Question 1

What is a fluorescence excitation and emission spectrum?

Accepted Answer

An excitation spectrum shows the wavelengths of light that a fluorophore absorbs, while an emission spectrum shows the wavelengths of light it emits after excitation. The emission spectrum is always red-shifted (longer wavelength) relative to excitation due to energy loss during the fluorescence process.

Question 2

What is the Stokes shift and why does it matter?

Accepted Answer

The Stokes shift is the difference in wavelength between the excitation and emission peaks of a fluorophore. A larger Stokes shift makes it easier to separate excitation light from fluorescence signal using optical filters, reducing background noise. DAPI has a large Stokes shift (~101 nm) while YFP has a small one (~13 nm).

Question 3

What is spectral crosstalk and how do I minimize it?

Accepted Answer

Spectral crosstalk (bleed-through) occurs when emission from one fluorophore leaks into the detection channel of another. Minimize it by choosing fluorophores with well-separated emission peaks, using narrow bandpass filters, and applying spectral unmixing algorithms during image analysis.

Question 4

How do I choose fluorophores for multiplex imaging?

Accepted Answer

Select fluorophores with minimal emission overlap. Space emission peaks at least 40-50 nm apart. Match excitation peaks to available laser lines or filter sets. Use the crosstalk matrix in this tool to evaluate overlap between your chosen panel.

Question 5

Why are the spectra shown as Gaussian curves?

Accepted Answer

Real fluorophore spectra have complex shapes with shoulders and asymmetries. This tool uses Gaussian approximations parameterized by peak wavelength and FWHM for fast, interactive visualization. For publication-quality spectra, consult manufacturer databases such as FPbase or Chroma.

Fluorophore	Ex Peak (nm)	Em Peak (nm)	Stokes Shift (nm)	Category
DAPI	360	461	101	Blue
GFP	488	509	21	Green
TRITC	547	572	25	Yellow

	DAPI	GFP	TRITC
DAPI	---	29.6%	0.1%
GFP	69.3%	---	3.2%
TRITC	0.0%	3.2%	---

Fluorescence Spectra Viewer

Fluorophores (3/6)

Blue

Green

Yellow

Red

Far-Red

Near-IR

Excitation & Emission Spectra

Fluorophore Summary

Crosstalk Matrix

Fluorescence Fundamentals

Multiplex Panel Design

Frequently Asked Questions

Next Steps

Fluorescence Microscopes

Filter Sets

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