Light BYTES: September 2016

More is Not Always Better

Look at the two images of fluorescent actin below. At first glance, they appear almost identical, but closer inspection reveals a significant difference. The first image was obtained using a SPECTRA X light engine® with a 475/40 (CWL/FWHM#) cyan excitation filter. For the second image, a 475/28 cyan excitation filter was used, instead. All other image acquisition conditions were identical.

The detected fluorescence levels in the first image are higher than in the second image, due to the increased excitation bandwidth. However, the background levels are also higher due to a small amount of excitation light bleed-through. Bleed-through is due to the bandwidth of the excitation filter being too wide, allowing a fraction of the excitation light to pass through the emission filter to the camera.

The first image shows the typical manifestation of a bleed-through — a uniform intensity increase in all pixels that is most clearly seen in the non-fluorescent background signal levels. A plot of the pixel intensity values across the image shows the effect more clearly. Although the second image is not as bright as the first, it has superior signal:background characteristics, ultimately resulting in lower detection thresholds for weakly fluorescent structures.

# Center wavelength/full width half maximum bandpass, in nm
 

Pixel intensities along image vectors marked by yellow lines plotted below.

Pixel intensities along image vectors marked by yellow lines plotted below.


 
Technical Details

  • Specimen: Bovine pulmonary artery endothelial cells labeled with Alexa Fluor 488 phalloidin.
  • SPECTRA X light engine, single band exciters 475/40 or 475/28 (CWL/FWHM), cyan channel, 20% maximum output intensity. Semrock FF409/493/573/652-Di01 polychroic and FF01-512/25 emitter.
  • Nikon Ti microscope, 40X, 0.75 NA objective, Andor Zyla 5.5 camera, 50 ms exposure.

 

Green arrows mark background region.

Green arrows mark background region.


 
Learn more about our SPECTRA X light engine, used in the example above.

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New Improved Liquid Light Guides

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SPECTRA X: light engine delivered output delivered through original (10-10084A) and improved (10-10084B) 3mm diameter liquid light guides (LLG).

Liquid light guides (LLG) provide flexible and convenient conduits for light delivery to fluorescence microscopes and other bioanalytical instruments. In addition to isolating the microscope from any heat and vibration associated with the light source, passage through a light guide acts to randomize spatial inhomogeneity in the light source output.

One disadvantage, however is that up to 30% of the original source output can be lost in the process of coupling into, and transmission through, the light guide. We have recently modified the design of our liquid light guides to minimize these losses. The result is liquid light guides that deliver about 10% more output to the microscope than their predecessors.

Click here, for more technical information on Lumencor’s liquid light guides, optical fibers and microscope input collimators.

New SOLA SE FISH Light Engines®

New SOLA SE FISH light engines

Lumencor’s SOLA SE FISH light engine® provides output that is spectrally optimized relative to the excitation characteristics of fluorescence in situ hybridization (FISH) probes, delivering sufficient intensity to generate readily detectable fluorescence from weak hybridization signals. The SOLA SE FISH light engine seamlessly combines the outputs of five solid-state light sources in two configurations, with ultraviolet (SOLA SE 365 FISH®) and violet (SOLA SE FISH®) source options.

The main differences from the standard SOLA SE and SOLA SE 365 light engines are the red shifted spectral outputs of the cyan and yellow component sources, resulting in better matching to the excitation spectra of SpectrumGreen™ and SpectrumRed™ labeled nucleic acid hybridization probes.

This sort of application specific lighting highlights Lumencor’s industry-leading capability in mating optimal excitation with a readily configurable set of sources. Lumencor promotes such “assay specific lighting” for FISH today.

We welcome your new requests for other assay-specific lighting applications as you find them! Make an appointment to talk with one of our talented representatives. Contact us at info@lumencor.com.

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14940 NW Greenbrier Parkway | Beaverton, OR 97006 USA | 503.213.4269 | info@lumencor.com