New strategies to enable Live Cell Microscopy with a stable temperature at the field of view
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- Pranjali Divekar (1), Philipp Tripal (1), Benjamin Schmid (1), Ralph Palmisano (1)
1. Optical Imaging Centre Erlangen (OICE), Friedrich-Alexander-Universität of Erlangen-Nürnberg, Erlangen, Germany
live cell microscopy, heated coverslips, primary cortical neurons, lysosomes, total internal reflection flurorescence microscopy (TIR-FM)
- Abstract text
Mammalian cells including primary cells and cell lines command immense importance in biological and medical research fields . Temperature is a significant parameter for survival of these cells in vitro which is accounted for while culturing mammalian cells. They typically are cultured under an environmental control such as a 37°C incubator, 100% humidity in the and 5% CO2 . Temperature is also monitored in live cell microscopy by imaging the cells in a similar 37°C incubation chamber supplemented with CO2. However, this still poses a major challenge. While the cell sample is imaged in an incubator at the optimum temperature, the temperature at the field of view is lowered. This occurs by heat transfer due to the immersion oil which acts a thermal bridge between the objective and sample . The intracellular environment is sensitive to these changes in temperature. The most obvious process affected by increase or decrease in temperature is growth rate. At temperatures higher than 40°C, growth is sharply inhibited. Protein synthesis is also inhibited at high temperatures and a heat shock response is induced when the temperature is lowered . Assembly of microtubules is drastically inhibited at temperatures above 39°C . Fast temperature dynamics and effective heat compensation would be required to address this issue.
Tracking of lysosomes within rat primary cortical neurons
An appropriate spatial organization of proteins is vital to polarized cells like primary neurons . Synaptic proteins and small molecule neurotransmitters have known to be synthesized locally within the metabolically active pre-synaptic axon terminal. Neurons, with their long axons rely on local protein biosynthesis. This local protein synthesis at sites away from the nucleus requires long distance transport of RNA granules whose traffic has been noted within the neuronal axons and dendrites. Until recently, the mechanism of how these RNA granules tether to the transport machinery was unknown. In 2019, a study carried out by Liao et al.  presented a mechanism for the transport of RNA which ‘hitch-hike’ on lysosomes via microtubule motor proteins by using annexin A11 present on the RNA granules (ANXA11) as a molecular tether.
Due to the importance of temperature for cellular processes, all experiments based on live cell microscopy, should be performed at constant temperature. Minor temperature variations at the field of view could have an enormous impact on the speed of cellular processes. In this study, we aim to identify cellular processes, which are influenced by varying temperatures. The stabilization of temperature at the field of view by using VAHEAT (Interherence GmbH) was established to ensure stable conditions for live cell microscopy. To study the effect of temperature on the lysosomal transport within axons of primary cortical neurons, live cell imaging was carried out with total internal reflection fluorescence microscopy (TIR-FM) by using VAHEAT to ensure dynamic temperature control at the field of view.
Methods and Materials
Primary cortical neuronal cells were isolated from E17-18 rat embryos in cooperation with the Department of Psychiatry, UK-Er (Prof. Dr. Fejtova). Neurons were seeded at a cell density of 1.25 x 105 cells/cm² with Dulbecco’s modified eagle medium (DMEM). DMEM was exchanged with Neurobasal maintenance media (NB; with glutamax and B27 supplement) after 3 hours. Lysosomes from the primary cortical neurons were stained with LysoTracker® (Molecular Probes©) and were tracked using the TIR-FM. Neurons were seeded in heated coverslips.
Results and discussion
Temperatures within a range of 30-41°C were tested. Lysosomal movement seemed to speed up with an increase in temperature by approx. 1-2°C. At 30-31°C, lysosomes were stationery and only showed an increase in speed at 37°C. At 39°C, velocity further increased but now displayed a substantial amount of Brownian movement and reached aplateauwhen temperature was increased up to 41°C. At higher temperatures i.e 40-41°C and also if held at 39°C for a prolonged period, morphological changes in the neuronal cell body and the axon were observed. These changes occurred to be irreversible.
The aforementioned TIR-FM data suggest that lysosomal velocity is dependent on temperature. However more data will need to be acquired for confirmation. Kymographs were used for data analysis. The multikymograph plugin from ImageJ was used but since this analysis is highly manual, it is likely to lead to a high standard deviation. In this regard we are investigating further approaches such as ‘particle tracking’ using Trackpy, a python-based software for semi-automated particle tracking.
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