White Paper
Dye Detection in DNR's Bio-Imaging Systems Bio-Imaging Systems
Date: September , 2006
In the biology fields, there is a growing need for highly specific, sensitive and safe dyes. Although the procedure by which a dye or a combination of dyes is used to color a given sample is fairly uniform and well-established, there are always innovations in the dyes' development.
The life science fields frequently use dyes to mark structures in biological tissues, examine bulk tissues (such as muscle fibers) and organelle/s within individual cells and detect nucleic acids or proteins.
The dyes which are detected by biological reaction emit light in a wide range of the photonic spectrum and can be grouped into the fluorescent, chemifluorescent, chemiluminescent and colorimetric categories.
Image courtesy of Molecular Expressions (http://micro.magnet.fsu.edu)
The fluorescent and chemifluorescent dye detection mechanism functions according to a typical excitation and emission spectrum.
Absorption and emission in molecules is a process by which the materials borrow energy from a source (i.e. photons of light).
The absorbed energy can then be released by heat or by the emission of a photon of light. Immediate release of the energy is called fluorescence.
The spectrum is divided into several wavelengths, whereby the higher the wavelength's energy, the shorter the wavelength. For example, the blue wavelength region (~420nm-~490nm) has more energy than the green wavelength region (~500nm-
~570nm). Infrared radiation can change the way molecules vibrate. Visible region and UV wavelengths are associated with changing the way electrons are distributed within the molecule.
Excitation spectrum is defined as a region where a molecule can be irradiated from a lower energy state to higher one. When compounds are irradiated with a wavelength of light near the spectrum's peak, they will emit more brightly than irradiation with a light wavelength near the spectrum's edge.
Emission spectrum is a region where an irradiated molecule in a high energy level, is returned to a lower energy level, by emitting light energy. During this procedure, photons from molecules in an excited state emit longer wavelengths (lower frequency) and are less energetic than the excitation photons.
The gap between the excitation and emission spectrum peaks' is termed Stokes shift.
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Image courtesy of Molecular Expressions (http://micro.magnet.fsu.edu)
The fluorescence process is when the molecule is excited from its ground state by the absorption of light energy. Thus, fluorescent molecules emit light only while being irradiated.
Chemiluminescence is a generation of electromagnetic radiation by the release of energy from a chemical reaction. It occurs when an exothermic reaction produces a molecule in an excited state. When it returns to the ground state, it releases energy as a photon of light.
Colorimetric dyes are detected via white light illumination. These dyes are visible to the human eye, but in order to get more accurate experimental results, the sample is detected by additional illumination sources in the unique Smart Dark Chamber of DNR
Bio-Imaging Systems.
Light Emitting Diodes
Light-emitting diodes (LEDs) are semiconductor devices which typically produce light with a bandwidth of 40-50nm.
This illumination source is mainly used for fluorescent dye excitation.
DNR's Bio-Imaging Systems employ the most powerful LEDs in the imaging field, the HeptaLEDs System. The broad spectrum range of biological excitation dyes covered by the HeptaLEDs
System provides brighter and more accurate experiment results. The LEDs employed by DNR's systems are:
Blue:
Dark Green:
Middle Green:
Light Green:
Yellow:
Red:
470nm
500nm
525nm
568nm
588nm
621nm
* More LED options are available apon request.

White Paper
Dye Detection in DNR's Bio-Imaging Systems Bio-Imaging Systems
Ultraviolet (UV) Illumination
UV light has a shorter wavelength than that of visible light and is used for fluorescent dye excitation. As it is known that contact with UV light is dangerous, all DNR systems are designed to protect the user from the detrimental effects of UV light by preventing any contact with UV radiation either inside or outside the bio-imaging system. This protected environment is created by the Smart Dark Chamber
Technology and built-in safeguard which automatically shuts down the UV illumination when the system's door or drawer is open.
DNR offers Bio-Imaging Systems which contain three powerful
UV illuminations:
254nm
312nm
365nm
* There is also an option of dual UV combinations.
White Light (WL) Illumination
This kind of illumination is mainly used for colorimetric dye detection, but can also be used for unique applications, such as the ECL Standard (patent pending).
Filters are materials which are interposed in the path of radiation to modify the spectral distribution of the radiation. Optical filters are used to select the wavelength of light desired, and allow the user to eliminate both scattered light and light from different kinds of dyes, which may be present in a given sample.
Two main parameters affect the performance of an instrument by a filter: the optical quality and the quality of the filter aligned in the light's path.
For example, placing a filter at a slight incline to the incoming light will alter the optical path of light passing through different parts of the filter.
There are several terms used to describe different kinds of filters including wide, narrow, cut-off, band pass, long pass, etc.
These terms refer to the shape of the curve when light is passed through the filter. For example, long pass filters allow light above a given wavelength to pass through, while cut-off filters only permit the transmission of radiation of wavelengths which are longer than or shorter than a specified wavelength.
In general, detection of marked biological samples in the DNR
Bio-Imaging Systems can be described as follows:
Marked biological sample is excited in a narrow wavelength, in accordance with the specific excitation spectra of the dye.
The marked sample absorbs the light energy, reaches an excited energy level, and starts to emit light in all directions.
The emitted light goes through a unique filter, which allows passage to specific wavelengths (in accordance with the dye emission spectrum).
The emission dye spectrum is detected by a scientific camera.
Image courtesy of Molecular Expressions (http://micro.magnet.fsu.edu)
In the chemiluminescence experiments, there is no light excitation.
The emitted light from the sample is caused by an enzymatic reaction, and every emitted photon is critical for getting the best, most reliable experimental results. As such, the detection is made only by the scientific camera, without any excitation source or filter.
The following table (next page), Stain Guide , presents the recommended illumination sources and filters to detect the most common dyes in life science.
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White Paper
Dye Detection in DNR's Bio-Imaging Systems Bio-Imaging Systems
Stain Guide
LumiBIS
(with HeptaLED system)
M-ChemiBIS MF-ChemiBIS
MF-ChemiBIS
(with HeptaLED system)
L621
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White Paper
Dye Detection in DNR's Bio-Imaging Systems
Lippincott-Schwartz J.; Patterson G. H., "Development and Use of Fluorescent Protein Markers in Living Cells"
Science 2003 , 300, 87 – 91
Guilbault G. G. (ed.), "Practical Fluorescence", Marcel Dekker, New York, 1990
Wampler E. "Instrumentation: Seeing the Light and Measuring It", in Chemi- and Bioluminescence , Burr J.G., ed.,
Marcel Dekker, New York, 1985.
Correspondence should be sent to: support@dnr-is.com
Bio-Imaging Systems
Bio-Imaging Systems
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DNR Bio-Imaging Systems | P.O. Box 34, Maale HaHamisha, Jerusalem, Israel 90835 | Tel: +972 2 570-0818
Copyright © 2006 DNR Bio-Imaging Systems. All rights reserved.
The information contained in this document represents the current view of DNR Bio-Imaging Systems on the issue discussed as of the date of publication. Because DNR Bio-Imaging systems must respond to changing market conditions, it should not be interpreted to be a commitment on the part of DNR Bio-Imaging Systems, and DNR Bio-Imaging Systems cannot guarantee the accuracy of any information presented after the date of publication.
This white paper is for information purposes only. DNR Bio-Imaging systems makes no warranties, express or implied, in this document.
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