Our laboratory uses the Zeiss LSM 510 laser scanning confocal microscope which employs krypton-argon, argon, and helium-neon lasers (488, 514, 543 nm excitation lines), coupled to an Axiovert 200 inverted microscope and a Windows®-based host computer running the NT operating system.
We use it mostly for observations of immunolabeled protein targets and the temporal changes in their localization and expression. It was important for us to have good equipment which allowed for the precise discrimination of different states and the most accurate visualization of each fluorophore.
Some novel and important characteristics of this model have helped in obtaining more reproducible results and should be emphasized.
The LSM 510 can collect data in up to 4 channels simultaneously utilizing a quad-photomultiplier 12-bit A/D detection system, converting the light signals into a digital image of 4096 brightness levels. A complementary transmission light channel is available and is equipped with a photomultiplier too. It is, therefore, possible to superimpose a multiple-fluorescence image onto a brightfield, differential interference or phase image.
If you have a multi-channel image, you may save a colored overlay image as raw data in tiff format (8 bits per channel) suitable for importing into other programs. But saving files in the LSM format has advantages: Not only you will keep note of all parameters used in the experiment, but also you can benefit from Zeiss Image Browser software which facilitates classifying and finding images.
The system is controlled by Zeiss LSM software which possesses routine and expert modes. Using the latter, you need to set all parameters and functions upon your own decision; this mode, therefore, provides you with the greatest flexibility of operation.
The advertised scanning speed is 2.6 frames per second at 512 x 512 pixels. This is a valuable feature for rapid and precise analysis of fluorescence modifications, for example, in time series. However, in spite of such performance, this type of confocal microscope is not designed for temporal resolution, as is, for example, a spinning disc confocal system.
Users can measure fluorescense intensity using the LSM 510 within any region of interest; data is presented as profiles of intensity vs. wavelength. It is important to note that comparing fluorescense intensity from one image to another requires internal normalization standards. This imaging system is especially suitable for FRAP (Fluorescence Recovery After Photobleaching), because one may bleach regions of interest of various shapes.
In a multi-labeling system, the combination of several fluorophores often (if not always) results in overlapping signals. To address this problem, I highly appreciate using the overlaid signals separation method available in the LSM 510 (spectral unmixing, in other words). In order to obtain the true image of a fluorescent label (i.e. to measure the signal intensity of each fluorophore – and only that fluorophore), it is only necessary to prepare slides with mono-labelings of each of the fluorescent labels which have been used in combination. The signal from each fluorophore is recorded at a given wavelength and stored by the LSM software and applied to the final image generated from the combined fluorescent labels. The signal of each label is only applied for the wavelengths where it generates maximum signal. This innovative technique really helps in recording clean images.
To summarize, I would be glad to recommend this model to anyone searching for a good confocal microscope.