The First Supercontinuum Confocal that Adapts to the Sample

With the new Leica TCS SP5 X and its white light laser source the user has complete freedom to choose the detection area in up to five spectral confocal PMT channels. The active acousto-optical beam splitter AOBS  allows the selection of up to eight simultaneous lines from anywhere in the white light spectrum (Figure 1). Optimal adjustment of the excitation line to the sample – in 1 nm increments – reduces cross-excitation and minimizes sample damage (Figure 2). The system precisely adapts to any existing or future fluorescent dye. With 200 nm of freely tunable excitation lines, it can be tuned to provide optimal excitation of any available dye. This flexibility is critical for core facilities that service hundreds of users with various samples and various dyes.

If comparing excitation spectra and available emission lines of conventional lasers, the gap is obvious: most dyes cannot be excited at the position of their maximum cross section. A common dye like Alexa 488, although it is designated as "488 nm" dye, has an excitation maximum at 500 nm, whereas the absorption at 488 nm is only 75 %. The Leica TCS SP5 X allows users to steplessly tune the excitation, no matter what the name of the fluorochrome is suggesting. So the fluorochrome can be excited at its maximum cross section, for Alexa 546 this would be 561 nm (Figure 3).

Nevertheless, this is not necessarily the best position of excitation. In order to prevent excitation light from entering the detector, a certain "security distance" has to be kept between excitation and the blue edge of the emission band that is collected. If the Stokes shift is rather low, then the residual window for emission collection might cut off a significant part of the available photons, which is not desirable. So it sometimes does indeed make sense to excite the dye somewhat off the peak in the blue range (Alexa 488 case) and compensate for the lower absorption by increasing the laser intensity.

The combined operation of tunable excitation and tunable emission can help to find the best setting for excitation and emission: a software tool is available that acquires images at incrementing excitation wavelength (excitation scan) and also adjusts automatically the blue cut off of the emission band, for example always 10 nm off the excitation to prevent reflected light from entering the detector.

A common challenge with multiparameter fluorescence is that excitation even by a narrow line will excite not only the targeted dye, but also other dyes in the sample. In most cases this is not wanted as the separation of various channels suffers from cross excitation making some applications, like FRET experiments, difficult to work with. Here again, the tunable wavelength of the white light laser source is an easy cure for cross excitation. The longer wavelength excitation can be moved out of the absorption of the blue dye. Here, an interactive optimisation for reduced cross excitation and efficient emission collection is easily done by dialing the excitation and adapting the emission band – online by a few scans only.

If separation is still not optimised, the system also offers sequential scan: that is recording a line with the first excitation and subsequently the same line with the next excitation for the next dye – and so on. Furthermore, the SP detector as a tunable device can be specified to collect sufficiently narrow bands for minimal crosstalk on the emission side. If the above procedures still do not provide optimal separation, linear unmixing for dye separation – emission or excitation – is also implemented.

As an example, a FRET AB (FRET by Acceptor Bleaching) experiment with the FRET pair Alexa 488 and Alexa 568 is described. This is only one out of a long list of possible FRET pairs, but commonly used. The bleaching of the acceptor is performed by the 543 nm HeNe laser line, if there is no better choice available. According to officially published data, the absorption of the donor is sufficiently low (<  2  %), to not interfere with the acceptor bleaching. The experiment was done as mentioned, and as a result, no increase of the donor was detectable, so one would conclude that the proteins are not "colocalised", and at least 10 nm apart. When measuring the excitation spectrum of the donor in situ by a supercontinuum confocal, it turned out that the absorption at 543 nm is indeed much higher, about 11  %. When the same experiment was done with laser light tuned to 580 nm for the acceptor, the donor emission increased significantly by approx. one third – corresponding to 33 % FRET efficiency. 580 nm is sufficiently away from donor excitation, so the donor is not bleached during acceptor bleaching and will emit more fluorescent light after the FRET partner is removed. The lack of increase in the previous experiment was due to bleaching of donor by the laser light applied for acceptor bleaching (Figure 4).

Excitation properties in situ

The white light laser source in combination with an AOTF as fast programmable line selector provides a very straight forward of generating excitation spectra from dyes in situ. The software allows for automatic incrementing of the excitation wavelength while the scanner takes images at each l-position. The standard operation will keep a preset emission band; the result is a direct measure for excitation dependence of the dye as a function of wavelength. A second operation mode will move the blue edge of the emission band synchronously with the excitation in order to always record the maximally available fluorescence. This is especially valuable for screening new or unknown dyes to find out the best excitation emission settings. Evaluation of excitation spectra is easily possible by just drawing regions of interest into the recorded images. The software will provide graphs of fluorescence for all regions. As the measurement is done in the sample directly, the results are much more reliable as compared to published data. Series of spectra with varying conditions of the solvent can be measured to find out about spectral changes, e.g. at various pH levels.

Fluorescent proteins excitation spectra in situ

A set of new fluorescent proteins was examined by excitation scans. The data may be used to optimise excitation and reduce crosstalk when recording images with those FPs. Also, this information is needed when planning FRET pairs. And in general, one can also study changes in fluorescence para­meters in living cells and under controlled conditions (Figure 5).

Fig. 5: Comparison of published data for excitation of mCherry and excitation scan with WLL (courtesy: Jalink K, Netherlands Cancer Institute, Amsterdam).

λ²-maps on cyanobacteria in Roman catacombs

As an example, the fluorescence spaces of vital cyanobacteria were measured. The specimen was a cyanobacterial film that was collected in a Christian catacomb of the Via Appia Antica in Rome. The microbial community is formed by different filamentous and chroococcal cyanobacterial species, together with bacteria. The spectral differences in the cyanobacterial cytoplasm are due to the intrinsic content in photosynthetic pigments of the three main types chlorophylls, phycobiliproteins and carotenoids. These pigments show species-specific variations in excitation and emission spectra, which help to identify types and physiology of the cyanobacteria. The cyanobacterial mats are identified as destroyers of Roman hypogean monuments.

Spectral correlation data were taken from a single optical layer by tuning the supercontinuum laser between 470 nm and 670 nm and recording emission spectra between 515 nm and 720 nm. Subsequent analyses of single individuals in regions of interest through the whole four-dimensional sequence reveal species specific intensity patterns. The intensities were normalised and color-coded to visualise the patterns: Very obviously, the two selected types differ in both excitation and emission properties. Upon blue excitation, Type A emits only at longer wavelengths, a very significant emission between 600 nm and 700 nm in Type B is rather absent in Type A. Many more details can be found and evaluated from data at higher spectral resolution (Figure 6).

Fig. 6: Excitation – Emission maps of fluorescence from biofilms. Two different species of cyanobacteria are presented and show variant patterns of fluorescence.

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