Maximize Fluorescence Performance with the Semrock Avant Filter Set Family

Each member of the Semrock® Avant™ Filter Set Family provides significant improvement in fluorescence performance for its corresponding short Stokes Shift fluorophore. We present the rationale behind this development, and how it was achieved.

Rationale

A fluorescence filter set as used in biological microscopy consists of the following:

●  The excitation filter, which limits the excitation light wavelength range
●  The dichroic beamsplitter, which reflects the excitation light towards the sample and transmits the longer wavelength emission light towards the detector
●  The emission filter, which passes a limited wavelength range of the emitted fluorescence to the detector

The excitation passband is placed over the excitation spectrum for the fluorophore, and the emission passband, over the emission spectrum. The issue here is the optimal placement of these passbands and how that can depend on the size of the Stokes Shift.

The Stokes Shift is the separation in wavelength between peak excitation and emission wavelengths. Fluorophores with large Stokes Shifts have widely separated excitation and emission peaks, and the filter passbands can be centered on the peak excitation and emission wavelengths to optimize for maximum emission signal and minimum excitation light bleedthrough into the emission channel. However, the popular short Stokes Shift fluorophores have filter passband edges placed close together, and compromises between signal and bleedthrough have therefore often been required, as shown below.

The usual arrangement of filter passbands for short Stokes Shift fluorophores is shown schematically in Fig. 1. The excitation and emission spectral edges lying closest to the crossover wavelength are known as the ‘critical edges.’ In current practice, the so-called Gap between the critical edges is usually located quite asymmetrically with respect to the crossover wavelength for the fluorophore. This asymmetry pushes the emitter passband out to longer wavelengths, resulting in less emission light capture by the emission filter and therefore reduced filter set performance.

Figure 1: Schematic excitation (dashed red line) and emission (solid red line) spectra for a fluorophore with corresponding excitation and emission filters (solid black lines).

 

The Gap has been difficult to reduce for two reasons. First, the narrow Gap associated with short Stokes Shift fluorophores has resulted in higher levels of excitation light bleedthrough, i.e., excitation light reflected from the specimen plane into the emission filter. Second, the two critical edges must not be placed too close together, as shifts in their positions due to variations in production could result in reduced blocking. Though Semrock filters have been offering best-in-class consistency of this edge placement within and between production batches, closer placement of the critical edges requires an unprecedented improvement in precision of edge placement in production.

Because the described performance deficit holds for a significant number of popular short Stokes Shift fluorophores, there is systematic underperformance of corresponding filter sets over the industry. As IDEX Health & Science has recently made strides in improving the Semrock optical filter coating performance and metrology capabilities, we set out to improve the performance of filter sets for short Stokes Shift fluorophores, i.e., those with less than 3% Shift relative to the crossover wavelength. These improvements are now available in the Avant Filter Set Family.

 

Paradigm

Three steps were used to avoid the limitations described above:

1. The critical edges were significantly steepened from their versions in standard filter sets.
2. Once the edges were steepened, the critical edges were moved closer together.
3. Deep complementary blocking (OD 10, Design Specification) was implemented in both exciter and emitter to suppress excitation light bleed-through.

Recent key, proprietary advances at Semrock made this possible:

●  Steeper critical edges were made possible by improvements in control of the filter coating process.
●  The placement of the critical edges with increased accuracy was made possible by the Precise Edge Placement capability, which results in tightened statistical distribution of edge positions.
●  The KolaDeep™ Spectral Measurement System, with its ability to measure OD down to OD 8 or better and to resolve edges steeper than 0.2% of the edge wavelength, was used to confirm critical edge characteristics.

Results

An example is shown in Fig. 2, which shows the modeled increase in fluorescence emission signal for the Cy5.5 fluorophore. Note the improved position of the Gap, now placed symmetrically with respect to the crossover point.

Figure 2: The Avant filter set for Cy5.5 (red lines) increase the signal compared to the Brightline (blue lines) filter set.

Modeling was performed using the SearchLight simulation tool using measured spectra of the new filter sets, summarized in Table 1. The fluorescence improvement is significant, as shown in the rightmost column, and the excitation light bleedthrough (shown in the column to the left of the fluorescence percent increase) has been effectively eliminated.

Fluorescent Probe	Excitation Source	Filter Set	Fluorescence Signal × 106	Excitation Light Bleedthrough × 109	Fluorescence Signal Improvement with Avant
Venus	510 nm LED (X-Cite NOVEM)	LED-Venus-A	3.53	1.15	29%
		Avant Venus-YFP	4.54	< 10-4
Cy3	555 nm LED (X-Cite NOVEM)	Cy3-4040C	2.79	0.65	> 50%
		Avant Cy3	4.63	< 10-4
Cy5.5	655 nm Lumiled LXM3-PD01	Cy5.5-C	1.76	1.2	31%
		Avant Cy5.5	2.3	< 10-4
Cy7	740 nm CoolLED	LED-Cy7-A	4.28	5.2	26%
		Avant Cy7	5.38	< 10-4

Early results from a Semrock optical filter customer using Cy5.5 showed an increase of greater than 40% in fluorescence signal over background when the Cy5.5 Avant filter set was used in comparison to an incumbent filter set.

 

SUMMARY

The Avant Filter Set Family delivers improved fluorescence signal and signal-to-noise ratio to application areas that prioritize efficiency, speed, and performance in single-band filter sets. In addition, this new Avant technology is now available for custom filter and filter set design. We welcome inquiries from all OEM developers of fluorescence-based tools.

 

Article Source:  https://www.idex-hs.com/news-events/stories-and-features/detail/maximize-fluorescence-performance