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Emission Filter Guide for Various Fluorescent Reagent
(Excitation and Emission wavelengths)



ドクターバイオイメージング  Excessive exposure to UV can result in harmful effects on human body.
With the advent of visible ray LED, we now have safer alternatives !
 To capture high contrast image, you need to select appropriate LED
to excite the reagent and separate the emission. Please refer following
instruction for selecting suitable LED light and filter for you.
Key factors for capturing quality image

Emission filter guideline  for agarose gel electrophoresis.Filter usage and combination for different fluorescent reagent (Excitation and Emission wavelengths)

1. Absorption (Ex) and emission (Em) wavelength of the reagent.
2. Wavelength characteristics of LED
*
3. Spectral characteristics of filter.
4. When Ex/Em wavelengths are close and overlaps, it is crucial to
   have effective filter to cutoff the skirt of wavelength.
 ■ Example with Ethidium Bromide (EtBr)
1. Excitation (Ex) and Emission (Em) wavelength
Figure shows excitation and emission spectral for
Ethidium Bromide (EtBr)
Excitation peak is at 300nm (UV). With enough LED power,
it can also be excited around 518nm (visible ray) as well.
2. Excitation with UV Transilluminator
In conventional practice, UV transilluminator with 254, 312, 365nm have been used for EtBr
UV Transilluminators has advantages such as high energy
and distinct Ex/Em difference. However, sample deterioration and harmful effects on human body can not be ignored either.
* SC60 filter only transmits emission wavelength and
cutoff unwanted light
leakage from the source.
3. Excitation with LED (Visible Ray)
As mentioned above, EtBr can be excited around 518nm too, so 470nm (blue), 505nm (cyan), 530 (green) can also be used.
However, absorption is too weak with blue, and green is too close to emission wavelength
(= make it difficult to separate).
 
In the left figure, triangle area below the point LED and filter lines merges represent background. Emission is much weaker in actuality than light source and so it has large impact on outcome.
 
4. Dichroic filter for eliminating background light
Even with use of the SC-56,
(cutoff short wl side and transmit emission as much as possible) we still can not ignore background light. And it is difficult to visually recognize weak emission with background light.
We can solve this background problem with dichroic filter which would cutoff long wl side of LED with steep angle.

* Our LED transilluminator is equipped with very high quality compound dichroic filter

LED505-TR60W
  Dramatic Effect of Dichroic Filter Effect ⇒

Filterwork Guide for Popular Fluorescence Reagent and LED ⇒ 
Notice : The guide is reference purpose only. It does not gurantee any visibility of certain emission.
       Result may vary depends on such factors as LED brightness, filter quality, and camera spec.
■ How to eliminate backgrand light when using UV Transilluminator

・Why background occur ?
When shooting fluorescence emission with digital camera, stripe patterned background often appears in the captured image. This is Infrared (IR) ray leakage from UV transilluminator and without the proper IR cut filter,
it would result in poor image contrast. * Most digital camera is equipped with IR cut filter but quality varies.

The UV transmission filter equipped with UV transilluminator cutoff most visible rays, however, it transmits
infrared to near-infrared light. In monochrome and Polaroid film era, the film only had sensitivity up to 630nm and there was no background problems. However, with coming of the digital era, the camera utilized for gel documentation these days have high sensitivity and captures even near-infrared light at 1200nm.
And now we have a background problem.
・ Solution 1
Utilize IR cut filter which would cutoff near-infrared above 650nm.
We have IR cut filter MFC65-52 for our Bio-Pyramid system (original gel documentation system)
       
       
・ Solution 2
Select UV transilluminator equipped with surface filter that contains Infrared (IR).
Our UV transilluminator MBP-UV312 is equipped with hybrid filter that cutoff most IR rays.
         





■ How to eliminate background light when using LED Transilluminator

・Why background occur ?
LED has narrow wavelength and it is usually considered to be suited ight source for excitation.
However it also has tendency to leave out wavelength skirt around the peak wavelength
and this skirt causes background. The fluorescence emission is often very weak and the background
makes it difficult to distinguish the fluorescence emission of the band. (Below figure has maximum 100% ,
however, backgounrd occer at the lower part where LED and emission wavelength overlaps)


Example with EtBr
 As left figure shows, wavelength overlap (=background) is large with 530nm (Green LED) / SC56 combination and the overlap still remain with SC60 as well.
 505nm (Cyan LED) and SC56 combination also leave out wavelength skirt.
 470nm is inefficient in this case in terms of its power for excitation.It requires extra high energy to acquire enough emission.
     
・ Solution 1
LED505-TR60W
Use Dichroic Filter which would cutoff long wavelength side of LED
light source. Our LED transilluminator LED505-TR60W is equipped with
compound dichroic filter.
・ Solution 2
Use extra high power light source and long exposure to capture image. (sacrifice short wavelengths side with SC60)
Our LED transilluminator LED470/505-TR60W is super high-power with unprecedented 60w level output.