In mammalian tissues, fluorescence excitation in the near infrared (nIR) wavelength region (>650 nm) is useful to avoid the confounding effects of visible-range autofluorescence. The image shown here, generously provided by Dr. Matt Kofron (Cincinnati Children’s Hospital Medical Center), exemplifies this advantage by utilizing excitation light from a Lumencor light engine. The specimen is a human kidney section, imaged at 20X magnification. The tissue exhibits high levels of autofluorescence from red blood cells excited at 445 nm, which is pseudocolored yellow in this image. White represents cytokeratin immunodetection using a secondary antibody labeled with the near-infrared fluorophore Alexa Fluor 750. Nucleii stained with DAPI are shown in blue.
Expanding the spectral range into the near-infrared also increases the number of targets that can be simultaneously detected by multicolor fluorescence imaging. A total of 5 targets plus autofluorescence are present in the image, making use of the full spectra range of the SPECTRA X light engine. Three of the targets are mRNA transcripts detected by single-molecule fluorescence in situ hybridization (smFISH) using the fluorophores Opal 520, Opal 570 and Opal 650. These are more clearly evident in an enlargement of the segment marked by the orange rectangle.
Technical Details
Specimen: Human kidney section
Microscope: Nikon Ti2 controlled by NIS Elements software
Beaverton, Oregon, (March 28, 2018) – Lumencor, Inc. investigated certain Excelitas products after their launch last year and concluded they infringe a number of Lumencor patents. The infringement pertains to light engines incorporating the high-intensity, luminescent-rod-based light sources that Lumencor calls “light pipes.” On February 2, 2018, Lumencor filed a complaint with the International Trade Commission (ITC) accusing Excelitas X-Cite Fire products of infringing Lumencor’s U.S. Patents 9,574,722, 8,493,564 and 9,395,055. These products were manufactured for Excelitas by their Canadian subsidiary Lumen Dynamics Group, Inc. On the basis of Lumencor’s complaint, the Commission instituted an investigation into violations of Section 337 of the Tariff Act by Excelitas.
Most recently, Excelitas has agreed to end importation of the accused X-Cite Fire products and also agreed not to import any light engines or light engine components which include a luminescent rod and infringe the Lumencor patents. On the basis of the agreement, both parties filed a motion for entry of a Consent Order and termination of the investigation. The Chief Administrative Law Judge of the ITC made an initial determination granting the motion on March 20. The Consent Order is expected to be issued by the Commission no later than April 19, 2018.
“Lumencor pioneered solid-state illumination for the life sciences with the development of high-intensity light pipe technology for light engines. Our technology and intellectual property are vital to the design, development and manufacture of our innovative light engines with best-in-class product performance,” said Steven M. Jaffe, Ph.D., Co-founder, President and CEO of Lumencor, Inc. “We are pleased that Excelitas decided to respect our intellectual property rights and stop the import and sale of the offending products that are accused of infringing our patents. We remain committed to enforcing our patents to preserve our exclusive rights in our patented light engine technology.”
About Lumencor, Inc.
Lumencor is leading the life sciences with light engines for bioanalysis. The company has developed innovative, powerful, pure, stable, durable and cost-effective lighting solutions designed for an array of instruments including fluorescence microscopes. Discrete outputs are available through the UV-Vis-NIR spectrum from a proprietary mix of independently controllable sources. Lumencor products provide more power than an arc lamp with the durability, stability, speed and flexibility of a solid-state solution. Lumencor products are available in OEM and off-the-shelf configurations.
The origins of bleedthrough and crosstalk, two common confounding problems in fluorescence microscopy, are quite often confused. The following three 40X images of FITC-labeled actin and Cy3-labeled mitochondria serve to demonstrate the different manifestations of, and remedies for, bleedthrough and crosstalk.
Image 1. SPECTRA X light engine®
Cyan channel excitation, 485/25 filter, Semrock LED-DA/FI/TR/Cy5-4X quad polychroic and emitter
Both bleedthrough and crosstalk are present in this image. Bleedthrough is manifested by the relatively high extracellular gray level in the image (compare with Image 2 where bleedthrough has been eliminated).
Image 2. SPECTRA X light engine
Cyan channel excitation, 475/28 filter, Semrock LED-DA/FI/TR/Cy5-4X quad polychroic and emitter
Bleedthrough has been eliminated by changing the excitation filter to one with a transmission band that does not intersect with those of the quad emitter. The cause of bleedthrough is transmission of excitation light through the emitter to the camera. Crosstalk, manifested by detection of perinuclear mitochondrial fluorescence, is still present in this image (compare with Image 3 where crosstalk has been eliminated to produce an actin-specific image).
Image 3. SPECTRA X light engine
Cyan channel excitation, 475/28 filter, Semrock single band FITC dichroic and emitter
Fundamentally, crosstalk is due to excitation and emission spectra of fluorophores such as FITC and Cy3 not being confined to discrete wavelength ranges. Even though Cy3 fluorescence is optimally excited by green (~550 nm) light, it is also excited by cyan (475 nm) light to a sufficient extent to be readily detectable in Image 2. The crosstalk signal is eliminated by changing the quad band polychroic and emitter for a single band dichroic and emitter to achieve increased blocking on the emission side of the detection system. This solution is a compromise, as it produces an increase in resolution at the expense of speed.
