BrightLine LED-Optimized Fluorescence Filter Sets

Feb 7, 2024 by IDEX Health & Science

Over the past decade, LED-based light engines have largely replaced gas-discharge lamps (or arc lamps) for fluorescence imaging because LEDs enable faster channel switching, deliver discrete output wavelengths, consume less energy, and offer significantly longer lifetimes. An important factor to consider in this change in excitation technology is that the spectral signature and energy distribution of LED light engines differ from those of arc lamps.

This is illustrated in Figure 1 (below).

Because traditional filter sets are tuned to match the output spectra of arc lamps, the mismatch between the illumination spectra of the new LED lamps with the transmission spectra of the traditional filter sets causes a reduction in the brightness of the illumination reaching the fluorophores. As an example, consider the case of the DA/FI/TR/Cy5-4X-B (traditional) quadband filter set. The excitation filters in this set are optimized for the output spectrum of Xenon (Xe) or Mercury (Hg) arc lamps. When the light source is changed to an LED light engine of similar brightness, the overall transmitted energy through the four excitation channels is reduced, and the excitation intensity is extremely low within the Cy5 band.

BrightLine LED-based Light Engine Filter Set Solutions

To address the loss of transmitted energy caused by spectrum mismatch, Semrock developed a new generation of filter sets that match the excitation filters to the unique spectral peaks of the most popular LED-based light engines on the market today. Take, for example, the Cy5 channel of the previously mentioned quad-band set. Figure 2 (below) shows the improvement of transmitted energy when, for an LED light engine, the selected excitation filter is changed from the FF01-650/13 (traditional Cy5 exciter) to the corresponding FF01-635/18 (LED Cy5 exciter). It can be seen that the peak for the FF01-635/18 has been centered over the LED peak.

Designing new excitation filters for the LED-optimized sets has resulted in improvement of the transmitted signal for the DAPI and TRITC bands by 35% each in comparison to the traditional sets. Most importantly, the signal within the Cy5 band is restored, with an improvement of 750%.

Figure 2: “SearchLight™ simulation comparing the transmission through a FF01-650/13 (Cy5 exciter of traditional multiband filter set) and a FF01- 635/18 (Cy5 exciter of the multiband set optimized for LED Light Engines) when illuminated by a typical LED light engine.”

The Semrock BrightLine Benefit

The new generation of LED-optimized BrightLine filter sets offers several benefits over traditional multiband filter sets:

• Excellent Signal Quality: Excitation filters are spectrally positioned to deliver the brightest signal and the best signal-to-noise ratio in the industry.
• Reduced Complexity and Cost: Common excitation filters can be used for both single-band and corresponding multiband configurations. This interchangeability provides a seamless transition between single-band and multiband imaging when configuring the excitation filters into the LED-based light engine and can also provide significant cost savings.
• Wide Selection: The BrightLine LED filter set family includes over twenty filter sets, supporting single-band, full multiband, Pinkel, and Sedat configurations. The filter sets include penta-band (5 channel), quad-band (4 channel), and triple-band (3 channel) configurations for the most popular fluorophores.
• Affordable Eight-Color Imaging: The penta-band LED-DA/FI/TR/Cy5/Cy7-5X-A and triple-band LED-CFP/YFP/mCherry-3X-A filter sets have complementary channels that provide an easy off-the-shelf solution to image eight fluorophores within a sample. The separation among all eight channels of the penta- and triple-band filters makes eight-color imaging easy and affordable (Figure 3).

Figure 3 (below): SearchLight™ simulation demonstrating the compatibility of the LED-DA/FI/TR/Cy5/Cy7-5XA (penta-band LED set) and the LED-CFP/YFP/mCherry-3X-A (triple-band LED set)