What is a Fluorescence Microscope?
A fluorescence microscope excites fluorescent molecules (fluorophores) with specific wavelengths of light and captures the emitted light at longer wavelengths. This allows you to see specific proteins, organelles, or structures invisible in brightfield or phase contrast.
If you're working with GFP (green fluorescent protein), RFP (red), DAPI (DNA), or Alexa Fluor dyes, you need a fluorescence microscope. Phase contrast and brightfield cannot see fluorescence.
How Fluorescence Microscopy Works
- Excitation: Light source passes through an excitation filter selecting a narrow wavelength band
- Dichroic mirror: Reflects excitation light toward the specimen while allowing emitted light to pass through
- Emission: Fluorophore absorbs excitation light and re-emits at a longer wavelength
- Detection: Emission filter blocks excitation light, camera captures only the fluorescent signal
Common Fluorescent Proteins & Dyes
| Fluorophore | Excitation (nm) | Emission (nm) | Filter Set | Used For |
|---|---|---|---|---|
| DAPI | 358 | 461 | DAPI (UV) | DNA/nuclei staining |
| GFP | 488 | 507 | FITC/GFP (green) | Protein tagging, gene expression |
| RFP/mCherry | 587 | 610 | TRITC/RFP (red) | Co-localisation, dual tagging |
| CY5 | 649 | 670 | CY5 (far-red) | Third channel, deep tissue |