In our laboratory, we routinely use fluorescent labels for visualization and localization of different protein targets, using a laser scanning confocal microscopy method. We most often use double or triple combinations of labels so it is important for us to have good equipment allowing the most sensitive visualization of each fluorescent probe, precise discrimination of different fluorescent labels, and reproducibility of our quantitative results. Also, the cross-talk necessarily present in multi-labelling should be limited as possible. That is why I am constantly monitoring technological advances in the field of confocal imaging, looking for the state of the art microscope that is most adapted to our needs.
Over a long period, I explored the capacities of the new spectral Nikon Eclipse C1si model equipped with four lasers: diode 405 nm, argon (488 nm), helium-neon green (543 nm) and helium-neon red (635 nm), and with three detection channels and one transmitted light detection channel. The system was coupled to inverted microscope Nikon TE2000-PFS. This confocal microscope allows two approaches: conventional and spectral, independent one from another.
The conventional module is a traditional one, which does not allow for recording of only a part of the spectrum of the fluorescent probe. But full spectrum recording is reliable when employed for a single label or even for two labels provided that their spectra are well separated. If you opt for the conventional mode, you gain more sensitivity, since all emitted light is captured on a single photomultiplier (PMT). By the way, the microscope can be bought without this module, thus saving money.
The spectral module is more interesting for use with multi-labelling systems. The Eclipse C1si possesses an innovative multi-anode PMT with 32 channels, which allows acquisition of a 320 nm wavelength range in a single pass, under the condition that you use a spectral resolution of 10 nm. This feature is interesting for living cells as it considerably limits the time of exposition to laser light, thus limiting cell damage. It is possible to visualize all 32 images (each issuing from a 10 nm-part of the spetrum recorder by one anode) superposed, or to “cut off” some parts of the spectrum from the final superposed image. Also, you can realize a spectral unmixing via a special algorithm to separate fluorescent labels used. On the other hand, if you need a higher spectral resolution (2.5 nm and 5 nm are also available), the wavelength range will be proportionally reduced, so you will have to program several laser passes depending on the range you wish to acquire. This also implies that you will have to manually create complete series of images from the previously recorded parts; this can be performed by the software, but is somewhat time-consuming.
Also, it is not possible to individually adjust the gain and offset depending on lasers which are used, because these parameters are common for all anodes of the spectral PMT. This means that it is impossible to program a “real” sequential acquisition on different parts of the spectrum. In this way, you can only adjust laser intensities to more accurately adapt acquisition parameters to the concentration and brightness of fluorescent labels, and an experienced user may rapidly feel uneasy with these restrictions. Otherwise, you should manually change all parameters (laser to use, wavelength range, intensity, PMT sensibility) between each acquisition, and this solution can only be envisaged if you use your confocal microscope on an ad hoc basis. An experienced user could also regret the presence of only four possibilities for fixing the pinhole diameter, which excludes the possibility of changing the pinhole diameter depending on the wavelength of the fluorescence.
But overall, this is an easy to use confocal microscope, appropriate for living cells and bright fluorescent labeling; it is especially suitable for beginners.