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Continuously Variable Filters in Spectroscopy and Fluorescence

Biochemists and chemists wishing to investigate the structure of unknown compounds often apply a technique known as spectroscopy. The word derives from the Latin noun spectrum meaning “an image” or “apparition” and the Greek verb skopein meaning “to look”. Spectroscopy involves submitting a sample to some form of energy in the form of radiation or light and examining how the sample interacts with that energy. Newton and Goethe, the great German scientist, philosopher and writer were among the earliest scientists investigating the spectral properties of light.

Spectrometers consist of four basic units:

  • Energy/light source
  • Wavelength selector
  • Sample holder
  • Detector

Here, we will focus on wavelength selectors. There are two major types of wavelength selectors:

  • Monochromators
  • Interference filters

Monochromators

Monochromators have two slits, an entrance and an exit slit through which light enters and leaves the device with a dispersing unit, either a prism or a grating, and mirrors to manipulate the light as it enters and leaves the prism or grating.

The spectral quality of light emitted by a monochromator is a function of the width of the slits and the dispersive capability of the prism or grating. For applications requiring the highest levels of performance, a double grating is used whereby the light from the first grating is passed through a second unit, reducing scatter and giving even greater resolution. However, each optical unit which the incident light impinges on results in a loss of energy. Hence there is a trade-off between wavelength resolution and the intensity of the emitted light.

Interference filters

Interference filters are considerably smaller and lighter than monochromators and offer technological benefits, in particular greatly increased potential grasp of energy ('light grasp') compared to monochromators. When properly designed, an interference filter is capable of collecting several hundred or even several thousand times the quantity of light collected by a monochromator with the same bandwidth. In general, interference filters are made of up to several hundred optical layers deposited on a transparent fused silica or glass substrate. The thickness of the optical layers determines the specific performance characteristics of the filter. Interference filters can be designed to meet the needs of spectroscopists, e.g., Continuously Variable Filters in which the wavelength of light transmitted and reflected from the optical layers changes continuously as it passes along the length of the filter. This is usually referred to as wavelength scanning and can be used for both excitation and emission scanning in fluorescence spectroscopy.

Continuously variable filters

A Continuously Variable Filter (CVF, a.k.a. Linear Variable Filter) is an optical interference filter whose spectral functionality continuously varies along one direction of the filter, compared to a traditional optical filter whose spectral functionality is intended to be identical at any location of the filter. The wavelength variation as function of the position on the filter is a design parameter and can be linear or for example exponential. The wavelength variation is achieved by an interference coating that is intentionally wedged in one direction, creating a continuous shift of the center or edge wavelength along the same direction of the filter (see figure below). The continuously variable filters made by Delta Optical Thin Film for example, are rectangular types where the wavelength characteristic changes along the longitudinal direction. Other manufactures have made circular variable filters where the variation is obtained by rotating the filters. In other designs, tunability can also be obtained by changing the angle of incidence.

Principle of Continously Variable Filters

Principle of Continuously Variable Filters

Delta Optical Thin Film has introduced a new combination of continuously variable filters. Delta Optical Thin Film offers Continuously Variable Long Wave Pass filters (CVLWP), corresponding Continuously Variable Short Wave Pass filters (CVSWP) together with a Continuously Variable Dichroic (CVD). Each of the filters can be used separately. Combining CVLWP and CVSWP enables the construction of bandpass filters that can be tuned continuously with center wavelengths from 320 nm to 850 nm, with the added benefit of tunable bandwidth. As CVF monochromator the filters are used, for example, in fluorescence microplate readers.

Besides setting new standards in transmission level and edge steepness (see Figure 2), the filters offer blocking better than OD3 over the complete reflection range (see Figure 3). It is possible to increase the blocking to beyond OD5 by placing another continuously variable filter in series. The filters are coated on single fused silica substrates for minimal auto-fluorescence and high laser damage threshold. All of Delta Optical Thin Film’s Continuously Variable Filters are coated with ultra-hard surface coatings (UHC) that are also used by Delta Optical Thin Film in traditional fluorescence filters. 

Measured Transmission of CVLWP filter

Measured transmission of CVLWP filter

Measured blocking of CVLWP filter

Measured blocking of CVLWP filter

Continuously variable filters for spectroscopy

The simplest implementation of CVFs for spectroscopy is a single tunable bandpass filter where the output wavelength is selected simply by the spatial position on the filter. A more flexible filter can be designed however by combining a CVLWP with a CVSWP filter to create a fully tunable bandpass filter. By moving both filters together the central wavelength can be continuously adjusted and by moving them relative to one another the bandwidth of the filter can also be tuned. In applications such as fluorescence spectroscopy this allows the user to optimize the filter perfectly to maximize the excitation efficiency and the fluorescence signal. Using two of these fully tunable bandpass filters together with an Continuously Variable Dichroic (LVD) makes it possible to design measurement systems that work after the epi-fluorescence principle. Because the CVFs have intrinsically high transmission efficiency, this technology allows maximum tunable power from a supercontinuum source with in excess of 100 mW of tunable light possible with the highest power supercontinuum lasers or other broadband light sources such as Xenon flash lamps or laser driven light sources (LDLS).

Complementing gratings in a single-monochromator spectrometer with CVFs

Spectrophotometers are available as single and double monochromator versions. Single monochromators have a strong signal but low signal-to-background ratio, while double monochromators have high signal-to-background ratio at the cost of strongly reduced signal strength and increased cost, size and complexity. The ideal combination of strong signal and high signal-to-background ratio is achieved by combining variable filter-based monochromators with a grating based monochromator. The figure below shows a tunable bandpass filter as a complement to a diffraction grating both in the excitation and the emission leg of the spectrometer. The primary wavelength selective elements are the gratings, and the tunable bandpass filters are adjusted to the wavelengths selected by the gratings. The very high transmission of the filters maintains the strong signal while the excellent deep broad out-of-band blocking suppresses the higher orders of the gratings and the stray light level in the spectrometer.

Typical setup of single-monochromator spectrometer.

Typical setup of a single-monochromator spectrometer. M: Mirror, L: Lens, S: Slit, DG: Diffraction Grating, F: New variable filter stage.

The figure below shows the results of Raman measurements of cyclohexane. The green curve for the filter solution combines the strong signal of the single grating monochromator with the high signal-to-background ratio of the double grating monochromator.

The results of Raman measurements of cyclohexane

Raman spectrum of cyclohexane measured at an excitation wavelength of 500 nm. The red curve (upper) denotes a spectrum taken at 1 nm spectral resolution without the use of variable filters. The green spectrum (middle) is taken with the variable filters in place. For comparison, the same measurement was repeated on a double-monochromator spectrometer (blue – lower). The signal is severely reduced.

Improved performance, reduced background levels

Three important points in regard to performance and background noise are worth noting:

  • A combination of linear CVLWP and CVLWP interference filters can be used to realize a variable bandpass filter. This filter can be scanned synchronously with the wavelength selected by the diffraction grating in a monochromator to strongly reduce the influence of higher order effects and stray light. Thus, the performance of the spectrometer is greatly improved.
  • The variable bandpass filter has the capability to reduce background levels by two orders of magnitude and increase the signal to noise ratio by up to a factor of five in various situations and at least a factor of two in the standard water Raman test.
  • A monochromator equipped with synchronous filters thus can achieve a background reduction comparable with a double monochromator system (which gives higher spectral resolution) without the cost of increased size and signal loss at multiple gratings.

Written by Dr. Ing. Oliver Pust, Director of Sales, Delta Optical Thin Film

Labels: spectroscopy,fluorescent measurement,continuously variable filters,continuously variable short wave pass,long wave pass,bandpass,signal to noise,monochromator,linear variable filter,

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