SubscribeDavid Will, President , Intracellular Imaging Inc. Dr. Eric Gruenstein, Professor, University of Cincinnati. The methodology for imaging intracellular calcium in individual living cells with ratiometric dyes such as fura-2 is now almost 20 years old.
During this period, many calcium signal transduction pathways have been found to involve a variety of subtle spatial phenomena, such as intra- and intercellular waves, as well as complex kinetic behaviors including repetitive spikes and oscillations. Systems to image and measure Intracellular Ca2+, pH, and a number of other signaling phenomena can cost as little as £18,000 and as much as £71,000. The differences in price are due to performance specifications of system components and software to integrate the components .
How fast you need to "go" and how brightly fluorescent your cells are will have a major impact on the specifications and cost of your system:
1. How fast are the changes you want to measure and how often do they occur? Changes that take 10 or more seconds from stimulus to peak are typical of the response of many cells to a wide variety of agonists, including hormones and cytokines, as well as growth factors. Measuring these changes does not require cutting edge technology. Measuring substantially faster changes canrequire more expensive and sophisticated components.
2. How brightly fluorescent are the cells you will be studying? Again it is fairly typical for many dye loaded cells to be easily seen on the microscope in a dimmed but not darkened room. Experiments with these cells do not generally require cutting edge cameras or very expensive objectives. Cells that are not bright due to small size or do not load dye well will require more sophisticated and expensive components.
What kind of system is required to do intracellular calcium measurements? All ratiometric calcium imaging systems, no matter how expensive, consist of essentially the same 5 components: a computer, a fluorescence microscope, a low light level camera, a device to change excitation wavelengths, and software to integrate them. Let’s consider each of these components in the context of the "typical" and "high speed" experiments.
Computer. The computer (sometimes grandly referred to as the "image processing unit") needs to be fast enough and have sufficient memory to rapidly process, display, and store the large amounts of data contained in each image. Fortunately, today’s PCs are so fast and memory has become so inexpensive that the cost of this component should be under £1,500.
Microscope. Nothing fancier than a simple inverted fluorescence microscope with phase optics and a camera port is required. Although vendors will often recommend large microscope stands with other features and options, these are, in the great majority of cases, not necessary and the price differential can be substantial. A complete, simple inverted fluorescence microscope should cost between £6,000 and £9,500. On the other hand, a large stand inverted fluorescence microscope similarly equipped is likely to cost between £14,500 and £21,500.
A "fluor" objective is one component of the microscope that deserves some more discussion. The brightness of an epi-fluorescence specimen is directly proportional to the 4th power of the objective’s numerical aperture and inversely proportional to the square of the magnification. For fluorescence work, our experience is that discretionary funds are better spent on a higher N/A objective as opposed to a larger stand microscope. Higher N/A objectives are essential when working with faster speed reactions or smaller cells that simply load less dye.
Video Camera. Capturing a sequence of fluorescence images for calcium measurement absolutely requires a low light level video camera. Usually this will be a CCD camera and the most important parameters to look for are the resolution (how many pixels/image), the number of bits/pixel (8, 10 or 12), whether the camera is cooled or not, and whether binning and sub-arraying of the images are available.
Increasing the number of pixels increases spatial resolution. Increasing the number of bits per pixel generally increases the light sensitivity and the dynamic range of the camera. Adding cooling permits exposure times of more than 3 seconds for detection of very faint objects. Binning increases the rate at which images can be acquired as well as the brightness of objects, but at the expense of spatial resolution.
Sub-arraying can dramatically increase the rate at which images can be acquired, but the size of the image and hence the number of objects detected is reduced accordingly. That said, for most types of experiments in the "typical speed" range on most types of cells, a 10-bit, uncooled camera with a resolution of 640 x 480 pixels is adequate. Such cameras are available for as little as £1,500 and very well equipped 12 bit cameras with binning can acquired for less than £3,500. Faintly illuminated cells, size of field of view, and fast reactions can necessitate more sophisticated and expensive cameras which can cost as much as £14,500.
Wavelength Changer. Finally, there is the question of which method to use for changing the excitation wavelengths. This feature is required for ratiometric measurement with dyes such as fura-2 for calcium and BCECF for pH. Two methods are available: filter wheels and monochromators.
Filter wheels are generally slower, being able to change between two wavelengths only a few times per second, but they are substantially less expensive. They are available for as little as £3,000. Monochromators can acquire pairs of wavelengths as fast as 250 times per second and they are somewhat more flexible in terms of selection of wavelengths, but they cost between £7,000 and £14,500.
So let’s take a look at data from an experiment conducted with typically responding cells on an economically configured system. Figure 1 shows image and kinetic data on intracellular calcium levels in human fibroblasts following stimulation with growth factors. Calcium was measured at one second intervals using a system consisting of a fast IBM PC with InCytTM calcium imaging software, a simple inverted fluorescence microscope, a 10-bit, 640x480 CCD video camera, and a filter wheel. Such systems are available commercially for as little as £18,000.
For applications requiring greater speed and improved image resolution, complete monochromator based systems capable of capturing image pairs 20 times faster than in figure 1 (i.e. 20 image pairs/sec) are commercially available for about £28,500 and up, depending on camera specifications. Data obtained from an experiment measuring calcium oscillations in rat cortical neurons using such a system are shown in figure 2.
Software. Finally, we come to the question of how to evaluate the software, the heart of the system. Your prime consideration should be ease of use because this, more than anything else, is likely to determine long-term productivity. You don’t want a system that only one person in your lab really knows how to use, because what happens when that person leaves?
The question then is how to determine how easy a system is to actually use. First, see how logical the arrangement of menus is and whether the terms used are clear and intuitive. A good rule of thumb to remember here is that flexibility is the enemy of simplicity. The more options you are given within each dialog box and the more dialog boxes there are, the more difficult the system is likely to be to use. Second, speak to other investigators using the same system.
A good company will give you the names of at least 5 or 10 people to contact. If possible choose people whose names you recognize. Fluorescence imaging of intracellular phenomena can be extra-ordinarily expensive, but it does not have to be. By understanding the requirements of your research, you can keep the cost of a system within a very moderate budget.
Figure Legends
Figure 1. Calcium concentration in serum stimulated human fibroblasts. Cells were loaded with fura-2AM, image pairs were obtained at 1 second intervals and analyzed using InCyt‘ ratiometric software from Intracellular Imaging Inc. (A) Pseudocolor montage of images of intracellular calcium concentrations as they change over time following stimulation with growth factors. (B) Kinetics of the calcium response, average of 3 cells measured individually in the same field of view.
Figure 2. Spontaneous calcium oscillations in rat cortical neurons in vitro. Cells were loaded with fura-2AM and intracellular calcium was measured at 50 msec intervals. The imaging system was similar to that of figure 1 except that wavelengths were alternated using a monochromator, images were acquired with a 12-bit CCD, and a microscope objective with a higher numerical aperture was used.
NB All prices are exclusive of VAT