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Opening up a new world of spectral imaging!


Fluorescent proteins are widely used as reporters of transcription in live cells observed with a light microscope. Additionally, proteins and other molecules of interest inside the cell can be tagged with fluorescent marker dyes. It can be very difficult to cleanly separate the signal from multiple fluorescent probes with

conventional fluorescence or confocal microscopes because of the overlap of their fluorescence emission spectra. Artifacts due to spillover can be especially troublesome in FRET microscopy where precise localization of the source of the signal is required.  To solve this problem, Nikon has developed exciting new technology to acquire the fluorescence spectrum at high resolution and to mathematically separate the signal from each probe and assign it to a discrete data channel free from confounding spillover. This technology is now available in a confocal laser fluorescence microscope system, the DIGITAL ECLIPSE C1si. This spectral imaging system 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. This flexibility cannot be realized with conventional imaging systems or with earlier generation spectral confocal microscopes. It opens up a new world of spectral imaging

Fluorescence unmixing of GFP and Alexa488
GFP is expressed in the nuclei of HeLa cells. Actin fiber is stained with Alexa488. Exciter wavelength is 488nm. This image is a projection of 32 images acquired with a wavelength resolution of 2.5nm, from 493nm thru 570.5nm
This graph shows the fluorescence spectrum of GFP and Alexa488. from 493nm thru 570.5nm at the wavelength resolution of 2.5nm per channel

The image below has been fluorescence-separated with EZ-C1 spectral unmixing software


Cells provided by Dr. Yoshihiro Yoneda and Dr. Takuya Saiwaki, Medical Department of Osaka University.



x-axis- Fluorescence Wavelength

y-axis-  Time (seconds)

z-axis- Fluorescence power (Pixel Value)



JC-1 is loaded to the nerve cells. The observed spectral change reflects the change of membrane potential of the mitochondria at intervals of 30 seconds. Excitation at 488nm/514nm. The figure shows an image at 0 seconds, which has been stacked with 32 images with a range of 530nm thru 607.5nm at the wavelength resolution of 2.5nm


 Image provided by Dr. Yasushi Okada, Cell Biology, Medical Dept of Graduate School, Tokyo University



• Variable spectral resolution (2.5nm, 5nm, 10nm), providing true high spectral resolution

• Single scan capture of the full spectral range

• Wide bandwidth, 400-750nm

• High precision 32 channel simultaneous acquisition, for fast spectral acquisition

• Point scanning spectral confocal imaging with Nikon’s legendary optics

• Enhanced sensitivity through unique & innovative optical and electronic design

• Polarization enhanced optical sensitivity using DEES

• Dual Integration Signal Processing (DISP)

• Superior data integrity

• Proven reliability of C1


The emission path of the DIGITAL ECLIPSE C1si spectral confocal microscope system, can be easily switched between a standard three channel the fluorescence detector and the high resolution spectral detector, allowing a single instrument to be used for the widest possible range of applications.

Spectral imaging focusing on brightness

The uncompromised optical design and signal processing of the DIGITAL ECLIPSE C1si places it among the most efficient spectral detectors available. The newly developed DEES (Diffraction Efficiency Enhancement System) technology (patent pending) and high efficiency fluorescence transmission technology (patent pending) both optimize the signal reaching the photomultiplier tubes. The newly

developed DISP (Dual Integration Signal Processing) technology (patent pending) eliminates digitization dead time. These technologies ensure the highest efficiency spectral imaging with the best signal to noise.

True spectral imaging

With DIGITAL ECLIPSE C1si, acquisition of accurate fluorescence spectra in true fluorescence colors is now possible at a spectral resolution as high as 2.5nm. This is possible because wavelength resolution does not depend on confocal pinhole size (patent pending). The DIGITAL ECLIPSE C1si spectral detector also employs a unique mechanism to prevent illuminating wavelengths from blinding the detector (patent pending) Sensitivity calibration among fluorescence detectors is performed using a NIST-traceable illumination source (patent pending), verifying the response of each detector element.

Wide band, high resolution spectral imaging

The high sensitivity and excellent signal to noise characteristics of the DIGITAL ECLIPSE C1si makes possible the high data acquisition rates necessary for live cell spectral imaging. Simultaneous 32 channel spectral image acquisition over a range of 320nm at 10nm spectral resolution is possible using either single or multiple laser excitation. Simultaneous 32 channel acquisition at 5nm or 2.5nm resolution is possible at the same speed at correspondingly reduced spectral range.

Imaging of unmixed fluorescent probes without crosstalk

The power of DIGITAL ECLIPSE C1si is that it can cleanly 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 these probes from auto-fluorescence is also possible.



Maximum Wavelength Range • 400-750nm, Wavelength Resolution • 2.5 / 5 / 10nm (switchable), Dynamic Range • 12 bits, Display Mode • 512 X 512 pixels (maximum) When acquiring spectral imaging , Speed of Spectral Acquisition • 2 seconds (minimum) When acquiring spectral imaging


Laser safety standard: Class III B Laser Product.  This product is controlled by EAR (Export Administration Regulations).  It should not be exported without authorization from the appropriate governmental authorities.

Micron Optics, 240 Cedar Knolls Road , Cedar Knolls, NJ 07927


973-267-5799 phone

973-267-6598 fax