Filter and Microscope Basics

Filter and Microscope Basics

Abbott Molecular's expanding line of single, dual and triple bandpass filter sets produce sharply defined, high contrast visualization of Abbott Molecular's reagents. By allowing simultaneous viewing of up to five different fluorophores, multi-bandpass filter sets enhance the utility of fluorescence in situ hybridization (FISH) procedures, enabling multiple results to be simultaneously obtained from a single assay. Each filter set has been designed and optimized for use with the Abbott Molecular fluorophores. Abbott Molecular filter sets are available for most common microscope models, including:

  • Olympus®
  • Zeiss®
  • Leitz/Leica®
  • Nikon® 
Contact Technical Service for pricing and availability of filters for your microscope.
 
 
 
 
Microscopy Basics

A properly configured, well maintained fluorescence microscope is essential for obtaining optimum FISH results. Be sure to closely follow the manufacturer's instructions in the operation and care of your microscope.

 
 
 
 
Excitation Lamp Source

The excitation lamp is the source of light that excites fluorophores to emit a fluorescence signal. The two key features of an excitation source are emission spectra and intensity. The lamp must emit within the absorption peaks of the given fluorophores in order to cause fluorescence. The stronger the excitation intensity, the brighter the fluorescence. However, the brighter the excitation intensity, the faster the photo-oxidizing or fading of the fluorophores. Any choice of lamp has to take into account trade-offs between the brightness of the image being reviewed and the extent of time that it remains visible. Vysis recommends using a 100-watt mercury excitation lamp source.

 
 
 
 
Recommended Objectives In Situ Hybridization
  • Plan objectives optimized for fluorescence microscopy; High numeric aperture (~1.3)
  • Objectives with the fewest lens elements Numeric aperture > or = 0.75
  • Not recommended; Objectives with many lens elements Numeric aperture > or = 0.75
 
 
 
 
Filter Sets

Filter sets are designed for viewing a specific fluorophore or a specific combination of two or more fluorophores. A filter set for fluorescence microscopy is composed of three elements: an excitation filter for selecting the wavelengths used to excite the fluorophore; a dichroicmirror (beam splitter or polychroic) for reflecting the excitation light down to the object on the slide, blocking much of the excitation light reflected back from the slide to the eyepiece, and passing the light emitted from the fluorophore in the in situ hybridization; and a final emission filter (barrier filter) for passing only the emitted light from the fluorophore while any other wavelengths outside that specified range are blocked.

 
 
 
 
Single Fluorophore

Filter Sets

Filters fall into three main classes: bandpass, long pass, or short pass. A bandpass filter transmits wavelengths within a region of wavelengths "cutting on" at a low wavelength and "cutting off" at a higher wavelength. Wavelengths are blocked below the cut-on or above the cut-off by absorption and/or reflection. The bandpass or bandwidth (BW) is the peak width in nanometers (nm) at 50% of the transmission maximum for a given peak. It may also be referred to as FWHM (full width half maximum) or HPBW (half power bandwidth). A long pass filter transmits wavelengths above a specified value and blocks wavelengths below this value.

 

A short pass filter does the opposite of the long pass and therefore transmits wavelengths below a specified value while blocking wavelengths above that specified value. Interference filters are made by vacuum deposition of thin films on a glass substrate. Thin films are comprised of alternating layers of metal ordielectric with high and low refractive indices. Interference filters can be designed to be short pass, long pass, or bandpass filters. Colored glass filters are made from dye impregnated glass and generally are long pass or bandpass. Filters can contain multiple elements of the colored glass and/or interference type stacked and cemented to each other with optical epoxy (laminated). If a filter contains any interference elements it is referred to as an interference filter.

 

Interference filters are highly desirable with their narrow bandwidths, high transmission efficiency and extremely sharp cut-ons and cut-offs. These filters are more expensive, less durable and generally have higher signal/noise (S/N) than colored glass filters. Colored glass filters generally have a wide bandwidth (i.e., 40nm for FITC exciter), lower transmission efficiency and trailing cut-on and cut-offs. Colored glass filters are used because they are readily available, less costly and more durable. 

 

Excitation filters are normally bandpass filters. Excitation filters can be of the colored glass or interference type. The greater the bandwidth the greater the excitation energy. As bandwidth increases there is the accompanying increase in emission intensity, but a greater chance of generating non-specific autofluorescence.

 

Dichroics are interference filters. Dichroics are manufactured using thin film technology, but since they need to be kept thin for high image quality they are not protected with a laminated cover glass. Unprotected dichroics are easily scratched and damaged. With Zeiss Axioline filters, the user must take care in mounting filter sets and only handle the dichroic by the edges with gloved hands.

 

Emission filters may be bandpass or long pass.

 

Emission filters can be the colored glass or interference type. Long pass filters pass the most light resulting in brighter, but noisier images with higher background and lower signal/noise (S/N). This increase in noise and background results from cell debris, the microscope slide, etc.

 
 
 
 

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