We have used Leica’s TCS SP2 Laser Scanning Spectral Confocal Microscope for more than a year. Our laboratory works with 3 lasers: an Argon laser (488 nm excitation), a Helium-Neon green laser (543 nm excitation), and a Helium-Neon red laser (633 nm excitation). These 3 lasers allow triple labeling using most common fluorescent labels which emit green, red or far red light. In our research, we often use triple labelings on sections of human or animal intestine. For example, we simultaneously analyze types I and II of neurokinin receptors and G proteinsd using Alexa Fluor 488, TRITC and Cy5 conjugated antibodies.
Since this confocal microscope is really complete, it is impossible to detail all of its available functions. I will, therefore, emphasize those features which distinguish this confocal microscope from others I have used.
The “Zoom In” function is particularly easy; it enlarges a selected region of interest (ROI) to the maximal size as determined by the display window size. The navigation in Zoom mode is very comfortable. You can directly choose a region to enlarge as well as easily return to the initial state via the “Undo Zoom” button. In this way, you will never feel the need to save an x/y position, although that feature is also available.
Different scan modes are abundant. In addition to the classical modes (xyz, time-series), you can access a plane image or a z-series with different wavelengths (lambda series). This is particularly interesting when applied to dye separation in lambda series, with or without the use of reference spectra as it allows you to simultaneously divide the recorded signal into different channels.
You have a choice of digitization with an 8 or 12 bit depth of the pixel. Eight-bit digitization is completely sufficient for most applications, nevertheless, for image recording with very high intensity dynamics (e.g. for material specimens with high reflectivity of the surface and occurrences of very dark areas at the same time), 12-bit digitization proves useful, resulting in 4096 grey levels per pixel. It is to be noted that the data volume doubles with 12-bit digitization and this format is rarely supported by software. I recommend using alternate image analysis software, such as Bitplane software.
Curiously, the features which can appear superficial prove extremely useful and ergonomic during long confocal sessions. For example, Leica’s acquisition software systematically records all parameter settings including zoom, gain, offsets; thus, the user is exempted from manually noting these parameters. Also, this simplifies using the settings from a previous experiment for recording a new experiment. This is especially valuable when you wish to compare the quantity of fluorescent signal from one experience to another. This lets you configure the settings for new image recordings with a single click with the scan parameters that have been optimally configured for a previous application.
At frequent points during image recording, various parameters have to be reset. For this reason, the control panel with 7 buttons (in front of the user) can be used to control functions quickly and directly. Nice feature: You can pre-program and save your own configuration of the panel buttons attributing the function that you prefer to each one (for example, gain, offset, zoom values). You can then later retrieve every single configuration by simply clicking its name. Given that 30% of time at a confocal microscope can be lost to configuring parameters, and not observation, such practical aspects become valuable!
Overall, the Leica TCS SP2 is a very nice piece of equipment, one which is easy to share with a team.