Filter Guide for Various Fluorescent Reagent (Ex/Em)
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Excessive UV exposure has harmful effects on human body.
With the advent of visible ray LED, we now have safer alternatives !
To capture high contrast image, it is important to select appropriate
LED to excite the reagent and separate the emission. Please refer
instruction below for selecting suitable LED color and filter for you.
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| Key factors to capture quality fluorescence image |
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1. Absorption (Ex) and emission (Em) wavelength of the reagent.
2. Wavelength characteristics of LED
3. Spectral characteristics of shap-cut filter
4. When Ex/Em wavelengths are close and overlaps, it is crucial to
have effective filter to cutoff the skirt of wavelength. |
| ★ Causes of most common problems and solutions |
| ■ Let's explain with most common reagent - Ethidium Bromide (EtBr) |
| 1. Excitation (Ex) / Emission (Em) wavelength |
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| ・ |
Left figure shows excitation
and emission spectral of EtBr
(Ethidium Bromide)
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| ・ |
Excitation peak is at 300nm (UV). With enough power, it can also
be excited around 518nm
(visible ray) as well. |
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| 2. Fluorescence excitation with UV Transilluminator |
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| ・ |
UV transilluminator with
254, 312, 365nm have been used for EtBr in conventional practice.
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| ・ |
UV has high energy and distinctive Ex/Em which enable to detect band with
more clarity. However, sample deterioration and harmful effects on human
body can not be ignored at the same time. |
* SC60 filter transmits only emission wavelength and
cutoff unwanted light
leakage from the source. |
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| 3. Fluorescence excitation with Visible ray LED Transilluminator |
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| ・ |
As mentioned above, EtBr can be excited around 518nm too and 470nm (blue),
505nm (cyan), 530 (green) LEDs can also be used. |
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| ・ |
However, absorption is too weak with blue LED, and Em wavelength is too
close with green LED,
making it difficult to separate. |
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| ・ |
Circled area in the left where wavelength curve of LED and filter overlaps would result in unwanted background glow. Emission is much weaker compare to LED intensity and has large impact
on outcome. |
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| 4. Dichroic filter for eliminating background light |
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| ・ |
Background glow can not be ignored with 505nm LED too
and weak emission may be difficult to detect especially when SC-56 (sharp-cut
filter) is used to cut off short wavelength side for the purpose of transmit
as much emission as it can.
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| ・ |
We have solved this problem with compound dichroic filter. It eliminates
unwanted background glow by sharp-cutting longer end of LED wavelength.
Enabled to detect even weak band with clarity |
* Mecan LED transilluminator is equipped with high quality dichroic
filter LC525 |
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LED505-TR60W
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Dramatic Effect of
Dichroic Filter Effect ⇒ |
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| Notice : |
The guide is reference purpose only. It does not guarantee visibility of
any certain emission. Result may vary depends on such various factors as
LED brightness, filter quality, and camera specifications, etc. |
| ■ How to eliminate backgrand glow when using UV Transilluminator |
| ・Cause of unwanted background glow |
| ■ |
When shooting fluorescence emission with digital camera, stripe patterned
glow is often captured
along in the background and impairs image contrast. Two factors associated
with this phenomena
are IR (Infrared) ray leakage of UV transilluminator lamps and also quality
of IR cut-filter that is
built in digital camera. The UV transmission filter equipped in UV transilluminator cutoff most visible rays, however transmits infrared to near-infrared light. Films used to only have sensitivity up to
630nm in old monochrome or Polaroid era and therefore did not create background
problems.
However, with coming of the digital era, the camera used 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 |
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Use IR cut filter and cut off near-infrared wavelength over 650nm.
IR cut filter MFC65-52 is available (optional item for our Bio-Pyramid gel documentation system) |
| ・ Solution 2 |
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Use UV transilluminator 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 glow when using LED Transilluminator |
| ・Cause of unwanted background glow |
| ■ |
LED is ideal light sourece for fluorescence excitation with its characteristic of narrow wavelength.
However, it also has small “wavelength skirt” around the peak and it can
cause background glow.
The emission can often be very weak and the background makes it difficult
to distinguish the band. (Below figure has maximum of 100%, however, backgounrd
occer at the lower part where LED and emission wavelength overlaps) |
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| ・ |
Example with EtBr
As in left, wavelength overlap (=background) is large with 530nm &
SC56 pair and it still remains even with SC60. 505nm & SC56 pair also
still leave out wavelength skirt. 470nm in this case is inefficient. It
requires extra high energy to acquire enough emission.
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| ・ Solution 1 |
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Use Dichroic Filter which would cutoff long wavelength side of LED light
source. |
| ・ Solution 2 |
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Use high power light source and long exposure to capture image together
with more or less
sacrifices short wavelengths side with SC60.. |
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Mecan's LED transilluminator LED470/505-TR60W has unprecedented 60w level
super high-power and equipped
with Dichroic Filter LC525.
It eliminates background glow
and enables to detect band with amazing clarity. |
LED505-TR60W
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|
Dramatic Effect of
Dichroic Filter Effect ⇒ |
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