Calcium Imaging in Plants

LSFM Allows Rapid and Long-Term Acquisition of Calcium Dynamics

  • Fig. 1: (a) LSFM principle: a thin sheet of laser light illuminates the sample, while the fluorescence from the whole plane is collected orthogonally to the excitation path. (b) Scheme of the LSFM for FRET imaging: a cylindrical lens (fCL = 50 mm) creates the light sheet from a single-mode fiber-coupled (FC) laser, and projects it on the back-focal plane of the 10X water-dipping illumination objective through a telescope (f1 = f2 = 50 mm). The seedling is held in a chamber filled with its physiological solution and can be moved by means of a translation stage. The 20X water-dipping detection objective sends the fluorescence signal to a relay system (f3 = f4 = 100 mm). A dichroic filter separates the image into the CFP and cpVenus channels, which are filtered by their specific emission filters. A tube lens (fTL = 180) creates the two images on the same CMOS sensor. M=mirror.
  • Fig. 2: Time-lapse imaging of root and root hairs growth for two different plants acquired at 20x (a) and 4x magnification (b). Time-lapse frames were obtained from the maximum intensity projection of 100 images LSFM stacks, acquired with 3 µm spacing. Every frame was then color-coded according to the acquisition time and merged into a single image: different colors represent different instants of the growth (colorbar).
  • Fig. 3: (a) Three-dimensional reconstruction of an Arabidopsis root, showing the arrangement of root hairs. (b) Calcium signals extracted from the analysis of 5 different root hair tips of the same plant. The periodic oscillations indicate a possible role of calcium in the regulation of plant growth.

One of the challenges of modern plant biology is to understand the specific functions of plant tissues in close-to-physiological conditions. In order to understand how plant perceive and transduce environmental and developmental signals we use Light Sheet Fluorescence Microscopy. This method allows one to observe the plant growth in vivo, follow cellular differentiation processes and link these events with the dynamics of signaling molecules, including calcium.

Light Sheet Fluorescence Microscopy (LSFM) is a fluorescence imaging technique highly suited to study living samples, particularly developing embryos, tissue cultures and plants [1]. In LSFM a biological specimen is illuminated in a single plane (light sheet) and the fluorescence emitted by the sample is collected along the direction perpendicular to the illumination plane. This approach offers two major advantages compared to conventional and confocal microscopy techniques. First, fluorescence images are collected using common widefield optics and CCD or CMOS cameras, that offer high acquisition rates: more than 100 frames per second are acquired routinely with LSFM. Second, only the plane of the specimen under observation is illuminated. This configuration greatly reduces the light dose and therefore photo-bleaching effects and photo-toxicity.

When imaging plants, such as Arabidopsis thaliana, LSFM offers a third advantage that makes it ideally suited for long-term time-lapse experiments. The sample can be placed in vertical position, the plant root can be grown directly in an agar based medium, eventually placed in a hydroponic solution and the leaves can be illuminated with an artificial light: this makes the measurement as close as possible to physiological. In these conditions, plant development can be recorded over several hours of growth [2] and the plant function, such as the calcium dynamics occurring in response to internal and external stimuli can be studied over large tissue volumes [3].

Working Principles of LSFM-FRET
Calcium imaging in plants can be achieved using several different fluorescent labels (e.g. Fluo-3) but for subcellular imaging, genetically encoded Ca2+ indicators (aecquorin, GCaMP, Cameleon) are the tool of choice.

Cameleons are Förster Resonance Energy Transfer (FRET) based calcium indicators in which two fluorescent proteins, CFP and YFP (or circularly permuted variants of YFP, such as cpVenus), are linked together by the calcium-binding protein calmodulin and a calmodulin binding peptide. Binding of Ca2+ to these calcium-responsive elements alters the distance between the two fluorophores. Hence, the efficiency of FRET varies, allowing a quantitative assessment of calcium dynamics.

Using LSFM one can image Arabidopsis transgenic plants expressing, for example, the nuclear or cytosolic-targeted Cameleon [3]. A typical setup consists in an illumination path to create the light sheet and a widefield detection path (fig. 1a), where two wavelength channels are simultaneously acquired for ratiometric FRET imaging. A laser at 442 nm coupled to a single mode fiber is collimated and used for excitation. A cylindrical lens focuses the light in a horizontal plane and a telescope (f1, f2) images the focal plane of the cylindrical lens in the back focal plane of a water dipping microscope objective (10X). As a result, a vertical light-sheet is created on the sample. The fluorescence is collected by a 20X water dipping microscope objective which, in combination with a lens (f3), creates an intermediate image. Calcium imaging with FRET requires the detection of the signal emitted by the two fluorescent molecules CFP and cpVenus. This task is achieved using two separate cameras or an image splitter placed in front of a single detector. Here, the intermediate image is split in two channels (fig. 1b) on the CMOS camera by a custom beam splitter. The plant root is mounted on a motorized stage for accurate and automatic scanning of the sample.

Live Imaging of Arabidopsis Development and Function

By moving the sample back and forth through the light sheet, the system creates three-dimensional recordings of the sample fluorescence and FRET signal, with an acquisition time of one or few seconds per volume. This rate is suited to study a large number of physiological processes in different plant cell types (and tissues) that occur with seconds or minute time scales, such as the outgrowth of root hairs or the growth of primary and later roots. Looking at the fluorescence, either in the CFP or in the cpVenus channel, one can observe the phenotype of the root and, even more importantly, follow the growth of the sample (fig. 2) under the microscope, for minutes, hours or even days. At the same time, FRET signals can be acquired and analyzed in real-time, in order to estimate the calcium concentration in the different compartments of the cells within the root cells or in root hairs. In these experiments, normally the ratio (R) between the signals emitted by the two fluorescent proteins is measured and eventually normalized to its initial value (R0), calculated at the beginning of the experiment (fig. 3).

Looking at the outgrowth of root hairs, spontaneous calcium oscillations appear specifically at their tips, being characterized by a period that ranges between few and tens of seconds.

Are these oscillations related to the growth of the root hairs? We think that LSFM can help to finally answer this question as many other questions related to the function, development and gene expression of plants.

Conclusions
LSFM combines the advantages of different microscopy approaches in a single technique: it provides single cell resolution, typical of confocal laser scanning microscopy and is fast enough to observe systemic responses, characteristic of wide field microscopy. Calcium signaling can be monitored in vivo, at high resolution, with negligible photo-bleaching over a large portion of the root tip, including a large number of root hairs, thus proving statistically relevant information for plant biology studies.

Acknowledgments
This project has received funding from the European Union‘s Horizon 2020 research and innovation program under grant agreement no 654148 Laserlab-Europe.

References
[1] J. Huisken and D.Y. Stainier: Selective plane illumination microscopy techniques in developmental biology, Development 136(12), 1963-1975 (2009) DOI 10.1242/dev.022426.
[2] A. Maizel, D. von Wangenheim, F. Federici, J. Haseloff and E.H.K. Stelzer: High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy, Plant J. 68(2), 377-385 (2011) DOI 10.1111/j.1365-313X.2011.04692.x
[3] A. Costa, A. Candeo, L. Fieramonti, G. Valentini, and A. Bassi: Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy, PloS one 8(10), e75646 (2013) DOI 10.1371/journal.pone.0075646

Authors
Alessia Candeo1, Fabrizio G. Doccula2, Gianluca Valentini1, Alex Costa2, Andrea Bassi1
1 Department of Physics, Politecnico di Milano, Milano, Italy
2 Department of Biosciences, Università degli studi di Milano, Milano, Italy

Contact
Prof. Andrea Bassi

Politecnico di Milano
Physics Department
Milano, Italy

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