?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.
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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.
? My PI just informed me our new project is to investigate the instantaneous effect of a compound on live cells. Some of the setup is intuitive but now I have run into issues I didn’t realize would be problematic. For instance in order to observe my cells what kind of containment is best to use? How do I expose the cells to an alternate media without compromising them? How do I precondition my media? How long does it take to acquire the expected changes? Who can help?
! Fortunately, you are not the first person to encounter these issues. As it turns out, this is a routine process used to investigate many cellular reactions. One by one in order, the type of containment vessel should be a parallel plate flow cell having a coverslip surface for imaging. There are several commercial ones available. The one most recommended is the Bioptechs FCS2.
When comparing to the others, the FCS2 was the only one that allows you to select or create any flow geometry you want including laminar flow instead of being rigidly confined to the manufacturers design. It also provided temperature support uniformly over the entire aperture of the chamber. It is compatible with all modes of microscopy you may intend to use and is easy to assemble. When it comes to perfusion you may be thinking it is a plumber’s nightmare but in fact it is already worked out. If you are working with two sources of media, one is just a nutrient media and the other has the variant factor. The plan is to image cells on the scope for about 30 minutes with nutrient media to demonstrate they are “happy” and not compromised in their foreign environment. Then, depending on experimental protocol, introduce the alternate media containing your variant factor while acquiring images of the effects.
If the expected reaction takes place slowly you can manually introduce the variant, but if the protocol requires a rapid introduction of the variant that is where it gets a little tricky and is best to automate the process. There are two sources of media coming together external to the optical cavity where the cells are plated. Therefore, there is a dead-volume between the adjoining flows and the cells, and a small diffusion gradient that occurs when one flow follows another. The goal is to get the variant factor to the cells at its source concentration ASAP to record the immediate reaction. To accomplish this there is a computer program that controls the flow rate of two independent, smooth flow, peristaltic perfusion pumps. The program allows you to; establish a maintenance flow of nutrient media for a predetermined amount of time that you control, then ramp down the nutrient flow while accelerating the flow of the variant factor, then sustain that rate for a for another determined period of time you chose to ensure the concentration of the variant media is at its peak. The Variant flow rate is then de-accelerated to a minimum rate for imaging. The process can then repeated to rinse the variant factor and restore the prior environmental conditions. This saves a lot of headache
making these transitions especially when it has to be consistently repeated many times for statistical reasons
If you can’t get away with hepes media, you will need to sustain a CO2 tension in the media to maintain proper pH. A number of CO2 systems are available but the one that makes the most sense starts with a 5% CO2 and air tank under pressure then reduces the pressure to ambient with a regulator. That way the gas mixture can be metered out by virtue of its volume with a simple peristaltic pump. If you place a 14 gauge tube into a flask containing media each bubble that rises is about 15 micro-liter. Therefore, all you have to do to establish the flow rate is count the bubbles per minute and do a little arithmetic!
The amount of time it takes to acquire results will depend on the concentration of the variant factor and your cells.
So you have everything planned perfectly; the lighting, media, a camera to capture the moment, and it all goes cold! What went wrong? Cells in vitro tend to be very moody about their environment and nothing kills the mood quicker than getting cold. Two important elements of imaging with high numeric aperture lenses that need addressed are, first the method of which the specimen being heated and secondly the heat sink factor of the objective.
The biggest problem with using peripheral heating systems such as stage heaters and stage top incubators is that it relies on the heat from the base to radiate to the specimen when a large percentage of that is absorbed by the microscope. Unsurprisingly, this causes a temperature variant across the specimen plane that heavily effects the cells growth and behavior. With such an inefficient heat transfer the re-equilibration time with changes in temperature or media introduction tends to be very slow. The thermograph below on the right indicates the disadvantage of peripheral heating. This is a thermal image of a 50mm culture dish in the center of a 100mm diameter uniformly heated, 3mm thick, aluminum plate with a 25 mm hole in the center. This image was acquired after 20 minutes of equilibration. Note the high temperatures of nearly 60° C, that it takes to reach 37° C in the specimen area. In this case heat that is not beneficial to the specimen is sunk into the stage causing Z-axis instability. In contrast the thermograph on the left shows the efficiency, accuracy and uniformity of a heating system that directs efficient heat to only the specimen (Delta T™ system). Notice the temperature of the stage adapter. It is nearly the same temperature as the room temperature background. The dotted oval shows where the edge of the stage adapter is in visible light. Only the specimen and media are heated. Power consumption is 0.9 watts because heat is only applied to the specimen area. There is no heat transmitted to the stage.
Once the specimen is being heated accurately there still remains the problem of the objective acting as a heat sink. In this instance the optical coupling medium (oil, glycerin or water) acts as a thermal coupling medium and draws heat away from the specimen. The thermal mass of a fluid coupled objective is overwhelming when compared to the thermal mass of the cells. To eliminate this thermal gradient, it is important to accurately and carefully warm your objective (Objective Heater). It may also be necessary to isolate the objective from the nosepiece turret for proper objective temperature regulation with a Thermal Spacer. When selecting an objective heater it is essential to select one that is referencing the temperature on the focal plane of the objective since this is the point that can affect the specimen. By using a system that is specifically designed to slowly heat the objective then hold the objective at the set point value you have eliminated the cooling factors that prevent cells from their natural behavior all while protecting your objective from damage of overshooting and inefficient heating.
Sample preparation has always been a notoriously time consuming task that tends to detract from the more essential functions of collecting and analyzing data. A few key factors come in to play with improving the process of plating cells for imaging and the time that it takes. Cell adhesion, media re-equilibration, an unobstructed path for free migration of cells are some of the important factors to improve plating efficiency. Cell adhesion is pendant on the surface the cells are being plated on. The chemical composition of the glass affects cell adhesion and all glass surfaces are not created equal. It is best to use glass that is alkaline free and designed for cell adhesion (check out the Bioptechs Delta T Culture Dishes, FCS2 coverslips, 30mm ICD coverslips, and Microaquaduct slides). Sometimes an ECM is required depending on the cell type and protocol, however, in all cases cell plating is improved with the use of Culture Cylinders. A unique attribute of using a Culture Cylinder for plating is the rapid, organic adhesion in a defined location on the dish. This ensures the best cells are in the most viewable position on your substrate for imaging. Culture Cylinders have made innovative improvements over pouring tripsinized cells into an entire dish of media and waiting. Loosely related to cloning rings; Culture Cylinders in contrast are autoclaveable borosilicate glass polished on one surface optically flat to create a hydrostatic seal to the substrate that cells are plated on so that grease is not required. This minimizes the volume of media used for cells to re-equilibrate to once tripsinized, thereby allowing cells to plate faster. Also by eliminating the use of grease there is no contamination induced by plating cells or obstructive surface to inhibit cell migration. As noted in a manuscript by S. Mathupala and A. Sloan (2009) using grease with cloning rings has its own set of inherent problems that have a clear effect on the plating process. By applying this method, cell plating has become more efficient and also enables a variety of new experimental configurations with multiple Culture Cylinders
Mathupala, S. P., & Sloan, A. E. (2009, April). An agarose-based cloning-ring anchoring method for isolation of viable cell clones. Retrieved August 19, 2016, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727865/