A new confocal detection technique in laser scanning microscopy opens up experimental possibilities that seemed totally unattainable in the past
Multicolour fluorescence microscopy is routinely used with biomedical samples to discriminate between multiple proteins, organelles, or functions in a single cell or animal and for the simultaneous visualisation of different structural or functional features.
Fret (fluorescence resonance energy transfer) can even approximate the physical relationships between individual proteins within the cell, and powerful techniques such as these have become invaluable in almost all biomedical research disciplines.
Traditionally, each fluorescent species is assigned a spectral band and sets of optical bandpass filters used to differentiate between the multiple fluorochromes.
However, even in simple systems where just two or three fluorochromes are used, spectral overlap or cross-talk can be difficult to eliminate, limiting the ability to distinguish one signal from another with any confidence.
The natural tendency is to assign very narrow bands within the emission spectra for detection.
However, this leads to a significant portion of the signal being discarded together with a much-reduced intensity.
Since only a small number of flurorochrome combinations can be efficiently separated with optical bandpass filters, there are limitations to the versatility of the technique.
Recently, a breakthrough approach to multi-fluorescence imaging has overcome these problems leading to the efficient separation of signals with extremely overlapping emission profiles.
Developed by Carl Zeiss and the California Institute of Technology, a new confocal detection technique in the LSM 510 Meta laser scanning microscope opens up experimental possibilities that seemed totally unattainable in the past.
So impressive is the new system's technology that R and D Magazine granted it one of this year's Oscar of Inventions in the R and D 100 Awards.
Emission fingerprinting.
The new technique combines innovative confocal detector technology with intelligent processing to capture the spectral signatures of up to eight fluorochromes.
Called Emission Fingerprinting, it differs from traditional methods in collecting the entire complex of emission signals originating from the multi-labelled sample rather than attempting to limit them in any way.
A multi-channel detector at the system's heart records not only the brightness distributions in the examined specimen but also the spectral composition of fluorescence light in each of the scanned object spots.
Using an optical diffractive element, the system splits the fluorescent light that has passed the confocal pinhole into its spectral components and projects it onto the detector, which consists of 32 photo-multiplier (PMT) elements capable of collecting photons across the whole visible spectrum.
Parallel recording produces a lambda stack - a three-dimensional image representing the complete spectral distribution of the fluorescence signal for every point on the confocal image.
These spectra are then separated, using digital deconvolution algorithms to perform linear unmixing and resolve the individual fluorochromes.
The LSM 510 Meta combines all the capabilities of the existing LSM 510 with a fast, sensitive, and reliable spectral imager, including robust linear unmixing functions integrated into the standard LSM system software.
The Meta hardware consists of a special grating as a dispersive element and the 32- channel PMT array to collect photons across the visible spectrum.
Neither the grating nor the detector moves; rather, turning on or off individual detector channels specifies the range of photons to be collected.
Thus, full flexibility of bandpass selection with solid reproducibility is possible and the system is robust and reliable.
The Meta spectral imager replaces a single conventional PMT in the LSM 510 scan head and can be used in combination with the other on-board PMTs, all with adjustable pinholes, for maximum flexibility.
In addition, current LSM 510 systems can be upgraded with the Meta detector, offering new technology to existing LSM 510 users.
The Meta can be used with all visible lines, plus UV and also multiphoton.
