Fluorescent Imaging Using Filters and Spectroscopy
Fluorescent microscopy systems are becoming more and more of an industry standard in the medical field. These systems typically
consist of a few entities — excitation, emission and dichroic filter, illumination, and an imaging sensor. The dichroic and
emission filter are the two most crucial elements. Together they prevent non-emission energy and stray light from reaching
the sensor. In this type of microscope system, the quantum dot in place absorbs the excitation energy and then converts it
to a radiating glow, or rather emission energy.
The important parameters of a filter include the center wavelength (CWL), minimum transmission percentage, optical density
(OD), and bandwidth, which at times can also be referred to as the full width at half maximum (FWHM). The CWL and bandwidth
work together and refer to the wavelength where maximum transmission is reached and the region to which 50% transmission is
still attainable. The minimum transmission dictates the amount of attainable fluorescence and excitation from the quantum
dots. This value should be as high as possible, with an industry standard at or around 85%. New and improved filters that
have increased blocking efficiencies are now achieving >93% minimum transmission. The OD refers to the filter's blocking efficiency
to light and wavelengths outside the desired bandwidth. With a higher OD there is improved reduction of noise and a sharper
cutoff between transmission and rejection, resulting in sharper colors and higher contrast. Lastly, the final element of importance
is the dichroic filter. These filters are specifically in place for cleanup of stray and excess light. Ideally, they will
possess an extremely crisp transition between maximum reflection and maximum transmission. The point of maximum transmission
will ensure minimal stray-light and a maximum signal-to-noise ratio which is crucial for fluorescence imaging.
The spectral characteristics of the quantum dot in place dictate which filters would work best in the fluorescent microscope
system. Compared to a typical fluorescent agent, quantum dots are proven to be much brighter and more stable, by a scale of
20× and 100× (3). Not only will quantum dots fluoresce and emit a much higher energy, but there is very little risk of photo-bleaching
by the illumination source as they are synthesized from semiconductor materials. The two key parameters are the peak excitation
λ (nm) which refers to the wavelength the quantum dot absorbs best, and the maximum wavelength that it then emits after absorption,
which is known as the peak emission λ (nm). A third parameter is the excitation range of the quantum dot, which typically
resides within the FWHM of the filters. Larger quantum dots have closely spaced energy levels, and therefore would emit a
small band of wavelength, whereas smaller dots have more widely spaced energy levels, and would ultimately emit a much larger
wavelength band (2).
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References
(1) C.T. Troy, "Multicolor quantum dots ID rare cancer cells." bioPhotonics
10 (2010).
(2) A.F. Van Driel, "Frequency-dependent spontaneous emission rate from CdSe and CdTe nanocrystals: Influence of dark states."
Physical Review Letters
95 (2005).
(3) M.A. Walling and S. Novak, "Quantum dots for live cell and in vivo imaging." International Journal of Molecular Science
441 (2009).
Edmund Optics
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