A tool for live-cell confocal imaging of temperature-dependent organelle dynamics

Microscopy, 2024, 73(4), 343–348 DOI: https://doi.org/10.1093/jmicro/dfad064 Advance Access Publication Date: 12 January 2024 Technical Report A tool for live-cell confocal imaging of temperature-dependent organelle dynamics Keiko Midorikawa and Yutaka Kodama * Center for Bioscience Research and Education, Utsunomiya University, 350 Mine, Utsunomiya, Tochigi 321-8505, Japan * To whom correspondence should be addressed. E-mail: Intracellular organelles alter their morphology in response to ambient conditions such as temperature to optimize physiological activities in cells. Observing organelle dynamics at various temperatures deepens our understanding of cellular responses to the environment. Confocal laser microscopy is a powerful tool for live-cell imaging of fluorescently labeled organelles. However, the large contact area between the specimen and the ambient air on the microscope stage makes it difficult to maintain accurate cellular temperatures. Here, we present a method for precisely controlling cellular temperatures using a custom-made adaptor that can be installed on a commercially available temperature-controlled microscope stage. Using this adaptor, we observed temperature-dependent organelle dynamics in living plant cells; morphological changes in chloroplasts and peroxisomes were temperature dependent. This newly developed adaptor can be easily placed on a temperature-controlled stage to capture intracellular responses to temperature at unprecedentedly high resolution. Key words: confocal laser-scanning microscopy, microscope stage, organellar dynamics, organellar morphology, peroxisome, temperature control Temperature influences various biochemical activities in living organisms, including plants [1,2]. For example, chloroplast function is temperature dependent: at low temperature and weak light irradiation, the lipid composition of chloroplast membranes changes, leading to reduced membrane fluidity, inhibited stomatal reactions and reduced activity of enzymes involved in a series of biochemical reactions [3]. Changes in membrane lipids also affect chloroplast morphology [4–7]. Indeed, in mutant plants with altered chloroplast membrane lipid composition, the chloroplast thylakoid membrane is highly curved and the envelope membrane is much rounder, resulting in balloon-like chloroplasts with large interstitial regions [4,6]. Peroxisomes are responsible for the photorespiratory metabolism associated with the photosynthesis of chloroplasts. Under weak light irradiation, peroxisomes are in close proximity to chloroplasts with morphological changes [8–10]. This suggests that organelle morphology and physical contact may alter metabolic processes in response to the external environment. Peroxisomes are highly dynamic organelles whose morphology and abundance immediately change in response to changes in the extracellular environment [11–15]. Therefore, investigating the details of the environmental responses of these dynamic organelles will increase our understanding of stress responses in plants. Maintaining proper temperature control in specimens under the microscope is important when observing living cells and organelles [16]. The temperature of the specimen between the microscope glass slide and coverslip is greatly affected by ambient air temperature, direct contact with the objective lens via the immersion medium and the coverslip and heating due to illumination [17]. Confocal laser-scanning microscopy is an excellent tool for high-resolution imaging of organelles in live cells, but accurate temperature control is challenging. To control the temperature under the microscope, several custom-made microscopic stages have been developed [18,19]. Among these, Peltier-based temperaturecontrolled stages appear to accurately maintain specimen temperatures, but complete customization is not easy for many researchers. Peltier-based temperature-controlled stages are also commercially available, but they may not be sufficiently adiabatic to protect the specimen from the ambient environment [17]. Here, we report on a custom-made adaptor (Fig. 1a) that can be installed on a commercial temperature-controlled microscopic stage (Fig. 1b). Installation of this adaptor allowed us to precisely control specimen temperature under a confocal microscope. We demonstrated its effectiveness by detecting temperature-dependent morphological changes in chloroplasts and peroxisomes in the liverwort Marchantia polymorpha, whose organelles are clearly observable by fluorescence microscopy [20,21]. This adaptor offers a new tool for capturing temperature-related processes in various organelles of living cells at high resolution. Transgenic M. polymorpha expressing OEP7-Citrine [20] or Citrine-peroxisomal targeting signal 1 (PTS1) [21] were maintained asexually and cultured on half-strength Gamborg’s B5 medium with 1% (w/v) agar at 22∘ C under 75 μmol m−2 s−1 continuous white light. OEP7-Citrine consists of the N-terminal 50 amino acids of Arabidopsis thaliana outer envelope membrane protein 7 (AT3G52420) fused to yellow fluorescent protein (Citrine) [20], and Citrine-PTS1 is Citrine fused to PTS1 (Ser-Lys-Leu) at the C-terminus [21]. The light intensity was measured using a light meter (LI-250A; Received 8 November 2023; Revised 19 December 2023; Editorial Decision 26 December 2023; Accepted 8 January 2024 © The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract 344 K. Midorikawa and Y. Kodama A tool for live-cell confocal imaging of temperature-dependent organelle dynamics LI-COR Biosciences). One-day-old gemmalings (immature thalli grown from gemmae) obtained from ∼1 month-old transgenic thalli [21] were used for microscopy observation. A confocal laser microscope (Leica TCS SP8X, Leica microsystems, Wetzlar, Germany) equipped with a hybrid detector and a flexible pulsed white light laser was used to observe Citrine and chlorophyll fluorescence. An HC PL APO ×40 dry lens was used as the objective lens to avoid contact with the coverslip. Citrine and chlorophyll were excited by a 513 nm laser. Emission signals were captured at 520–570 nm for Citrine and 650–750 nm for chlorophyll. When observing Citrine fluorescence, chlorophyll fluorescence was blocked Fig. 1. The custom-made adaptor for the temperature-controlled microscope stage. (a) Design of the adaptor. The adaptor is made of copper. Thermal compound is applied between the adaptor and stage to increase thermal conductivity. Dimensions are in millimeters. (b) Exterior of the adaptor. (c) Schematic diagram of sample (specimen) setting in the adaptor. The x–y cross section is shown in ( (...truncated)