When we felt a need to buy a new confocal microscope in our laboratory, I was charged to select a new one from recent models available on the market. We use our confocal microscope mostly for qualitative observations of fluorescent labeling of different protein targets, and for quantitative analysis of any temporal changes in their localization and expression. It was important for us to have good equipment allowing precise discrimination of different states, the most sensitive visualization of each fluorescent probe and reproducibility of our quantitative results. I had the benefit of a long test period for exploring the abilities of a new spectral Leica model, the Leica TCS SP5, equipped with an AOBS device (acousto-optical beam splitter). The model I tested was equipped with four lasers: diode laser (405 nm), Argon (laser lines 457, 476, 488 and 514 nm), diode laser (561 nm) and helium-neon red (laser line 633 nm), and with four spectral detection channels. The system was coupled to inverted microscope DMI 6000 Trino.
Some important characteristics clearly distinguish this confocal microscope from rival models. First, the patented AOBS device, which considerably enhances the sensitivity of the system, should be mentioned. Indeed, compared to dichroic mirrors which can cause a notable part of the emitted signal (up to 30%) to be lost, this device ensures a much better transmission. Moreover, being a single optical tunable element, it allows immediate employment of a new dye or laser line, thus avoiding acquisition of supplementary filters or dichroic mirrors.
In a multi-labelling system, the combination of several fluorophores often (if not always) results in overlapping signals. In this model, the specificity of signal recovering is guaranteed by the presence of up to five spectral detectors, and the presence of a prism-based Leica SP-detector (spectrophotometer-detector), which allows selection with high accuracy of the part of the spectrum to be recovered on each detector channel. Also important, the photomultipliers (PMTs) are refreshed to room temperature, so that their gain can be adjusted up to 1200V without increasing electronic noise, further improving the sensitivity of the system. Combined with AOBS, these features ensure a very good signal recovery.
Another novel feature is the SuperZ stage which permits distortion-free recording of large 3D stacks, due to its large 1.5 mm range. This stage is also available with XY motorization, and thus, it is possible to automatically create a mosaic of the entire region of interest via a dedicated mosaic wizard.
The interface is very friendly, and you do not have to think about the hardware configuration (dichroic mirrors assembly) necessary for your particular fluorescent marker combination.
This model offers the possibility of realizing a spectral analysis with steps of 3 nm, through a 400 to 800 nm range, and to realize spectral unmixing with or without previously recorded spectral data for markers used.
Also, supplementary modules are available to complete a basic version of acquisition software. Of particular note are the 3D reconstruction module (however, this module lacks rapidity and cannot compete with specialized programs such as Imaris) and the LCS physiology module with wizards for easy realization of FRAP and FRET experiments and ratiometry measurement for such applications as calcium imaging.
Also, it is now possible to record images with resolutions up to 8192 x 8192 pixels, combined with a new scanner concept allowing high frame rates (up to 250 frames per second), It offers a larger image field without turning to annoying mosaic procedures.
To summarize, I gladly recommend this model to anyone searching for a good state of the art confocal microscope.
Scientist
Cellular Imaging Department
Biovays