SPECTRA III light engine versus SPECTRA X light engine: Increased Brightness For Faster Image Acquisition
The metric most important when evaluating light intensities required for widefield fluorescence microscopy is the irradiance (or power density) at the sample plane, expressed in mW/mm2.
Our data table shows irradiance generated by SPECTRA III® and SPECTRA X® light engines in the four principle excitation bands used in multicolor fluorescence microscopy. Irradiance required for widefield fluorescence microscopy is typically on the order of 10–100 mW/mm2. Clearly, such levels are provided by the SPECTRA X light engine; prompting the question, why would the higher irradiance levels provided by the SPECTRA III be useful?
Higher irradiance allows exposure times to be shortened while maintaining the number of fluorescence photons detected. Shorter exposure times provide increased temporal resolution in time-lapse image sequences and reduce the time required to acquire multicolor z-stacks or slide scans.
Lumencor’s SPECTRA III light engine provides the best of spectral breadth, brightness, and speed for even the most demanding imaging and high throughput applications.
Power measurements made with Coherent FieldMax II–TO power meter with PM3 thermopile detector. Nikon Ti microscope with Semrock FF409/493/573/652-Di02 quad dichroic.
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.
Specimen: Human kidney section
Microscope: Nikon Ti2 controlled by NIS Elements software
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.
AURA light engine®, Serial Number 1106, has the distinction of being the first production model 4-color light engine manufactured by Lumencor. In February 2009, it was shipped to the laboratory of Michael W. Davidson at Florida State University, where it provided eight years of maintenance-free service. Michael Davidson, renowned for his Molecular Expressions website and his contributions to the development of fluorescent protein labeling and super-resolution microscopy among other things, passed away in 2015.
With the generous collaboration of Eric Clark (Florida State University), AURA 1106 is now back where it began, at our Beaverton factory. We took the opportunity to measure the power output of AURA 1106 for comparison with the original benchmarks measured in 2009. The results show that nearly nine years after it was manufactured, the performance of AURA 1106 is essentially as good as new.
Integrated Array of Eight Powerful Solid-State Light Sources
More Power ● More Colors ● More Control
The next generation of solid-state illumination is here. In Lumencor’s SPECTRA III light engine, eight individually addressable solid-state light sources deliver unprecedented performance. Each color band provides on the order of a half a watt of optical power at the end of a liquid light guide. The constituent light sources include LEDs, Lumencor’s proprietary luminescent light pipes and lasers. The outputs of the sources are refined by bandpass filters and merged into a common optical train directed to the light output port on the front panel. The light output port has built-in adapter for connection to microscopes and other bioanalytical instruments through a standard, 3mm diameter liquid light guide, LLG. TTL trigger inputs are provided for all eight sources for applications requiring fast (10 microsecond) switching.
The SPECTRA III light engine delivers substantial increases in output power compared to its SPECTRA® and SPECTRA X® predecessors. The advantages are clear: YFP and Cy7 excitation outputs are increased five-fold; GFP and Cy5 exception outputs are doubled. Not only are the outputs more intense but they are sustained by active stabilization. An onboard feedback loop continuously monitors the light output and maintains constant light output over time. SPECTRA III is not only bright but undeniably reliable, stable and consistent.