EX (Excitation Wavelength) Calculators

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In fluorescence, the excitation wavelength (EX) is the wavelength of light that is absorbed by a fluorophore to elevate it to an excited electronic state. The fluorophore then releases this energy as fluorescent light at a longer wavelength — the emission wavelength (EM). The difference between EX and EM is the Stokes shift. Selecting the correct EX wavelength (matched to the fluorophore's absorption maximum) and EM wavelength (matched to its emission peak) maximizes signal and minimizes background. Understanding EX/EM pairs is essential for fluorescence microscopy, flow cytometry, and plate reader assays.

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Fluorescence Principle

Photon absorption at EX wavelength → electron elevated to excited singlet state → vibrational relaxation → photon emission at EM wavelength (longer than EX due to energy loss). Stokes shift = EM_peak − EX_peak (in nm). Large Stokes shift allows better separation of excitation and emission light, reducing background.

Common Fluorophore EX/EM Pairs

  • DAPI: EX 360 nm / EM 460 nm (blue — nuclear stain)
  • FITC/Alexa Fluor 488: EX 490 nm / EM 525 nm (green)
  • TRITC/Alexa Fluor 555: EX 543 nm / EM 572 nm (orange/red)
  • PI (propidium iodide): EX 535 nm / EM 617 nm (red — dead cell stain)
  • Cy5/Alexa Fluor 647: EX 650 nm / EM 670 nm (far red)
  • GFP: EX 488 nm / EM 507 nm (green)
  • mCherry: EX 587 nm / EM 610 nm (red)

Filter Sets in Fluorescence Microscopy

A fluorescence filter cube contains: excitation filter (bandpass or shortpass — only passes EX wavelengths); dichroic mirror (reflects EX, transmits EM based on wavelength cutoff); emission filter (bandpass — only passes EM wavelengths to detector). Proper filter matching to the fluorophore maximizes signal and minimizes bleedthrough into other channels.

Spectral Overlap and Multiplexing

When using multiple fluorophores simultaneously, spectral overlap (bleedthrough) of one fluorophore's emission into another channel must be minimized by: choosing fluorophores with well-separated EX/EM pairs; spectral unmixing algorithms; and compensation controls in flow cytometry.

Glossary

Excitation Wavelength (EX)
The wavelength of light absorbed by a fluorophore to reach an excited state; should match the fluorophore's absorption maximum; paired with emission wavelength (EM) longer than EX by the Stokes shift.
Stokes Shift
The difference between excitation and emission wavelength maxima (λ_EM − λ_EX) in nm; larger Stokes shift allows better separation of excitation and emission light; reduces background.
Fluorescence Filter Cube
A set of three optical components (excitation filter, dichroic mirror, emission filter) matched to a specific fluorophore's EX/EM; used in fluorescence microscopes to separate excitation and emission light.

Frequently Asked Questions

The excitation wavelength (EX) is the wavelength of light a fluorophore absorbs. Absorbing a photon promotes electrons to a higher energy state. After rapid relaxation, the excited fluorophore emits fluorescent light at a longer (lower-energy) wavelength — the emission wavelength (EM). The Stokes shift = EM_peak − EX_peak. Example: FITC has EX peak ≈ 490 nm; EM peak ≈ 525 nm; Stokes shift = 35 nm. The EX must be close to the fluorophore's absorption maximum for maximum brightness; the EM filter must capture the emission peak without collecting scattered excitation light.

Match excitation filter to the fluorophore's absorption spectrum peak, and emission filter to the emission peak. Rule: excitation filter must overlap with the fluorophore's excitation spectrum; emission filter must overlap with the emission spectrum; these two filters should not overlap with each other (dichroic mirror divides them). Most microscope manufacturers provide filter sets matched to common fluorophores (DAPI, FITC, TRITC, Cy5). For custom fluorophores, consult the spectra viewer (Thermo Fisher Fluorescence SpectraViewer, BD Biosciences) to select optimal filters and check spectral overlap with other fluorophores in your panel.

The Stokes shift is the difference (in nm) between the absorption (excitation) maximum and the emission maximum of a fluorophore. Stokes shift = λ_EM − λ_EX. Typical values: FITC = 35 nm; DAPI = 100 nm; quantum dots up to 200+ nm. Larger Stokes shift: easier to separate excitation and emission light with filters; reduced background from scattered excitation light; better signal-to-noise. Small Stokes shift fluorophores require very precise filters and are more prone to excitation light leaking into the emission channel. When designing multiplex panels, choose fluorophores with large Stokes shifts and well-separated EX/EM from each other.

Bleedthrough (spillover) occurs when a fluorophore's emission extends into the detection channel of another fluorophore. This happens because fluorophore emission spectra are broad (10–100 nm FWHM) and often extend far into longer wavelengths. Example: FITC emission spills into the PE (phycoerythrin) channel in flow cytometry. Solutions: (1) Choose fluorophores with well-separated EX/EM profiles. (2) Use compensation controls (single-stained controls) to measure and subtract bleedthrough in each channel — standard practice in flow cytometry. (3) Use narrow bandpass emission filters. (4) Apply spectral unmixing algorithms for microscopy multiplexing.