In our laboratory, we routinely use fluorescent labels for visualization and localization of different protein targets, using laser scanning confocal microscopy. 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 experiments should be limited as much as possible. This is why I am constantly monitoring technological advances in the field of confocal imaging, looking for a state of the art microscope adapted to our needs.
During a long period, I explored the capabilities of a new spectral Zeiss model, the LSM 510 Meta MK4, equipped with four lasers: diode (405 nm), Argon (458, 477, 488 and 514 nm), diode (561 nm) and helium-neon red laser (633 nm), with one spectral detector META, two conventional detectors and a detector of transmitted light. The system was coupled to inverted microscope Axio Observer Z1, and directed via acquisition software optimized for Windows Vista installed to a user PC.
First of all, it should be noted that the Meta MK4 (Mark 4) is the most recent version of the LSM 510 model. It has extended capabilities of real-time electronics to control multiple scan systems and imaging modes (useful for example, for monitoring calcium imaging when you build your ratiometric curves simultaneously with the acquisition of images).
This model offers a very interesting configuration for multifluorescence analysis, because it combines the spectral META detector (a 32-anode photomultiplier, generating a lambda series of images in one pass for the entire fluorescence spectrum) with two other single, conventional detectors. Thus, you can configure the range of the spectral detector to the best fit for your marker, simultaneously using other detectors to maximize the signal yield from other markers. However, it should be mentioned that there are some limitations for marker combinations, due to the configuration of dichroic mirrors. Thus, if you have three fluorescent labels, it will always be the one with the shortest emission wavelength to be sent to the first (conventional) detector, the one (or more) label(s) with the intermediate emission wavelength(s) will be sent to the META detector, and the one with the longest emission wavelength, will be sent to the third (conventional) detector.
Another feature that differentiates this confocal microscope from many other models is the presence of three pinholes (whereas most often, there is only one pinhole): one for each fluorescence detector. The diameter of these pinholes is adjusted automatically, depending on the wavelength of the fluorescence to be captured, which amends the specificity and optimizes the signal acquisition. Nevertheless, there is one imperfection in this conception: when you send more than one signal to your spectral detector, the pinhole diameter will be of the mean value of all markers captured by this detector.
To resume, all fluorescent label combinations are suitable for the Zeiss 510 Meta MK4 configuration and the only two limitations cited above will not hamper your experience. You may rely on the exceptional quality of this model and be sure that all its features are worthy of the fame of Zeiss equipment: Z-stage with 10 nm-resolution, excellent optics, real-time data management, friendly acquisition software with image resolution up to 8192 x 8192 pixels and a variety of supplementary software modules.