Low-temperature resistance of plants is highly diverse, ranging from chilling susceptible species in tropical biomes to extreme freezing resistant species of the boreal zones that even survive immersion in liquid nitrogen in the dormant state. Stress caused by low temperature limits global plant distribution, threatens crop survival and by this can lead to tremendous economic damage [1]. A challenging factor for frost survival is ice management, that is where it initially forms, how it propagates, and how it aggregates in certain places and is generally accommodated within the plant body [2]. Investigation of ice management has largely been neglected when compared with other aspects of freezing resistance [2]. Presumably, this is due to difficulties in visualising ice in plant tissues as we lack simple and unambiguous detection techniques. Nevertheless, knowledge of ice accommodation in plant tissues will be essential for the fundamental understanding of frost survival of plants within the whole range of freezing resistance capacities (<0 °C to -196 °C). Additionally, this might be the basis for the applied sciences to create improved arrangements for frost protection of crops in the future [2].
For the investigation of ice management processes in plant tissues various methods have been employed [1, 3]: differential thermal analysis (DTA) [4, 5], infrared imaging [6-18] and in particular infrared differential thermal analysis (IDTA), nuclear magnetic resonance (NMR) / magnetic resonance imaging (MRI) [1, 19-23], microscopic observations [17, 20, 24-36], indirect observation by freeze-substitution EM [37], cryo-scanning electron microscopy (cryo-SEM) [32, 36, 38] and X-ray phase contrast imaging [3]. All these approaches differ largely in the obtained resolution, the necessary expenses, time requirements and the gained information: ice is either directly visualised or indirectly detected by measurement of freezing exotherms or assessed by the remaining amount of liquid water. Not all these methods allow control of cooling rates. Rates greater than 3 K/h can result in artificial supercooling [39] which is critical for frost injury.
Precise temperature measurements can detect ice formation, as the phase transition from liquid water to ice releases measurable heat, the so-called freezing exotherm. Based on this principle DTA compares the sample temperature with a reference temperature and allows to identify freezing, but cannot detect the initial site of ice nucleation or the direction of ice propagation [1]. In contrast, IDTA allows visualising the spatiotemporal ice propagation. This enables to detect the point of ice nucleation, the propagation pattern and its speed [6, 7]. As the release of heat is not quantified, both methods only provide a qualitative statement about the intensity of freezing [40]. Although the sensitivity of infrared signal detection improved rapidly, the localisation of ice inside of samples requires invasive sectioning [20].
The remaining liquid water in partially frozen plant samples can be quantified by NMR spectroscopy [21, 22], while NMR micro-imaging or magnetic resonance imaging (MRI) localises liquid water [1, 19, 20, 23]. Both are noninvasive techniques. However, it remains unclear, whether the loss of proton signal is due to phase transition from solid to liquid or due to desiccation. In contrast to IDTA, MRI visualises the spatio-temporal pattern of unfrozen water, which shows supercooled and non-frozen areas. With MRI, a maximum resolution of 20-100 µm can be achieved, however detection of rapid phenomena at high resolutions is difficult [20, 41]. Application of MRI to study freezing behaviour in florets and buds is established [20, 42], but for leaves there is limited research available [43]. MRI is a powerful tool for investigating freezing behaviour of plants, but there have been only a few high-resolution studies because of the limitation of machine time and expense [20].
Ice crystals can be visualised and located inside of plant tissues by cryo-SEM and/or light microscopy. Microscopic methods require sectioning at a certain point in time; this entails the potential risk of ice crystal formation during incision. Further, temporal monitoring of ice formation in a specimen is not possible [20]. However, microscopy is a standard tool for detection of ice in plant tissues. Light microscopy was mainly applied to identify extracellular ice whereas cryo-SEM in addition can detect intracellular ice [36]. Cryo-SEM is also a valuable tool for monitoring freezing responses of plant tissues or cells [38]. As fixation requires very low temperatures (-160 °C) [32] a direct observation of naturally formed ice crystals, which have formed prior to the fixation process, is not possible. Only indirect observation of naturally formed ice is possible as it can be distinguished whether ice crystals have formed before or after the fixation process [38]. By observation under vacuum conditions around -110 °C, there is a risk of ice crystal formation and deformation [20]. In former studies, sectioning was performed by fracturing under vacuum conditions which produced irregular surfaces and structures difficult to interpret [44]. However, techniques to gain flat surfaces were developed by which ice can be detected unambiguously [44]. Nonetheless, a trained eye is crucially important. Overall, the cryo-SEM procedure seems to be elaborate and requires expensive technical equipment.
To directly detect and observe ice in plant tissues, optical light microscopy techniques have been used since the 19th century [30]. Continuous improvements of resolution and particularly image quality gained more detailed insights. Transmitted-light microscopy in temperature-controlled conditions [45-48] and frozen specimens, which are incised and microscopically observed in reflected light [20, 29, 49], are the most common approaches. For transmitted-light microscopy specimens (organisms to cross-sections) are mounted on microscopic slides in liquid water. Indeed, freezing of this water may simulate extracellular ice, which allows monitoring of cellular responses to the presence of ice. Undoubtedly, the amount of frozen water and the location of ice masses inside the tissue are unrealistic and can cause misleading interpretations. To directly localise ice crystals, plant organs can be cut and inspected in reflected-light microscopy. The identification of ice crystals is based on the optical appearance in terms of colour and shape. Unfortunately, this can be a challenging task, as it does not work equally well for all plant tissues. In certain tissues, differentiation between ice crystals, liquid water or resin can be impossible as surface light reflections interfere and cause a similar appearance. Although light microscopic methods are easy and comparably cheap, ambiguousness of ice detection might have triggered the deployment of the much more complex methods presented before.
To improve visibility of ice in plants, a prerequisite is a reliable distinction between the solid and liquid aggregation state of H20. The optical property “birefringence” is a feature of ice but not of liquid water. While water in the liquid state is known to be optically isotropic, ice crystals are optically anisotropic [50]. However, plant tissue constituents can also have birefringence, but if so, they can be detected in unfrozen samples. Birefringence can be assessed by polarised transmitted-light microscopy, which is a standard tool in mineralogy studies for material characterisation in thin sections or powders [51]. A good hint that birefringence properties of ice can be used to visualise ice gives the image of a frozen Cornus officinalis bud, which was photographed with polarised light [52].
In this study, we tested whether ice can unambiguously be distinguished from liquid water using polarised light in reflected-light microscopy. To rule out disruptive effects during preparation of thin cross sections and the necessity of a liquid sample carrier for transmitted-light microscopy, we used reflected-light microscopy to inspect frozen plant organs immediately after cutting. Inspections were performed in structural different plant organs from three freezing resistant species: in leaves of an evergreen shrub (Buxus sempervirens), in leaves of an herbaceous species (Galanthus nivalis) and in overwintering buds of conifers (Picea abies).