Maximize Fluorescence Performance with new Avant™ Filter Set FamilyIDEX Health & Science

    Maximize Fluorescence Performance with the Avant™ Filter Set Family

    Each member of the 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 fluorescent filter set 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

    In a fluorescence filter set, the excitation passband is located over the excitation spectrum for the fluorophore, and the emission passband is located over the emission spectrum of the fluorophore.

    Our ability to set the two filter passbands for best results depends on the Stokes Shift, which is the separation in nm between the peak excitation and peak emission wavelengths of the fluorophore. A significant number of popular fluorescent probes have small Stokes Shifts, less than 3% of their wavelength. Though the excitation and emission filter passbands for these probes would ideally be placed close together, this is not consistently the case.

    The more typical pattern for these short Stokes Shift fluorophores is shown in Fig. 1. The closest excitation and emission spectral edges to the crossover wavelength are known as the ‘critical edges.’ The so-called Gap between the critical edges is located quite asymmetrically with respect to the crossover point for the fluorophore, where the normalized excitation and emission curves cross.

    Figure 1: The "Gap" Gap between the critical edges is located quite asymmetrically with respect to the crossover point for the fluorophore, where the normalized excitation and emission curves cross

    Figure 1: Simulation of absorption (dashed red line) and emission (solid red line) spectrum for a fluorophore with corresponding excitation and emission filters (solid black lines).

    This asymmetry pushes the emitter passband out to longer wavelengths, and the result is less emission light capture by the emission filter and reduced filter set performance. The Gap has been difficult to reduce for two reasons: (1) A decreased Gap results in higher levels of excitation light bleed-through, i.e., excitation light reflected from the specimen plane into the emission filter, and (2) manufacturing variability limits our ability to place the two spectral edges in the narrow Gap without risking collision between the two filter edges at deeper OD.

    Because this 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 our Semrock optical filter coating performance and metrology capabilities, we set out to improve the performance of filter sets for short Stokes Shift fluorophores. This has resulted in the Avant Filter Set Family.

    Paradigm

    Three steps were used to work around 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 spectral edges were made possible by improvements in control of the filter coating process.
    • The placement of the spectral 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 9 and to resolve edges steeper than 0.2% of the edge wavelength, was used to confirm spectral edge characteristics.

    Results

    An example is shown in Fig. 2, which shows the modeled increase in fluorescence emission signal for the Cy5.5 fluorophore.

    Figure 2: the modeled increase in fluorescence emission signal for the Cy5.5 fluorophore

    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 the table below. The fluorescence improvement is significant, as shown in the rightmost column, and the excitation light bleed-through (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
    Avant Venus-YFP
    3.53
    4.54
    1.15
    < 10-4
    29%
    Cy3 555 nm LED (X-Cite NOVEM)
    Cy3-4040C
    Avant Cy3
    2.79
    4.63
    0.65
    < 10-4
    >50%
    Cy5.5 655 nm Lumiled LXM3-PD01
    Cy5.5-C
    Avant Cy5.5
    1.76
    2.30
    1.20
    < 10-4
    31%
    Cy7 740 nm CoolLED
    LED-Cy7-A
    Avant Cy7
    4.28
    5.38
    5.20
    < 10-4
    26%

    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 applications areas that prioritize efficiency, speed, and performance. In addition, Avant technology is now available for custom filter and filter set design. We welcome inquiries from all OEM developers of fluorescence-based tools.

    Learn More about Avant

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