INTERNATIONAL BIOTECHNOLOGY LABORATORY / JUNE 2002
20
APPLICATION NOTE
Fully enclosing chambers for environmental
control in live cell microscopy
DERRICK ATKINS
B
iological research capabilities have expanded considerably in recent years with the introduction of live cell
microscopy. A variety of image contrast enhancement
techniques, such as Differential Interference Contrast,
Hoffmann Modulation Contrast, fluorescence and confocal microscopy have all proved important. Combined with the
use of specialised dyes to highlight certain types or parts of the
cell or with the use of electrodes or perfusion techniques to
mimic in vivo stimuli, these techniques have created a rapidly
expanding area of research into the fundamentals of cell and
molecular biology.
In cloning and microgenics, the removal and replacement of
nuclei and the addition of cellular material using laser microbeam
or optical tweezer techniques are all commonplace. Meanwhile,
outside of the research world, developments in in vitro fertilisation now means that intra-cytoplasmic sperm injection (ICSI) can
be used to inject a single sperm cell directly into an egg. In order
for these studies to provide useful information, it is essential that
the cell’s host environment is reproduced as closely as possible,
whilst at the same time ensuring the cells are accessible to
microscopic examination.
Environmental control for live cell microscopy is therefore central to the study of cell growth, life and death, as well as to the
many cell manipulation techniques and high resolution imaging
work involving live cells. The longer the period of observation, the
more precise the control must be — with the most demanding
time-lapse studies being carried out not just over minutes or
hours, but in some cases, over days. The laboratory environment, however, can be a place of considerable heat differentials
and drafts, with the high-power laser light sources used in fluorescence and confocal microscopy being a major source of heat.
This demands the use of air conditioning systems, which
although designed to stop the lasers overheating, do not take
into account the sensitivities of live cells. Adequate control in
such an environment can obviously prove a challenge.
Since the introduction of live cell microscopy, a number of
approaches have been developed to control the environmental
conditions for a specimen under observation. While warm stages
have a heated plate attached to the microscope stage, perfusion
chambers can provide a heated ‘jacket’ around the living cells, or
in some cases, provide heating below. Stage top incubation
chambers using circulating warm air provide a closed vessel
across the full width of the stage, while full enclosure incubation
chambers use circulating air to surround the stage, nosepiece
and a large part of the frame.
Each of these approaches has certain characteristics that
must be understood in order to choose the best solution for a
particular application and budget.
Heated Stages
Using the heated stage approach, the cells rest on top of the
heated zone and receive heat through the face of the culture vessel. There are, however, three drawbacks to this. Firstly, vessels
positioned on a microscope stage are generally not placed in
direct contact since it is likely that the vessel will become
scratched. There is typically a gap of 0.5 mm between the vessel
Figure 1
The Solent Scientific chamber provides full enclosure of the
microscope objectives and stage for control of
temperature and environment to maintain cell viability.
and stage. Secondly, the space directly beneath the part of the
specimen being examined will not be warmed, since this is open
to allow microscope observation. In the case of cells in multiwell
plates, for example, the well under observation at any particular
time is not actually on a warmed part of the stage. Thirdly, plastic is a poor conductor of heat. The consequence of these
effects is that there will be temperature gradients across the culture vessel, from the warmest areas in contact with the stage
around the periphery, to the area above the observation area in
the centre. Temperature gradients of up to 2ºC have been
observed across a 35-mm Petri dish and there is equivalent
uncertainty about the precise temperature in any part of the
growth medium. For vessels that can be returned to the incubator after quick examination or for basic low resolution
microscopy procedures where cells can be placed on a peripherally heated microscope stage, this may be acceptable.
However, for more advanced investigations using high resolution
imaging techniques, these temperature gradients represent a
significant threat to cell viability.
Perfusion Chambers
Perfusion chambers provide living cells with a flow of culture
medium, which in many cases, is the primary reason for their
use. As well as removing waste products from the cells, the
technique also allows experiments to be performed that involve
the introduction of reagents to alter cell physiology. While the
pumped supply of fresh medium helps maintain temperature
and control pH, it has not been designed specifically for this purpose, and as a consequence, heat loss and consequent temperature gradients can be excessive for many applications.
Stage Top Incubation
A stage top incubator system provides improved control
because the circulating warm air eliminates heat losses above the
growing cells. Although it is also possible to control pH by recirculating a fixed proportion of CO2 in the air, heat loss through the
observation hole in the centre of the stage still remains. This
means that the part of the specimen under observation will be at
a lower temperature than that at the periphery of the stage or culture vessel, and as such, temperature differences across the
specimen can be as much as 1ºC. An additional problem with the
use of circulating air is that evaporation can cause heat loss at the
surface and therefore lead to loss of medium.
Heat Drain
In high NA imaging using water immersion or oil immersion
objectives, the microscope objective is coupled to the coverslip
or in some cases directly to the medium in which the specimen
sits. The objective then acts as a heat sink. Clearly there is a considerable heat drain via the objective to the microscope nosepiece and frame. If the objective is not heated, it can create
temperature differences across the field of as much as 5ºC.
Full Enclosure
A full enclosure incubation chamber resolves many of these
problems since it maintains the space above and below the
specimen at a steady temperature. Filtered circulating air warms
the space within the chamber but also warms the microscope
frame so that heat isn’t taken from the specimen. Temperature
control can be optimised for an individual microscope frame,
while CO2 enrichment and humidity can also be incorporated to
maintain growth medium viability.
For confocal microscopy, the issue of cell viability may be secondary to the drift in focus when changes in temperature cause
expansion of the stage or frame. Shifts of the order of 40 µm in
focus position have been observed. When microscopy at this level
is required, the only realistic solution is to keep the whole microscope warm.
Incubation Chambers in Practice
Scientists in the Department of Biochemistry and
Immunology at St George’s Hospital Medical School use a range
of molecular and cellular approaches to study different aspects
of vascular and pregnancy-related diseases. Under the direction
of Dr Guy Whitley and Dr Judith Cartwright, the researchers are
investigating the regulation of trophoblast and endothelial cell
motility and survival in projects funded by the Wellcome Trust
and the British Heart Foundation.
Failure of trophoblasts to invade the uterus has been implicated in diseases of pregnancy such as preeclampsia. This may be due to changes in their ability to invade or
in their response to apoptotic factors. Endothelial cell motility,
which is important in repair of blood vessel injury and diseases
such as atherosclerosis, is the focus of a second project using
these techniques.
Fluorescence and time-lapse digital image microscopy in
these studies were carried out using an Olympus IX70 microscope and the images then analysed using Image Pro-Plus software from Media Cybernetics. A fully enclosed chamber was
used to maintain cell viability during these studies by controlling
the temperature and pH, while a heated stage within the chamber was initially tested but found to make temperature regulation
more difficult. The researchers originally worked with a home-
Figure 2
The Solent Scientific incubation chamber provides the precise
environmental control required for optimum cell viability durling
live cell imaging studies.
made incubation chamber that held single tissue culture dishes.
Constructed from an aluminium base with a plastic side ring and
an optical glass top, the culture dish was placed inside and then
surrounded by a heated cabinet. The team then moved to a
motorised stage system that allowed multiwell plates to be used
and so increase the number of experiments to be performed.
The CO2 gas was introduced through a hole in the side of the
plate although a new heated cabinet was required to house the
larger stage (Solent Scientific, Portsmouth, UK). According to
Dr Whitley, the cells remained healthy and viable for the duration
of the three-day time lapse microscopy experiments and so
allowed a large amount of data to be generated over a relatively
short period of time.
Cell Migration Studies
At the University of Liverpool’s Department of Physiology, Dr
Peter Noble is researching the inter-communication of cells in
the gastric epithelium, and in particular, how signals from the
hormone, gastrin, regulate cell movement. Cell movement is
critical in the gastric epithelium because it is involved in the normal healing processes and maintenance of structure. Aberrant
cell movement is also an important feature of gastric cancer.
Dr Noble uses an inverted epifluorescence microscope with
a motorised stage that is fitted within a fully enclosing Solent
Scientific incubator to maintain temperature and environment.
The group uses both a gastric cancer cell line with gastrin
receptors, as well as cells without the gastrin receptor expressing GFP, which are used in single and mixed populations in a
variety of assays to look at changes in morphology and cell
migration. Relatively long time-lapse periods of up to 24 hours
have been used to study cell movement.
In a mixed cell population, those cells without the gastrin
receptor (which are easily distinguished by their fluorescence)
also show gastrin-initiated movement. It is therefore clear that
there is a secondary factor produced by the gastrin receptor
cells and further work will look at primary cell cultures containing representatives of all cell types in the gastric epithelium.
According to Dr Noble, one of the advantages of using a fully
enclosing incubator is that a computer-controlled moving stage
has been easily incorporated within the chamber. This allowed
them to carry out simultaneous time-lapse studies of cell migration with, for instance, twelve concurrent time-lapse experiments recorded routinely from a single multiwell plate. Using
this system, the cells were still viable after three days but equally
importantly, the stability of the system in terms of focus was
also excellent. The researchers found that they could observe
the cells for long periods without any need to re-focus.
Derrick Atkins is Development Engineer, Solent Scientific Ltd,
The Enterprise Centre, Quartremaine Road, Portsmouth PO3
5QT, UK; tel: +44 23 9236 3377; e-mail: [email protected]