|
Live-Cell
Microscopy Environmental Control for Mammalian Specimens
by Daniel C.
Focht
One
of the most exciting aspects of biology today is found in
the small world of mammalian live-cell microscopy (MLCM).
The ability to observe and quantify mammalian live-cell
phenomena is made possible largely due to the recent
advancements in technological tools for imaging. These
advancements include microscopes, computers, detection
systems, dyes, reagents and micro-observation techniques.
We all know there is an unlimited wealth of dynamic
information just waiting to be discovered relative to cell
behavior, physiology, and morphology.
Typically,
live-cell experiments can be classified into two
categories; developmental studies to establish natural
behavior, or induced change to study the effects of a
controlled factor. Nearly all disciplines of cell research,
from the university level to pharmaceutical companies,
have embellished the benefits of this dynamic form of
live-cell imaging. There is, however, one area of
live-cell imaging which, until recently, has not seen
technological advancements contemporary with the other
imaging technologies.
This area
is the environmental control of the specimen with respect
to the limitations of optical microscopy. Frequently
investigators equip themselves with the most advanced
microscopes, computers, imaging programs, detectors, mass
storage devices, and exotic peripherals they can afford,
then cobble together a marginal micro-observation system.
Because the correlation between in vitro and in vivo
phenomena is paramount for mammalian live-cell biology, it
is of the utmost importance to accurately simulate the
host conditions of the isolated specimen during live-cell
microscopy. Therefore, it follows that the accuracy of the
data and thus the strength of the data's support should
not be weakened through the data acquisition process. This
article describes several new and contemporary
technologies which will greatly simplify the
micro-observation process for nearly all mammalian
live-cell experiments.
In
applications where single cell analysis is appropriate,
the specimen must to be contained in an optical enclosure.
The two most commonly used methods of micro-observation
are the open culture dish and closed-system observation.
Open
Dish Observation
In applications where cells are being observed with
low numeric aperture (N.A.) objectives, an open culture
dish is frequently used. This is optically very similar to
the observation of cells typically using phase microscopy
in a plastic culture flask. The sophistication of today's
experiments are far more demanding on the cell's
environment, especially as it relates to microscopy. For
example, during long-term developmental or induced change
studies it is necessary to accurately simulate the host
conditions on the microscope stage without limiting the
resolution of the microscope and maintain the simultaneous
flexibility to change variables. This simulation should
include control of temperature, medium, pH and
environmental toxicity. Medium and pH can be controlled by
regulated perfusion. The toxicity of the artificial
environment has to be evaluated against compatibility with
the specimen. Presuming the artificial environment is
non-toxic or biologically inert, one of the most difficult
factors to control has been temperature. Even with all the
relevant papers on the importance of accurate temperature
control and its effect on data, it should be readily
apparent that temperature is a critical factor in most
mammalian live-cell experiments.
When
using high N.A. applications most biologist have had to
rely on peripheral stage warmers and the use of carefully
supported coverslips to attain optical compatibility and
simultaneous temperature control. Many times this meant
sacrificing the accuracy of the thermal control, and/or
adequate perfusion of the cells resulting in either
inaccurate data, due to compromised cells or poor images.
The following outline demonstrates the limitations of the
traditional methods of open dish micro-observation.
- Conventional culture
dishes are made of plastic which is known to have the
following characteristics:
- Non-uniform
optical surface which degrades the image.
- Strain in the
optical surface due to the injection molding
process which prohibits the use of polarization
optics.
- Thickness of the
bottom surface prohibits the use of high N.A.
objectives.
- Inefficient heat
transfer from the source to the cells resulting in
long temperature stabilization cycles.
- Traditional culture
dish warmers are peripheral heating devices that must
radiate heat through two, poor, thermally conductive
mediums (air & plastic) therefore resulting in:
- Thermal inaccuracy.
- Non-uniform
temperature distribution.
- Excessive thermal
transfer time during thermal recovery (on the
order of minutes).
- Thermal expansion
of the metal surface causing vertical displacement
which is most apparent during 3D imaging or
confocal applications.
One
solution to the above problems is to observe cells in
culture maintained in a patented Bioptechs Delta T
live-cell, micro-environmental system. This new device
utilizes a thin-film coating of a transparent,
electrically-conductive coating applied to the bottom
surface of a glass substrate which is then incorporated
into a hybrid disposable culture dish structure optimized
for live-cell imaging. This technique provides temperature
control directly to the cells through a thermistor
feedback loop which applies an electrical current through
the coated underside of the glass substrate. The thermal
response using this technique is as fast as 1 degree C/sec.
The Bioptechs controller utilizes special, safety
circuitry that protects the cells and regulates the
control current intelligently within seconds making it
possible to compensate for temperature changes that occur
in the dish due to surface evaporation or perfusion. This
technique offers high resolution imaging capabilities
through a uniform glass surface free of strain and
available in a variety of thicknesses, including the
popular No.1.5 coverglass for high N.A. applications. The
dish environment is compatible with all modes of
microscopy including, but not limited to, brightfield,
darkfield, phase, DIC, fluorescence, reflection
interference and confocal.
The Delta
T technique provides the basis for many adaptations of the
basic principal. After becoming familiar with the accuracy,
ease of use and superior optical images obtainable in a
Delta T system, it is easy to envision and put into
practice numerous derivations for specific applications
other than isolated cell culture. The following are
examples of some of these uses:
-
In
tissue slice, natural or artificial membrane
observation, the specimen can be easily suspended at
an optical reference plane in the dish at the
appropriate focal distance from the objective for ease
or observation.
-
This
approach also works well with isolated solutions for
ion channel detection or cell migration studies.
-
The
Delta T can also be used for brain slice observation
on an inverted microscope with a brain slice adapter.
With this device the brain slice can be observed in a
temperature controlled optical chamber which can be
continuously perfused during high resolution
microscopy. This is of particular importance for
induced change and toxicology studies.
-
The
Delta T is also very useful for micro-observation of
smooth muscle cells in conjunction with the Kent
Scientific Rectilinear Force Transducer. With the Kent
transducer a section of muscle tissue can be attached
to the transducer and lowered into the Delta T,
enabling simultaneous reading of the force value and
correlating with optical observation on an inverted
microscope.
As you
can see, this system was designed with the live-cell
microscopist in mind and readily becomes an indispensable
addition to the microscope. There are a wide variety of
adapters and specimen carriers available that enable Delta
T users to take advantage of its superior capabilities.
Closed-System
Chambers
A closed system chamber is required when there is a
need for complete isolation of the specimen from the
outside world or the ability to perform more advanced
modes of microscopy than can be accomplished in an open
dish. In this case, cells are placed in a temperature
controlled, perfusable, optical cavity or plated onto a
coverslip which is part of this cavity for microscopy.
There are several traditional configurations of these
closed system chambers commercially available. Nearly all
of them utilize the same basic characteristics.
Traditional
closed system chambers provide two optical surfaces
separated by a perfusion ring sealed with gaskets. This
"sandwich" is then clamped together by several
other structures. With this type of enclosure the
perfusion rate, volume and optical suitability for various
modes of microscopy are interrelated. In most cases,
perfusion with this configuration results in a turbulent
jet stream which tends to dislodge the cells and there is
a trade-off for optical compatibility with all modes of
microscopy. Furthermore, temperature control is usually
achieved by the use of peripheral heaters or warmed air
blown across the stage. In either case temperature control
is not reliable nor controlled within an acceptable range.
When selecting a closed-system chamber the following
factors need to be considered:
- Optical compatibility
for the intended modes of microscopy.
- Temperature control,
including uniformity.
- Volume of chamber.
- Perfusion.
- Volume exchange rate
for perfusion.
- Cell surface shear.
- Imaging aperture.
- Thermal effect of
optically coupled (oil) high N.A. objectives.
A
solution to these problems is provided by the patented
Bioptechs microaqueduct perfusion technique. Microaqueduct
flow, the basis of the Bioptechs FCS2 closed-system,
live-cell micro-observation chamber, is achieved by
incorporating perfusion grooves into one of the optical
surfaces which defining the optical cavity, thereby
eliminating the perfusion ring common to most other
chambers and defining the optical cavity with only one
gasket separating the perfusion slide from the coverslip.
The physical configuration of these grooves produce a
laminar flow region in the optical cavity. The single
gasket design allows the user to define the size, volume,
thickness and shape of the optical cavity. In addition,
microaqueduct perfusion provides large aperture flow
inputs and outputs eliminating the problem of volume
exchange rates. To further enhance the performance of this
design, Bioptechs also includes the
electrically-conductive, thermal control coating on the
microaqueduct slide thus adding thermal uniformity to the
chamber even during experiments with period of no flow.
This
allows the specimen, adherent cells on a coverslip, to be
maintained safely in a temperature controlled optical
environment which is compatible with all modes of
microscopy, including but not limited to low and high
N.A., transmitted, brightfield, darkfield, phase, DIC, and
reflected modes of fluorescence as well as confocal.
High
N.A. Objectives
In high N.A. imaging it should be noted that the
optical medium used to couple the objective to the
coverglass will act as a heat sink. If the temperature is
not also regulated, this will result in a temperature
gradient across the field of as much as 5 degrees. The
microscope manufacturers have not yet met the live-cell
researchers' needs by providing integrated temperature
control for the objective so an external objective heater
must be used.
In order
to accurately control the temperature of the objective, it
is necessary to overcome the constant drain of heat from
any thermally conductive mass such as the nosepiece or
microscope frame. The thermal characteristics of all
objectives and microscopes vary considerably making it
necessary to provide an efficient transfer of heat to the
objective with an intelligent controller. Such a system
would sense the temperature of the objective at a point
close to the specimen and regulate the heat applied to the
objective while taking into account the thermal mass of
the objective and the ambient conditions. A patented
device for this purpose, which partially surrounds the
upper portion of the objective central tube with a heating
band and measures the thermal transfer in a gap formed
between the ends of the heating bands is available from
Bioptechs, Inc.. The controller regulates the heater
current in such a manner that it maintains the temperature
to within 0.1 degree C. Additionally, storing the
objective in a 37 degree C enclosure when not in use will
reduce the detrimental effects of thermal cycling between
physiological and room temperatures. Bioptechs also
provides enclosures of this nature.
Perfusion
Perfusion is necessary for one of the following two
reasons; either experiments on live cells take place over
a time span greater then cells will tolerate without fresh
media or the experiment is one in which chemical change is
induced through perfusion. In both cases it is necessary
to have a method of perfusing cells in their
micro-observation environment which will not impede the
experiment or the acquisition of electrical, chemical or
optical data. Due to the small volume of live-cell
chambers, the diaphragm affect produced at the unsupported
aperture of the coverslip and the sensitivity to shear
stresses of the cells, selection of a perfusion system
requires great care and in some cases a trade-off.
There are
three basic methods of perfusion -- gravity flow, manual
injection with a syringe or mechanical pumps. Gravity flow
is very inexpensive but difficult to control at flow rates
necessary for microscopy. Manual syringes are ideal for
adding growth factors, inhibitors or other periodic small
volume fluids. Mechanical pumps are the most reliable and
are available in two popular forms, motorized syringe and
peristaltic. The syringe pump is limited in volume for
long term experiments and subject to flow variations on a
micro flow scale due to temporary sticking of the plunger.
Before deciding which type is the most appropriate for the
application consider the following:
-
Flow
rate.
-
Uniformity
of flow rate.
-
Range
of flow rate.
-
Quality
of flow.
In view
of these factors, Bioptechs recommends the Micro Perfusion
pump for perfusion during microscopy. The Micro Perfusion
pump is a peristaltic pump small enough to be held in the
palm of one hand. It uses a tachometer regulated DC motor
and a multi-stage, step-down gear box to drive the roller
spindle resulting in a flow profile free from sudden
pulsations typical with most peristaltic pumps. It is
equipped with an internal speed control. It can also be
interfaced to a computer through an analog interface. The
pump can be configured to provide single tube perfusion
for closed chambers or dual tube for continuous
self-leveling perfusion in an open chamber.
Summary
The micro-observation approaches described in this
article demonstrate the commitment of Bioptechs, Inc. to
providing state-of-the-art solutions for live-cell
microscopy environmental control. Bioptechs techniques are
rapidly becoming the standard for live-cell observation.
Bioptechs live-cell observation products have wide ranging
applications in cancer research, pharmaceutical studies
and basic science, as well as direct medical uses in IVF
and clinical pathology. Bioptechs welcomes your questions
and suggestions.
About
the Author
Daniel Focht, founder and president of Bioptechs, Inc.
is an opto-mechanical/electronic engineer. He began his
career in optics when affiliated with the Carl Zeiss
Corporation, then furthered his experience in live-cell
microscopy when employed by Carnegie Mellon University to
develop automated microscopy systems for live-cell
imaging. Dan is currently designing and developing new and
more advanced systems for live-cell research.
|
