Several factors contributing to poor fluorescent signals may include the inappropriate choice of filter cubes for the fluorescence microscope. This rationale could not be simpler: the excitation filter should correspond exactly to the dye excitation wavelength; the emission filter should contain the emission wavelength range, and finally, the dichroic mirror should effectively split the beam.
Matching Principle: Spectra are the sole standard—align the excitation filter with the “peak” (excitation peak), place the dichroic mirror at the “valley” (Stokes shift), and let the emission filter catch the “echo” (emission peak).
Core Principle of Selecting Filters: Spectral Matching
The transmission bands of the filters must overlap with the dye’s excitation and emission peaks while avoiding the emission ranges of other dyes as much as possible.
| Parameter | Meaning | Why It Matters |
| Excitation Peak | Wavelength used to excite the dye | Must overlap with excitation filter |
| Emission Peak | Fluorescence emitted by the dye | Must overlap with the excitation filter |
| Stokes Shift | Must pass through the emission filter | Larger shifts are easier to separate |
How to Choose Filters for Multi-Color Fluorescence: From Dye Spectra to Hardware Matching
Step 1: Clarify the Experimental Scenario
- Single Channel Imaging: Choose narrow-band excitation filters (10-20nm) for best SNR.
- Multi-channel Imaging: A combination of dyes having spectral differences (DAPI->FITC->TRITC->Cy5) should be chosen with narrow band+long pass combination filter sets for minimal cross-talk.
- Type of Excitation Light Source: Lasers (488nm, 633nm) for narrow-band EX, mercury lamps/LEDs for wide-band EX.
Step 2: Identify Dye Spectral Characteristics and Parameters (Essential)
- Before selecting, you must identify two core parameters (from dye manuals, Thermo SpectraViewer, or databases): Peak Excitation and Peak Emission.
- Many dye names provide clues; for example, Alexa Fluor 488 has an excitation peak near 488 nm.
Step 3: Match the Excitation Filter (EX)
The bandwidth of the excitation filter should span the wavelength corresponding to the maximum excitation of the dye.
- EX: CWL = EX_max ± 5 nm, BW = 20-40 nm;
- DM: Cut-on = (EX_max + EM_max) / 2;
- EM: CWL = EM_max ± 10 nm, BW = 10-30 nm.
Narrow-band/Bandpass: Best suited for multicolor labeling experiments, as the excitation wavelength does not excite other dyes.
Wide-band/Longpass: Generates high-intensity signals but has more background noise.
Step 4: Match the Dichroic Mirror (DM)
This value for the cut-off wavelength should be between the peak wavelengths for the excitation and emission spectra. The selection is usually made at the junction where the excitation spectrum ends and the emission spectrum starts.
Step 5: Match the Emission Filter (EM)
- The emission filter should coincide with the fluorescence of the dye.
- Key Idea: The transmission spectrum of the EM must not overlap with the transmission spectrum of the EX filter; otherwise, the background will become completely white.
Common Fluorescent Dyes and Matching Filter Cubes
| Fluorescence Channel | Common Dyes | Ex Peak / Em Peak (nm) | Excitation Filter (EX) | Dichroic Mirror (DM) | Emission Filter (EM) |
| Blue (UV) | DAPI, Hoechst 33342 | 350 / 461 | 360/40 nm | 400 nm | 460/50 nm |
| Green (Green) | FITC, GFP, Alexa 488 | 494 / 518 | 480/40 nm | 505 nm | 535/40 nm |
| Orange (Orange) | TRITC, Cy3, Alexa 555 | 550 / 570 | 545/25 nm | 565 nm | 605/70 nm |
| Red (Red) | Texas Red, mCherry, AF594 | 596 / 615 | 560/40 nm | 600 nm | 630/60 nm |
| Deep Red (Far Red) | Cy5, Alexa 647 | 650 / 670 | 620/60 nm | 660 nm | 700/75 nm |
| Near Infrared (NIR) | Cy7, Alexa 750 | 750 / 770 | 740/40 nm | 760 nm | 800/100 nm |
DAPI → DAPI Filter Cube
DAPI fluorescence occurs at a wavelength in the blue spectrum, and it is used for nuclear stain applications. Example setup:
- Excitation Wavelength: 350–400 nm
- Dichroic: ~405 nm
- Emission Wavelength: 420–480 nm
Frequent Problem: Substituting a standard UV filter for a DAPI-specific filter results
FITC / Alexa Fluor 488 / GFP → FITC Filter Cube
These dyes have their excitation and emission wavelengths in the green fluorescence spectrum and are considered the most widely used channels in biological laboratories. Configuration:
- Excitation wavelength: 470-495 nm
- Emission wavelength: 510-550 nm
Compatible with: FITC, Alexa
TRITC / mCherry / Alexa 555 → TRITC Filter Cube
It is used to image orange-red fluorescence. Mis-matching normally leads to considerable signal crosstalk from the channel into adjacent channels.
Typical parameters:
- Excitation wavelength: 540 – 560 nm
- Emission wavelength: 570 – 62
Cy5 / Alexa 647 → Cy5 Filter Cube
Cy5 falls into the category of far red/NIR fluorescence. It has high sensitivity to light transmittance efficiency. Despite improvements in cameras, many old microscopes continue to have high light absorption in NIR wavelengths. Standard settings:
- Excitation Wavelength: 620 – 650 nm
- Emission Wavelength: 660 – 720 nm
In case your Cy5 is very dim, you should consider the following:
- Camera QE in the wavelength of 650-700 nm
- Transmittance in objective lenses for IR light
