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Transcript of Hyper-g Microscopy LIVE CELL IMAGING UNDER HYPER ... Hyper-g Microscopy LIVE CELL IMAGING UNDER...

  • 8 | B U L L E T I N D E C E M B E R 2 0 1 2

    H y p e r - g M i c r o s c o p y

    L I V E C E L L I M A G I N G U N D E R H Y P E R - G R A V I T Y

    C O N D I T I O N S

    YOUSSEF CHEBLI1, JACK J.W.A. VAN LOON2,3 AND ANJA GEITMANN1

    INTRODUCTION

    Long-term space missions and implementation

    of permanent bases on the Moon and Mars

    will require the detailed understanding of the

    functioning of biological processes under altered

    gravity conditions. Investigations at cellular level

    generally rely on or comprise microscopic studies

    - be it to investigate cellular structures, transport

    processes, expression of proteins or localization of

    cellular components. To study the effects of altered

    gravity conditions at cellular level, researchers

    have resorted to installing microscopes at the

    International Space Station (to operate under

    micro-gravity conditions) or to tilt microscopes

    to allow for imaging of objects such as plant

    roots positioned parallel to Earth's gravity vector

    [1-5]. Both set-ups are associated with certain

    technical challenges, but their long-term use does

    hardly pose a mechanical risk to the microscope

    during its operation. The third experimental

    condition that is traditionally used for gravity

    research, hyper-gravity, is more challenging in

    this regard. At least two technical solutions can be

    envisaged to perform microscopic imaging under

    hyper-gravity conditions: either a microscope is

    built specifically to spin around itself exposing

    the specimen to centrifugal force, or an adapted

    conventional microscope is placed into a large

    centrifuge that is rotated to apply centrifugal

    acceleration on the entire device. The former, a

    light microscope unit mounted on a centrifuge

    plate has been built in the 1990s for experiments

    ranging between 1 and 5g [6]. However, this

    particular microscope (the "Nizemi" or "slow

    rotating centrifuge microscope") is only able

    to acquire images in bright/dark field, phase

    contrast, and differential interference contrast. It

    was not conceived for fluorescence imaging. To

    enable fluorescence imaging, we opted to place

    a conventional epi-fluorescence microscope in

    the Large Diameter Centrifuge (LDC) at the

    facilities of the European Space Research and

    Technology Centre (ESTEC) of the European

    Space Agency (ESA) located in The Netherlands

    [7]. In the following we describe the experimental

    set-up and challenges as well as the biological

    system and the motivation for this project. To

    our knowledge this is the first time that live

    cell imaging using fluorescence has been used at

    hyper-gravity conditions.

    MICROSCOPE SET-UP IN THE LARGE

    DIAMETER CENTRIFUGE

    The LDC has four arms, each of which can

    support up to two gondolas with a maximum

    payload of 80 kg per gondola (Figs. 1A,B). The

    total diameter of the device is 8 m and g-forces of

    up to 20g can be achieved. An optical microscope

    (inverted Zeiss Axiovert 10) equipped for epi-

    fluorescence was fixed in one of the payload

    gondolas. During rotation the gondolas swing out

    so that the vertical axis of any object placed inside

    remains positioned parallel to the acceleration

    vector resulting from the centrifugal force and

    1 - Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal, Montréal, Québec, Canada

    2 - Department of Oral and Maxillofacial Surgery & Oral Cell Biology, Academisch Centrum Tandheelkunde Amsterdam (ACTA), University of

    Amsterdam and Vrije Universiteit Amsterdam, Research Institute MOVE, Amsterdam, The Netherlands

    3 - Life & Physical Sciences Instrumentation and Life Support Section (TEC-MMG), European Space Agency (ESA), Noordwijk, The Netherlands

    Corresponding author: anja.geitmann@umontreal.ca

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    H y p e r - g M i c r o s c o p y

    Figure 1. Large Diameter Centrifuge and experimental set-up (A) The LDC is located at the European Space Research and Technology Centre of the European Space Agency in Noordwijk,

    The Netherlands. It is composed of four arms supporting a total of up to 6 gondolas. (B) An inverted Zeiss Axiovert 10

    microscope equipped with a mercury lamp was fixed inside one of the gondolas allowing live observations of growing pollen

    tubes in Ibidi® cells (C).

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    H y p e r - g M i c r o s c o p y

    Earth's gravity. The microscope was equipped

    with a digital camera (Leica DFC 300 FX) for

    brightfield and fluorescence imaging. Since the

    weight of the whole system increases linearly with

    the applied level of centrifugal acceleration, and

    since the microscope used was a conventional

    model, we had to ensure that the system resists

    hyper-g conditions. Several major challenges had

    to be met when preparing the microscope for

    operation during centrifugation runs.

    1. Increased centrifugal acceleration caused

    a displacement of the condenser requiring

    refocusing of the samples during time-lapse

    imaging. To be able to perform this adjustment,

    the x-y position of the stage and the focus of

    the microscope could be remote controlled

    from an outside control room.

    2. Due to the centrifugal acceleration force, the

    optical and electronic equipment had to be

    firmly attached and reinforced. The objectives

    used here had spring-loaded retractable ends,

    which had the tendency to retract away from

    the sample at hyper-g. Fixation measures were

    applied to tightly fix the spring retractable

    end to the objective barrel and prevent any

    displacement.

    3. A conventional microscope slide/cover

    glass sample mounting would have risked

    dehydrating the sample. To ensure that the

    sample would not dehydrate or squeeze out

    during centrifugation, it was placed in tightly

    sealed wells of a 0.4 µm Ibidi® cell (µ-Slide VI

    0.4, IbiTreat) (Fig. 1C).

    4. Centrifuge operation always causes slight

    vibrations and made imaging difficult at high

    g-levels and high magnifications. To decrease the

    transfer of the vibrations from the gondola to the

    microscope, we suspended the whole microscope

    within the gondola using bungee cords (Fig. 1B).

    This set-up eliminated perceptible vibrations at

    acceleration levels of up 13g.

    THE BIOLOGICAL SYSTEM:

    POLLEN TUBE GROWTH

    The availability of ambient air, sustainable

    food supply and treatment of human waste are

    crucial for long-term space mission. All these

    requirements can be fulfilled through cultivation

    of plants on board the spacecraft or in the

    permanent bases on e.g. Moon or Mars. Because

    of their multiple roles, plants will play a primordial

    role in future space missions and understanding

    the plant metabolic and morphogenetic responses

    to altered gravity conditions is indispensable for

    the development of space craft ecosystems or

    long-term planetary colonization [8]. Cultivation

    of plants on orbital platforms affects growth of

    organs and individual cells as was shown in many

    plant species [9].

    Plants perceive the magnitude and orientation

    of gravity using at least two different principles:

    statolith based perception is based on the

    sedimentation of small starch filled bodies inside

    the cytoplasm that are of higher density than

    the surrounding cytosol - the statoliths. This

    mechanism is known to operate in specialized

    tissues such as the root cap. Hormone signaling is

    used to transmit the signal from the root cap to the

    rest of the plant and trigger a growth response in

    the growing portions of the organs. Interestingly,

    the majority of plant cells are not equipped with

    statoliths but are nonetheless known to respond

    to a change in the magnitude or direction of

    gravity using a different mechanism: statolith-

    less perception of gravity occurs in most plant

    cells, but the mechanical principle is not well

    understood. It is assumed that the protoplast is

    compressed under its own weight and/or that the

    cytoskeletal arrays are deformed [8,10].

    To assess the effect of altered g-force on plant cell

    growth and metabolism in a statolith-free system,

    we chose the model system pollen tube. The pollen

    tube is a protuberance formed by the pollen grain

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    upon contact with a receptive stigma. It carries

    the male gametes, the sperm cells, from the

    pollen grain to the female gametophyte nestled

    deep within the pistillar tissues of the flower.

    Since speed is a direct selection factor, pollen tube

    growth is the fastest cellular growth process in the