Fluorescence Filter Guide for Gel Imaging (Excitation / Emission)
Excessive UV exposure can be harmful to both users and biological samples. With the advancement of high-power visible LED technology, safer and highly effective alternatives are now available for gel documentation and fluorescence imaging.
Key Factors for Capturing
High-Quality Fluorescence Images
- Excitation (Ex) and emission (Em) spectra of the
fluorescent reagent. - Spectral output characteristics of the LED or UV light source.
- Cut-off and transmission characteristics of the emission filter.
- When Ex and Em wavelengths are close or overlapping,
precise filtering is critical to suppress background noise
and improve contrast.
Popular Fluorescent Reagents and LED / Filter Combinations
For reagent-specific combinations such as EtBr, SYBR Green, SYBR Gold, GelGreen, GelRed, GFP and related fluorescent dyes, please refer to the detailed filter-work guide.
FILTER-WORK GUIDE REAGENT LIST
Example: Ethidium Bromide (EtBr), a Common Gel Staining Reagent
1. Excitation (Ex) and Emission (Em) Wavelengths
EtBr has a strong excitation peak in the UV region, around 300 nm. It can also be excited in the visible range, approximately 480 to 520 nm, when sufficient light intensity is applied.
2. Fluorescence Excitation with a UV Transilluminator
UV excitation provides strong fluorescence and good spectral separation. However, UV light can damage DNA samples and also presents safety concerns for users.
3. Fluorescence Excitation with Visible LED Transilluminators
However, blue LEDs may provide lower excitation efficiency for EtBr, while green LEDs are closer to the emission range and can be more difficult to separate cleanly.
Overlap between the LED output and the emission wavelength range can create unwanted background glow. Since fluorescence emission is often much weaker than direct LED leakage, even a small overlap can significantly affect image contrast.
4. Dichroic Filter for Reducing Background Light
This enables clear detection of weak fluorescence signals with significantly improved contrast.
Dramatic Effect of Dichroic Filtering
Dichroic filtering helps suppress LED leakage and improves fluorescence
contrast,
especially when the excitation and emission wavelengths are close.
How to Reduce Background Glow When Using a UV Transilluminator
Cause of Unwanted Background Glow
When fluorescence emission is captured with a digital camera, stripe-patterned background glow may appear and reduce image contrast. This is often associated with infrared (IR) or near-infrared leakage from UV transilluminator lamps, as well as the IR-cut performance of the camera. UV transmission filters used in transilluminators block most visible light, but some may still transmit infrared to near-infrared wavelengths. Traditional film-based systems were less sensitive in this region, but modern digital cameras can detect longer wavelengths, which may appear as background noise in gel images.
Solution 1: Use an IR-Cut Filter
The MFC65-52 IR Cut Contrast Filter is
available as an optional item for the Bio-Pyramid gel documentation system.
Solution 2: Use a UV Transilluminator with an Improved Surface Filter
with a surface filter designed to reduce
infrared leakage.
The Mecan MBP-UV312 UV Transilluminator
uses a hybrid filter that reduces most
IR leakage and improves image contrast.
How to Reduce Background Glow When Using an LED Transilluminator
Cause of Unwanted Background Glow
LEDs are excellent light sources for fluorescence excitation because they
provide relatively narrow wavelength output. However, LEDs still have a
small spectral tail around the peak wavelength. When this tail overlaps
with the emission filter transmission range,
it may appear as background glow.
Fluorescence emission is often much weaker than direct excitation light. Therefore, even low-level LED leakage can make weak bands difficult to distinguish from the background.
The overlap can be significant with a 530 nm LED and an SC56 filter pair, and some background may remain even with SC60. A 505 nm LED and SC56 pair may also leave residual wavelength leakage. A 470 nm LED can be cleaner in separation but is less efficient for EtBr excitation and may require higher intensity or longer exposure.
Solution 1: Use a Dichroic Filter
Solution 2: Use High-Power LED Illumination with Optimized Filtering
This combination helps improve contrast and enables clearer detection of weak fluorescence bands.