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
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
Traditionally, each fluorescent species is assigned
a spectral band and sets of optical bandpass filters used to
differentiate between the multiple fluorochromes.
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
The natural tendency is to assign very narrow
bands within the emission spectra for detection.
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
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.
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.
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.
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
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.
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.
spectra are then separated, using digital deconvolution
algorithms to perform linear unmixing and resolve the individual
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.
hardware consists of a special grating as a dispersive element
and the 32- channel PMT array to collect photons across the
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.
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.