FAQ

• What is the primary purpose of this optical spectra viewer?

  • The primary purpose of SearchLight is to provide a free, web-based tool for modeling and evaluating the spectral performance of components in fluorescence microscopy and optical systems. Learn more about the key features here.

• What types of spectral data can it visualize (e.g., absorption, emission, reflectance)? Can I simulate spectral overlap between fluorophores and filters?

  • Yes, you can simulate spectral overlap with SearchLight. SearchLight can provide you with the following data points:

    • Overlay Spectra: You can visualize excitation and emission spectra of multiple fluorophores alongside the transmission curves of filters and dichroics.

    • Quantify Crosstalk: The tool calculates how much signal from one fluorophore might bleed into another channel, helping you assess spectral separation.

    • System Performance Metrics:

      • Signal-to-noise ratio

      • Crosstalk coefficients

      • Filter efficiency

    • Radar View: After submitting your configuration, the radar chart shows the top-performing filter combinations based on overlap and separation.

    • Advanced Controls: You can tweak parameters like numerical aperture, autofluorescence, and detector sensitivity to refine the simulation.

• How do you use a spectral viewer?

  • Choose Your Components

    • Select one or more fluorophores from the database.

    • Add excitation and emission filters to see how they interact with the fluorophores.

    • Include a dichroic mirror to simulate beam splitting.

    • Optionally, add a light source and detector for full system simulation.

  • Overlay Spectra

    • The tool will display all selected components on a wavelength graph.

    • You’ll see how well filters match the fluorophore spectra and where overlap or bleed-through might occur.

  • Adjust Parameters, tweak the following:

    • Filter angle of incidence

    • Numerical aperture

    • Autofluorescence

    • Detector gain or sensitivity

  • Analyze Performance

    • Good spectral separation between channels

    • High transmission at desired wavelengths

    • Minimal overlap between excitation and emission path

  • Export or Save

• How can I add a spectrum to SearchLight?

  • To add a spectrum to Semrock's SearchLight viewer, you can use the “MyData” feature, which allows you to upload custom spectral datasets for components not included in the built-in library. Use the following steps to add a spectrum to SearchLight.

    • Log in to SearchLight- Visit https://searchlight.semrock.com and log in to your account. If you don’t have one, you can register here: https://www.idex-hs.com/sign-up

    • Navigate to “MyData”- In the interface, locate the “MyData” section—in the group of tabs to the left of the main graph at the bottom under “Detectors”. Click “Add”

    • Upload Your Spectrum File- Supported formats typically include tab-delimited TXT files or CSV files.

      • Your file should contain two columns:

        • Wavelength (nm)

        • Intensity, transmission, or other spectral value

    • Assign Metadata- After uploading, you can label your spectrum (e.g., fluorophore name, filter type) and categorize it appropriately.

    • Plot and Analyze- Once uploaded, your custom spectrum will appear in the plotting area and can be analyzed alongside built-in components.

    • Save or Share- You can save your session, export plots, or share with collaborators using the built-in tools.

• Which file formats are supported for importing spectral data (e.g., CSV, FITS, JSON)?

  • The preferred file type is .CSV

  • Files to upload should be ASCII text data with two tab-delimited columns. The first column should contain wavelength values (in nm), and the second column should contain spectrum values (between 0 and 1). Text headers above the column data will be retained for your reference. Comments should be preceded by a "%"

• Can users zoom, pan, or select specific regions of the spectrum interactively?

  • Yes, SearchLight offers interactive features for spectral plots. Users can zoom in on specific wavelength ranges to examine fine spectral details. Panning across the spectrum is supported, allowing exploration of broader or shifted regions. Users can adjust axis limits to focus on specific spectral windows. This is particularly useful when comparing overlapping spectra or evaluating filter performance near cutoff wavelengths.

• Is the viewer web-based, desktop-based, or available as a plugin/library?

  • The SearchLight viewer by Semrock is primarily a web-based tool. It is accessible through a browser at https://searchlight.semrock.com and does not require installation on a desktop system.

• How can I find the right optical filter set for my fluorophores?

  • Go to searchlight.idex-hs.com and choose your fluorophore from the menu on the left. To add the fluorophore the graph, click the arrow on the row that appears when you click on the fluorophore name. Once the fluorophore is added to the graph, click the hamburger icon ≡ next to the fluorophore name in the right-hand navigation and a drop down will appear including “Find compatible products”. This will give you an indication of which optical filter set is compatible with your fluorophore. We also have a reference chart on our website: https://www.idex-hs.com/fluorophore-reference-for-semrock-filters

• What filter combinations work best for my custom life science instrument?

  1. Deciding on a set of optical filters for a new fluorescence-based product is a complex task that requires careful planning to ensure a successful outcome. Use SearchLight to simulate optical filter combinations and spectral overlap. If you have any questions, please reach out to one of our experts: https://www.idex-hs.com/contact/contact-us/custom-optical-filters

• Can I compare transmission spectra of different filters?

  1. Yes, you can compare transmission spectra of different optical filters using Semrock’s SearchLight Spectra Viewer. Here are a few key features SearchLight can help you with:

     • Overlay Spectra: View multiple filters’ transmission curves on the same graph to compare bandwidth, edge steepness, and blocking regions.

     • Customize Filter Roles: Assign filters as excitation, emission, or dichroic to see how they interact with your fluorophores.

     • Zoom and Pan: Focus on specific wavelength regions to inspect overlap or separation.

     • Add Fluorophores: Overlay fluorophore excitation/emission spectra to see how well each filter matches.

     • Export Graphs: Save your spectral comparisons for documentation or presentations.

• What customization options are available for the plots (e.g., color schemes, axis scaling)?

  • Semrock's SearchLight offers several customization options for spectral plots to help users visualize and analyze optical system components effectively. Here's a breakdown of the key customization features:

    • Color Schemes- Each spectrum (e.g., fluorophore, filter, light source) is plotted in a distinct color. When modeling variations (e.g., angle of incidence or polarization), the modified spectrum appears in the same color but with a dotted line, helping distinguish it from the original.

    • Axis Scaling- Users can zoom in/out and adjust wavelength ranges to focus on specific spectral regions. Also, the intensity or transmission axis can be scaled to better visualize overlapping spectra or subtle differences.

    • Overlay Multiple Spectra- You can plot multiple spectra simultaneously, including catalog components and custom uploads. This helps in comparing filter sets, fluorophores, and system configurations side-by-side.

    • Custom Data Uploads- Through the “MyData” feature, users can upload tab-delimited TXT files or modeled spectra.

    • Simulation Options- You can simulate filters with specific AOI (Angle of Incidence), CHA (Cone Half Angle), and polarization states.

    • Interactive Plot Legend- The legend updates dynamically and allows toggling visibility of individual spectra for cleaner views.

• How do I reduce bleed-through in multi-channel fluorescence imaging?

  1. Reducing bleed-through (also called spectral crosstalk) in multi-channel fluorescence imaging is crucial for accurate signal separation and clean data. Here’s a guide to help you minimize it:

     • Choose Fluorophores with Well-Separated Spectra- select fluorophores with minimal overlap between excitation and emission spectra. Use SearchLight to visualize and compare spectra before committing.

     • Use Narrow Bandpass Filters

       • Excitation Filters: Target the excitation peak tightly to avoid exciting off-target fluorophores.

       • Emission Filters: Use narrow filters that isolate emission peaks and block adjacent channels.

     • Optimize Dichroic Mirrors- choose dichroics with steep transition edges and high reflectance/transmittance in the correct bands.

     • Adjust Imaging Parameters

       • Sequential Imaging: Capture each channel separately to avoid simultaneous excitation.

       • Laser Power: Reduce excitation intensity to minimize spillover into other channels.

       • Detector Gain: Balance gain settings to prevent saturation and artificial bleed-through.

     • Simulate Before You Build- use tools like SearchLight to simulate your full optical path and evaluate for crosstalk coefficients and signal-to-noise ratios

• How do you calculate signal to noise (SNR) ratio?

  1. SearchLight’s System Calculators allow a user to compare the fluorescence signal and signal-to-noise ratio or contrast of varying filter sets. To see the signal-to-noise ratio output, enter the filter, fluorophore, light source, and detector information into the fields. Then click submit. In the results table, look for the “Signal-to-Noise Ratio” data point. Higher SNR equates to better signal clarity and lower background interference.