Nikon's new Digital Eclipse C1si, aims to meet the need for more complete spectral information to allow the detection of more fluorophores and to facilitate the detection of a specific fluorophore.
When observed with a light microscope, fluorescent proteins are widely viewed as reporters of transcription in live cells. Most commercial confocal microscopes currently have the ability to collect two or three pre-specified colours simultaneously.
Background fluorescence from endogenous fluorophores or from interfering exogenous fluorophores can severely reduce detection or interpretation of the image signal.
With multiple labelled samples, the signal from one fluorophore is often much stronger than another and can spill over to an adjacent channel.
The Digital Eclipse is a confocal laser fluorescence microscope system, which can simultaneously acquire the fluorescence spectrum over a 320nm wide range in wavelength at 10nm spectral resolution or over smaller ranges at 5nm or 2.5nm spectral resolution.
New FRAP and FRET macros make it easier for scientists to perform fast Fluorescence Recovery After Photobleaching studies, and Fluorescence Resonance Energy Transfer techniques using its spectral unmixing capabilities.
"The use of multiple fluorescent labels has long been commonplace in the study of fixed specimens, and is now becoming established for in vivo and live cell studies," said Stan Schwartz, marketing vice president, Nikon Instruments.
"The Digital Eclipse C1si eliminates expensive custom filter sets and provides a time efficient, and cost effective, approach for acquiring optimum wavelength coverage," he added.
One feature of the new product is its ability to separate the signals of fluorescent proteins including CFP, GFP, YFP, DsRed, even in combination with closely overlapping antibody conjugates such as Alexa488, Cy3, and others.
Clean separation of the signals of probes from autofluorescence is also possible. Not so long ago only three fluorophores were in widespread use (fluorescein, rhodamine and DAPI), but now there is a wide range of fluorophores available, each with its own unique spectral characteristics.
This has generated a considerable problem for fluorescence microscopists because many different filter sets are required for double or triple labelled samples.
The problem at the moment is that filter sets use expensive interference filters and dichroic mirrors, which are often difficult to interchange. Collecting the entire spectrum allows the fluorophore to be identified and separated computationally for easy analysis.
Other features of this product worth mentioning are its optical design and signal processing capabilities. Nikon's Diffraction Efficiency Enhancement System (DEES) and fluorescence transmission technology optimise the signal reaching the photomultiplier tubes.
The Dual Integration Signal Processing (DISP) technology eliminates digitisation down time. Together, these technologies ensure the highest efficiency spectral imaging possible with optimum signal to noise ratio.
With the Digital Eclipse, acquisition of accurate fluorescence spectra in true fluorescence colours is now possible at a spectral resolution as high as 2.5nm.
Opening the confocal pinhole does not compromise this high resolution. The technology's spectral detector employs a mechanism to prevent illuminating wavelengths from blinding the detector.
Sensitivity calibration among fluorescence detectors is performed using a NIST-traceable illumination source verifying the response of each detector element.
Currently it can be very difficult to cleanly separate the signal from multiple fluorescent probes with conventional confocal microscopes because of the overlap of their fluorescence emission spectra.
Artifacts due to spillover can be especially troublesome in FRET microscopy where precise localisation of the source of the signal is required.
The C1si solves these problems by acquiring the fluorescence spectrum at a high resolution, mathematically separating the signals from each probe, and assigning it to a discrete data channel free from confounding spillover.
The result is a "stack" of images of the same object or scene, each at a different spectral narrow band or colour.
Spectral imaging is usually non-destructive which means it is easy to acquire the spectral characteristics of complex biological materials including living or prepared cells or tissue.