently high power density to damage the cell wall. The damage seen is much less than and different from the microcraters formed subsequent to ion bombardment. We speculate that the microcraters may originate from the explosive release of gas generated in the biomaterial by ion bombardment.

Metal ion bombardment of onion skin cell wall

Ion bombardment of living cellular material is a novel subfield of ion beam surface modification that is receiving growing attention from the ion beam and biological communities. Although it has been demonstrated that the technique is sound, in that an adequate fraction of the living cells can survive both the vacuum environment and energetic ion bombardment, there remains much uncertainty about the process details. Here we report on our observations of onion skin cells that were subjected to ion implantation, and propose some possible physical models that tend to support the experimental results. The ion beams used were metallic (Mg, Ti, Fe, Ni, Cu), mean ion energy was typically 30keV, and the implantation fluence was in the range 1014–1016ions/cm2. The cells were viewed using Atomic Force Microscopy, revealing the formation of microcrater-like structures due to ion bombardment. The implantation depth profile was measured with Rutherford backscattering spectrometry and compared to the results of the TRIM, T-DYN and PROFILE computer codes.

Experimental Study on Freezing of Onion Skin Cells. Intracellular Ice Formation and Dehydration with Extracellular Solution.

Microscopic observations of the freezing process of onion skin cells have been made using a directional solidification stage, in order to study the freezing mechanism of living tissue. Experiments conducted under low cooling rates show that intracellular ice formation occurs due to surface-catalyzed nucleation (SCN) at the plasma membrane and proceeds cell by cell because the membrane blocks the growth of ice crystals. Since the probability of SCN increases with increase in the surface area in contact with the external ice, the tissue freezes at higher temperatures when the cell has a larger area in contact with the adjacent frozen cells. Dehydration and cell shrinkage were also observed in experiments with physiological saline simulating the effects of the vascular system in the organs. It is confirmed that a decrease of the cooling rate raises the dehydration rate and reduces the fraction of frozen cells but increases the amount of extracellular freezing.

Negative Turgor Pressures in Plant Cells

AbstractNon‐osmotic moisture stress can be simulated by applying to a plant tissue a solute that cannot penetrate the cell wall. The external reduction in water potential caused by such a non‐penetrating solute is compensated for by a reduction in pressure within the cell. The pressure is ambient throughout a cell that has been plasmolysed by a penetrating osmoticum. A supplementary non‐penetrating osmoticum must also be compensated for by lowering the internal pressure, i.e. a negative turgor pressure must develop. Onion skin cells can withstand a resulting mechanical stress of up to about 10KPa (1/10 Bar) before collapsing.

The response of Plant Cells to Different Forms of Moisture stress

A non-osmotic moisture stress can be simulated by applying a solute that cannot penetrate cell walls of plant tissue. The reduction in the external water potential by this non-penetrating solute is compensated for by a reduction in the hydrostatic pressure in the cell. In a cell that has been plasmolysed by a penetrating osmoticum such as NaCl, the pressures are ambient in the external medium, in cell wall pores, in the protoplast-free volume of the cell and in the protoplast. Compensation to a supplementary non-penetrating external osmoticum must be a lowering of the hydrostatic pressure in cell wall pores and inside the cell. The resulting negative turgor exposes the cell to a severe stress and causes its rapid collapse. Onion skin cells collapsed within a few seconds, epidermal cells of Rhoeo within minutes.

Scanning coherent anti-Stokes Raman microscope

We have constructed a spatially scanning coherent anti-Stokes Raman spectroscopic (CARS) apparatus that allows us to image the distribution of distinct chemical species in a microscopic sample region. Images of onion-skin cells have been obtained by using the CARS signal produced by the 2450-cm(-l) band of deuterated water. Future applications will be discussed.