Green light for quantitative live-cell imaging in plants Guido et al.

, 2018

Plants exhibit an intriguing morphological and physiological plasticity that enables them to
thrive in a wide range of environments. The imaging of plant cells entails a number of
specific challenges, such as high levels of autofluorescence, light scattering that is caused by
cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in
plants therefore requires adapting or developing imaging techniques, as well as mounting and
incubation systems, such as micro-fluidics.
Fluorescence-based microscopy has revolutionized various fields of plant biology, such as
large-scale organ morphogenesis, Ca2+ signaling during sexual reproduction or membrane
protein dynamics during pathogen attack (Barbier de Reuille et al., 2015; Fernandez et al.,
2010; Shaw and Ehrhardt, 2013).
The need to image live plant cells in intact tissues raised the demand for developing novel
experimental tools and setups (Shaw and Ehrhardt, 2013). The greatest challenge is the
inherent autofluorescence of most plant cells, which is largely caused by the presence of
chlorophyll and carotenoids in plastids, as well as by lignin and other phenolic compounds in
cell walls (Shaw and Ehrhardt, 2013).

Applying two-photon excitation fluorescence lifetime imaging microscopy to study

photosynthesis in plant leaves Koen et al., 2009
The best non-invasive optical imaging technique for measuring photosynthetic systems in
leaves is multiphoton fluorescence microscopy, because it allows imaging up to a depth of
500 ⴅm in living plant tissue (Williams et al. 2001; Zipfel et al. 2003).

Using spontaneous photon emission to image lipidoxidation patterns in plant tissues Simona
Birticet al 2011
Living organisms, including plants, spontaneously emit light. This spontaneous photon
emission, also called ultra-weak photon emission, biophoton emission or auto luminescence,
occurs at wavelengths from visible to near infrared, without any external excitation or
administration of chemiluminescence agents (Abeles, 1986; Devarajet al.,1997; Havaux,
2003)
In general, photomultiplier tubes and photon counting are employed for this purpose, and
spontaneous photon emission has been measured in a variety of plant samples, including
roots (Mathew and Roy, 1992; Makino et al., 1996; Hossu et al., 2010), leaves (Havaux,
2003; Kobayashi et al., 2007; Winkler et al., 2009), seeds (Suzuki et al., 1991; Ohya et al.,
2002) and isolated cells and organelles (Hideg and Inaba, 1991a; Rastogi and Pospisil, 2010).
It is generally assumed that one of the possible sources of spontaneous photon emission from
living organisms is lipid peroxidation (Abeles, 1986; Sies, 1987; Halliwell and Gutteridge,
1989; Havaux, 2003). Highly sensitive charge-coupled-device (CCD) cameras have been
used to improve autoluminescence imaging in plants and in other organisms including
humans (e.g. Takeda et al., 2004; Van Wijk et al., 2006; Kobayashi et al., 2009).

Light microscopy of whole plant organs A.C.J. Timmers al., 2016

Thinner sections of living material are technically possible, but inevitably will lead to the
death of cells that are cut. Indeed, under 25μm almost no plant tis-sue can be cut without
complete destruction of all cells with in the section. Information obtained from sections has
been and will continue to be important for many studies, an alternative way of obtaining
sections from thick plant samples became widely available with the introduction of the first
commercial confocal microscope in 1982 by Ox-ford Optoelectronics Ltd. (Sheppard, 2003).

Some plant organs are so thin that there is no restriction for whole organ imaging by means
of fluorescence microscopy. This is the case for a number of lower plants like
stoneworts(Charales)andmanymosses.However,evenforthinrootslikethe ones of A.thaliana,
image quality decreases with scanning depth (Haseloff, 2003) and a complete 3D
reconstruction of the whole root at high resolution is not achieved with confocal laser
scanning microscopy or light sheet microscopy (Maizeletal.,2011).
Ideally, functional and developmental imaging should be performed on living whole plants in
combination with a minimum of disturbance. For relatively thin organs like roots of A.
thaliana considerable resolution deep within the tissue can be achieved with light sheet
microscopy (Maizelet al., 2011) and functional imaging has been reported (Costaet al., 2013).

Recent advances in plant endomembraneresearch and new microscopical techniques

Yonglun Zeng ey al., 2023
Electron tomography (ET) generates 3D information by back-projection of 2D images taken
at a series of tilting angles (McIntoshet al.,2005). Combined with HPF/FS, ET in room
temperature (RT-ET) is able to resolve subcellular structures within single or serial sections
with4 nm lateral resolution and 6–8 nm Z resolution (Staehelin & Kang,2008). HPF can
effectively cryo-fix plant tissues up to 200 μm close to the native states. RT-ET is especially
good at resolving complex membrane morphologies such as membrane connections during
membrane remodelling, progression, fission, or fusion within the cellular or tissue
environment (Zhuanget al.,2017; Rothet al.,2019; Huanget al.,2022). Recently, scanning
transmission ET (STET) was applied to analyse plastid inner-membrane remodelling in
Arabidopsis cotyledons (Lianget al.,2022). Cryo-ET has recently thrived and succeeded in
resolving in sit membrane architecture and macromolecular complex structure at sub-
nanometer resolution (Erdmannet al.,2018).

Advances in Imaging Plant Cell Walls Yuanyuan Zhao et al., 2019

Cell wall imaging can considerably permit direct visualization of the molecular architecture
of cell walls and provide detailed chemical information on wall polymers, which is becoming
one of the hot topics in contemporary botanical research.
Label-free techniques based on Raman spectroscopic imaging, SRS in particular, can provide
label-free dynamics and quantitative microanalysis of chemical compositions of living plant
cell walls with a characteristic molecular vibration in situ, which has opened exciting new
avenues for cell wall imaging.
Further studies integrating advanced label-free imaging techniques, using super-resolution
microscopy, along with real-time studies of structural changes, will be conductive to refine
current molecular organization and better understanding of cell wall architecture.

Advances in plant imaging across scales Pamela S. Soltis

In plant biology, observations from satellites (e.g., Deneu et al., 2021; Cavender ‐Bares et al.,
2022) and airborne instruments (e.g., Sun et al., 2021) are providing new insight into the
distribution of botanical diversity, species abundance, and ecosystem productivity.