Over the past decade, with the advent of powerful new microscope systems and a wide range of specific fluorescent probes, laser-scanning confocal microscopy has become an indispensable tool in cell biology. This is particularly true in the stem cell field, where it is now possible to visualize the expression and location of the key molecules that regulate cell fate and to track cell differentiation at very high resolution. For this reason, the YSCC has established a Cell Imaging Core. The scientific supervisor of YSCC's Cell Imaging Core is Dr. Michael Nathanson, Director of the confocal microscopy core of Yale's Center for Cellular and Molecular Imaging since its inception in 1989. Mr. Thomas Ardito manages the Cell Imaging Core, and Mr. Ardito is available on an ongoing basis for scientific, technical, and administrative assistance.
The Cell Imaging Core is equipped with a Leica TCS SP5 Spectral Confocal Microscope, 405UV which is located in Room 333 of the Amistad Building. Conventional confocal microscopes use dichroic mirrors and filters to select the laser line(s) for excitation, and the emission band for the fluorophores used. This means that a separate set of filters needs to be inserted for each different fluorophore or combination of fluorophores. When new sets of fluorophores are developed, such as new GFP variants or the new Alexa dyes, a new filter set has to purchased, and sometimes designed. This can be a substantial roadblock, since even preliminary tests need to wait for production of a prototype filter set. For imaging two or more fluorophores, complete separation can be obtained if either the excitation or emission spectra are completely separated, although in most cases the colors need to be imaged separately, after switching dichroics. Most combinations of fluorophores have overlapping spectra, and one always will have bleed through. The Leica TCS confocal provides an elegant alternative to dichroics: user tunable filters. The fixed filter sets are completely eliminated from the confocal operation. Instead, the new system is based on three tunable filters:
- An acousto-optical filter (AOTF) replaces the excitation filter. In a typical operation the excitation is from two lasers. An argon laser has 4 lines in the blue: 458, 476, 488, 514 nm, of which the 488 nm line is the one used to excite most fluors. A He/Ne laser provides lines in the red (633 nm) and green (543 nm). The AOTF can select any one of these lines or any combination of the six for simultaneous excitation of different fluorophores. The AOTF can be switched between spectral lines very rapidly, much faster than the scan rate, permitting overlaid scans with two excitation wavelengths. Also of major importance, the AOTF is used to attenuate the laser to minimize photodamage during imaging, or opened to full power for photobleaching.
- An acousto-optical beam splitter (AOBS) replaces the dichroic mirror. Again the AOBS can be tuned by the user to reflect and pass different wavelengths, and rapidly switch between them as desired. Three or more excitation lines can be selected at once and the emission from all of them sent to the prism detection system.
- The Leica SP prism spectrophotometer detection system is a powerful replacement for dichroic emission filters. Instead of using filters to separate the emission, it uses a prism. The emission fluorescence from the specimen passes through a pinhole and then through a prism that separates it into its spectral components. Up to three separate spectral components can be measured simultaneously. The ability to simply dial the desired band of emission to be collected is an enormous advantage over previous confocals, which rely on dichroic filter sets to do this. It should be noted, however, that the detection system is limited to collecting bands of the spectrum defined by the slits, and it will not always be possible to separate emission spectra that overlap.
The microscope is also equipped with a controlled environment cell culture chamber, necessary for extended live cell imaging under physiological conditions. This is another rare feature and will directly benefit stem cell researchers.
The YSCC also has a Leica DMI6000 B inverted fluorescence microscope with adaptive focus control for phase contrast, DIC and epi-Fluorescence. The adaptive focus control system allows for automated focusing for imaging live cells during long-term culture so that the microscope can remain in focus and not drift due to changes in cell culture or room temperature. The Leica AF6000 digital imaging software system allows for live timelapse microscopy, and includes a computer and dual monitors as well as a Cool Snap HQ2 Digital Camera. In order to maintain live cells over time, the microscope has an attachable Tokai Hit Stage Top Incubator, with CO2 and Temperature Control. This Leica microscope shares the same microscope base (DMI6000), as our Leica SP-5 confocal, as well as the same Leica Application Suite software as that of the confocal, so that users need only to learn one software platform to use on either microscope.
Access: The Core strives to provide easy access to all stem cell researchers in the State of Connecticut. The services are provided based on a “first come, first serve” basis. If the number of requests exceeds the capacity of the Core, requests are reviewed and prioritized by Mr. Ardito. The following principles of prioritization are applied to the users: (1) Connecticut State-funded research receives the highest priority; (2) among them, research on non-federally approved lines will have higher priority; and (3) among CT-supported research on non-approved lines, first-time users will have the highest priority. It is the YSCC's goal to provide everyone working on hESCs access to the Core. The access is controlled by card scanning system and Mr. Ardito authorizes access to users.
Scheduling and Fees: A web-based booking system is used for researchers to schedule their time on the system. Users interested in scheduling time on the system need to obtain access from Mr. Ardito. The current charge for using the Confocal microscope is $40 per hour and the Inverted Fluorescence microscope is $25 per hour. Recipients of Connecticut Stem Cell Research Fund awards and YSCC members will be charged a discounted rate.
There is no charge for use of the workstations, nor for the training.
Michael Nathanson, Professor
Thomas Ardito, Research Associate