? I need to do fluorescence microscopy of mammalian, neuronal monolayers that are undergoing electro field stimulation at physiological temperatures in a nourishing environment. Is there a way to create the conditions to conduct this experiment while having direct access to the specimen?
! Let’s look at the physical constraints of your experiment. Since you need direct physical access to your specimen you will need an open dish system. You will want the highest resolution images you can get so the specimens should be on a glass #1.5 coverslip. The most efficient means to maintain temperature is direct heating of the coverslip where the cells are adhered. Note: you don’t want to heat the stage and should only provide heat to the specimen. In this case your best choice is the Delta T Culture Dish System. It is a coverslip bottomed, open dish micro-environment that heats only the media and specimens in the dish with a very fast, closed loop feedback system to ensure precision temperature control. This system is designed to be used with numerous accessories that customize it to conform to your experimental constraints. The following configuration would be ideal for your experiment: Cells can be plated in a Delta T Dish and have an electrical field introduced to them by a fixture containing bipolar platinum electrodes that are attached to a flip in / flip out support mechanism mounted to a dish retainer. A supplemental Hinged Perfusion Adapter is used to support perfusion needles for irrigating and aspirating the dish to facilitate long term-experiments. The hinged accessories allow you to easily exchange dishes, maintain registration and re-establish your desired conditions.
If you are imaging with a high numeric aperture objective, which requires an optical coupling medium, you will also need to warm the objective to prevent it from acting as a thermal sink to your specimen. When warming an objective it is critical to apply heat to the most efficient thermally receptive location on the objective and incorporate the objective as part of the control loop. These characteristics are exclusive to the Bioptechs Objective Heater to prevent overshoot and establish accurate control. To reliably and continually perfuse the dish the Delta T micro-perfusion pump is highly recommended. It will deliver media to the dish at a rate slower than it is removed so the dish cannot overflow. You simply adjust the height of the pickup needle to the level you want in the dish. This simple configuration is easy to set up, easy to maintain and will offer years of reliable data collection.
When it comes to live cell imaging, planning and preparation is the key to a successful experiment. While planning the equipment configuration is not glamorous, it is fundamental to success.
First things first: Specimen type
Whether working with adherent cells, suspended cells, tissue, or artificial membranes, each specimen type can be imaged in a variety of ways depending on the type of microscope and experimental protocol. These tips will get you pointed in the right direction.
Know your scope
A scope, like an environmental control system, is made up of a base configuration and a number of optional accessories.
You may have an upright scope providing a birds eye view or an inverted scope providing a worms eye view. Each has a choice of optional optical components that define its functionality.
For upright scopes live specimens can be positioned on the scope using the following methods:
Adherent cells can be plated either on the bottom of a dish so that they are laying down and observed with either a low magnification long working distance lens or a higher resolution lens (water dipping lens or immersion) can be used providing there is a coverslip over the specimen.
If cells are to be perfused in a discrete flow channel, they should be plated on a coverslip that is incorporated into a flow cell where the cells will be in a hanging orientation with media flowing immediately under them.
Alternatively, cells or tissue can be plated on the bottom surface of a fluid optical cavity where media flows over the cells and observation occurs through the coverglass above the cells, and through the media in flow.
If no flow is required, cells plated on a coverslip can be placed face down on a spacer in a dish so that media is contained between the bottom of the dish and cells on the coverslip.
Cells in suspension usually need to be contained by either a parallel barrier such as two coverslips with the specimen in between to limit the range of motion or a thixotropic media, and are typically observed with low magnification lenses.
Tissue in any form such as natural or artificial membrane can be imaged in a variety of ways. The simplest is having the tissue resting directly in the bottom of a dish within media being observed with a dipping lens. A dry lens can be used. However, can run the risk of condensation on the lower element if the specimen is warmed. A Coverslip Lid can be used as a barrier providing the objectives being used have a long enough working distance.
Another popular method is placing the tissue on a nutrient membrane surface such as a Corning Snapwell™ membrane.
The Delta T dish provides the optical, thermal and fluidic containment and the membrane provides a porous surface for nutrient exchange. [A small tube can be introduced into the side of the Snapwell to supply fresh nutrient media to maintain long term viability of the specimen.
Note: In some cases it is best to isolate and contain the atmosphere in the dish when using dipping lenses. Atmospheric Control Barrier Rings are used for that purpose.
For Inverted scopes, there are more options to view live specimens because of the obvious advantage of gravity. Most cells will preferentially plate toward gravity thereby giving the objective a worms eye view of the “footprint” of the cells on a flat optical plane. There are many configurations to choose from depending on the experimental protocol. If a discrete well defined flow of media is not required for cells, the following configurations are preferred:
Adherent cells in a dish open to the atmosphere (usually short term imaging less than 20 minutes)
Adherent cells in a dish sealed from the atmosphere (longer imaging time but usually not days)
Adherent cells in a dish sealed from the atmosphere while being perfused.
(much longer imaging time, but media has to be pre-equilibrated prior to entering the dish)
Adherent cells in a 5% CO2 controlled dish without perfused
In some cases where the inherent fluorescence of media contributes to background fluorescence, a coverslip affixed to a sleeve can be lowered near the top of the specimen plane to reduce the volume of media above the specimen.
There are two ways of observing specimens in a FCS2.
Note if usingfluid coupled lenses with physiologically warmed specimens it is essential to also warm the objective to prevent a 5-7 degree temperature drop at the specimen. To avoid excess heat radiating from a lens heater that can over heat the specimen by convection, heat must be applied to the objective at the most efficient location and method while measuring the heat propagation through the objective. This method measures the heat propagation inclusive of external factors and regulates to the expected temperature at the specimen plane. The most harmful technique is to use an inefficient heat transfer device that only regulates itself independent of the objective. This method does not and cannot compensate for the continual heat sinking effect of the nosepiece.
There may be times a sophisticated environmental system is not needed. For instance, when doing short term imaging. It is still a necessity to prevent the specimen from cooling down. In this instance a peripheral warmer will suffice. The most common warmer is simply a heated metal plate resting on/or in the stage so that the specimen is heated. Unfortunately, the specimen is displaced in the Z axis due to the thermal expansion of the plate, not to mention the additional drift due to the absorption of heat by the stage. This effect is now unnecessary. An advanced design to eliminate this problem is accomplished by placing the specimen in a peripherally heated structure where both the specimen plane and the support surface for the heated structure are in a common nodal plane so the heated structure is resting on a Z axis stable surface that insulates heat from the scope. The Stable Z is the only peripheral warmer to employ this technique.
Depending on protocols, it might be necessary to supply 5% CO2 to media in order to maintain the pH of the media that is being perfused through an atmospherically closed chamber or dish. This can be easily facilitated with the following technique:
Start with a 5% CO2 tank equipped with a demand regulator (a demand regulator converts the tank pressure to ambient pressure). This is a great first step because now it is simple to move the gas from the regulator to water (or media) by virtue of its volume using a simple, inexpensive peristaltic pump. To adjust the flow rate, set the pump to deliver one bubble every 3 – 10 seconds. Each bubble is about 15 microliter. Then multiply the bubbles per minute by 0.015. This is a lot easier and less expensive than a precision pressure regulator.
Note: make sure the media is pre-equilibrated in a CO2 incubator before starting this process. Keep in mind that the purpose of this configuration is to maintain pH not establish it.
Two protocols that achieve these results:
5% CO2 is bubbled directly into media that will be transferred to a closed system cell chamber.
When providing gas to an atmospherically regulated environment, such as a lidded dish, the incoming gas must be 100% humidified. This prevents a shift in osmolarity by displacing the 100% humidity already in the dish with a dryer incoming gas. It is necessary to set up the CO2 delivery as above but deliver the gas to a micro-humidifier before the gas goes to its destination. The Bioptechs Micro-Humidifier bubbles the incoming gas through heated water in a closed tube like cylinder. The air or gas space above the water becomes saturated with water vapor. Finally, the outflow gas is captured in this saturated airspace and transferred back through the heated water and short coupled to its destination so that the vapor does not have a chance to condense. 10ml of water can last up to 2 weeks.
5% CO2 at 100% humidity must be the levels that arrive at the enclosed airspace above the media in the dish where cells are plate.