Excited filter

Unless a dual-excitation filter cube is used, in combination with a excitation switcher. 

Note that this is always the case for wide-field and confocal determination where D and S are collected simultaneously using the same excitation filters or lasers. In case three separate filters are used, care should be taken to match the filters so as to fulfill Eq. (7.A4). 

The basic layout of Raman sensors is similar to fluorescence probes. The common sensor form is that of a fibre optic probe, with excitation and collection fibres. As the excitation light comes from a monochromatic source no excitation filter is required, but a spectrally matched emission notch filter blocking the excitation wavelength is almost always part of the sensor head. 

Signals for methyl paraben were monitored with UV detection at 254 nm. The signal for rhodamine 110 chloride was monitored via fluorescence detection with an excitation filter of 482 nm (35 nm bandwidth) and emission filter of 535 nm (40 nm bandwidth). A gradient method (same as the one in Figure 6.16) was used. The compositions of mobile phases A and B were 5 95 H20 CH3CN with 0.1 HCOOH and CH3CN with 0.085% HCOOH, respectively, with a total flow rate of 300 fiL/ min (corresponding to 12.5 /rL/min for each column). 

Fluorophores can be visualized in fluorescence microscopy using special filter blocks that are composed of the excitation filter, dichroic mirror and emission filter. The excitation filter must select wavelengths of light from a light source that fall in the maximum absorption region of the fluorophore. The emission filter must pass the fluorescent wavelengths but not the excitation wavelengths. The dichroic mirror... 

Excitation filter selects wavelengths of light from a light source that fall in the maximum absorption region of a specific fluorophore. 

Fig. 3.161. (A) Zone electrophoresis patterns of FITC-labelled transferrin samples by fluorescence detection. The unbound dye (providing a main peak and several minor ones) was not removed from the samples. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length 41 cm) X 75 pm i.d. injection of samples 100 mbar x s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut-off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 13 pm (1 mg/ml) Tf and (a) 0.01 mM FITC, (b) 0.1 mM FITC, and 1 mM FITC. (B) Zone electrophoresis patterns of an FITC-labelled transferrin sample by simultaneous fluorescence (upper trace, left axis) and UV detection (lower trace, right axis). The unbound dye shows several peaks with both detections. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length fluorescence 41 cm, UV 50.5 cm) X 75 pm i.d. injection of samples 100 mbar X s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 6.5 pm (0.5 mg/ml) Tf and 0.1 mM FITC. Reprinted with permission from T. Konecsni et al. [199].

Apparatus. The HPLC instrument used was a Water s Associates model 6000A pump for the solvent supply, a U6K septumless injector and a radial compression module with standard Radial Pak columns. Immediately after the column a low dead volume tee was inserted and another 6000A pump was used to deliver a solution of OPT for the post-column derivatization of histamine. Twenty feet of 9 thousandths (id) coiled stainless steel tubing was used as a mixing chamber and held at 60 C in a water bath. The reaction mixture then passed through a Water s 420 fluorescence detector which was connected to a recorder. The detector was equipped with a 340-nm excitation filter and a 440-nm emmission filter. 

Fluorescence microscope equipped with appropriate excitation filter, dichroic mirror, and barrier filter. For this example, an Olympus BHT compound microscope equipped with a BP490 excitation filter, BH-2DM500 dichroic mirror, and a LP515 barrier filter is used. 

Fig. 1. Comparisons of the wide-field, flying spot, pinhole detector, and pinhole confocal microscopes. Components include an excitation light source (V), an excitation filter (E), a dichromatic mirror (DM), an emission barrier filter (B), an objective lens (n), a detector (D), and a pinhole (P).

The detection performance of an LIF photometric device is governed by the emission filter(s), excitation filter(s), detector type, the excitation source and the detection scheme. The selection of optical elements and device configuration as it relates to the detection performance is further described by expanding the collection efficiency term in Equation 11.3 ... 

In UV fluorescence photography, the fluorescence of a substance excited by UV illumination is captured. The source of ultraviolet radiation filtered with an ultraviolet transmission filter, or excitation filter, is aimed at the subject in a completely darkened room. The subject reflects the ultraviolet light, but can also emit a visible fluorescence. The ultraviolet light is then prevented from reaching the film by a barrier filter that only allows visible light to be transmitted to the film. 

Thacker [24] reported the design of a miniature flow fluorimeter for liquid chromatography. The body of the fluorimeter was machined from a block of aluminium and contained a low-pressure mercury lamp, an excitation filter, a quartz flow cell, an emission filter, a photomultiplier tube and a photoconducter in order to compensate for fluctuations in lamp intensity. Fluorescence was examined at a direction perpendicular to that of the excitation light. The cell was small enough for it to be attached directly to the end of the column with a minimum dead volume. 

Fig. 37. Power spectrum of the excitation, filter function, and rate distribution for the filtered experiment in Figs. 35 and 36...

Fig. 7 Set-up for time-resolved imaging of 96-microwell plate formats, a Fast gateable CCD-camera, b Optical emission filter, c Light-guiding adapter, consisting of 96 optical fibers, d 96-microwell plate (black with transparent bottom), e Optical excitation filter, f Pulseable LED array with 96 light emitting diodes...

Fig. 6 x 100 fluorescence image of dextran fluorescein coated gold nanoparticles. The sensing particles were suspended in aqueous solution. A 470-490-nm excitation filter, a 505-nm dichroic mirror, and a 515-nm cut-on emission filter were used to collect the image. The exposure time is 0.5 s. The nanosensors are uniform in size and evenly coated. 

The Microplate algal toxicity test, another popular small-scale bioassay, is equally employed for WASTOXHAS applications (see Chapter 3, volume 1 of this book). In our case, rapid endpoint determinations of growth (72h-IC50s) are made with a microplate fluorescence reader, where algal biomass is indirectly measured via chlorophyll a fluorescence (excitation filter 440 nm - emission filter 640 nm). 

The fluorescence intensity of GFPuv was measured in the eluted samples in a spectrofluorophotometer (RF-5301 PC Shimadzu, Kyoto, Japan), with an excitation filter of 394 nm and an emission filter of 509 nm. Known amounts of purified recombinant GFPuv (standard GFPuv Clontech) diluted in buffer (10 mM Tris-HCl, pH 8.0 1.0 mM 0ME 0.1 mM PMSF) were used to generate a standard curve (Eq. 1) in order to relate the protein concentration with fluorescence intensity of the TPP-extracted/ HIC-purified aliquots. 

Examine as soon as possible with a fluorescence microscope (excitation filter No.l (BG12) and barrier filter No.47). 

Figure 16.5 (a) Diagram of a fluorescence microscope assembled by the addition of an excitation filter... 

In the epi-illumination method, a dichroic beam splitter comprises two parts one acting as an excitation filter and the second as an emission filter (Figure 16.6). 

An exploded view of the filter cube is shown in Figure 2. The excitation filter is shown in yellow and the emission filter in red to describe a typical bandpass Texas Red filter set. 

Another strategy is to utilize single multiband dichroic mirrors and emission filters and separate exciter filters either in an external slider or filter wheel. This will preserve the image registration and reduce mechanical vibrations, but the trade offs are a reduced brightness of the fluorescence, limitations on how many different probes can be separated, and reduced dynamic range and sensitivity due to the necessary color CCD camera. 

Epifluorescence Microscope. The diagram shows the working of an upright epifluorescent microscope. The excitation beam (black ray) from the arc lamp passes through the excitation filter and dichroic mirror and directed toward the specimen. The return beam of emitted fluorescence wavelength (red, green and black rays) is reflected of the dichroic filter, emission filter, ocular and goes to the detector (eye or camera). Courtesy of Prof. J. Paul Robinson, Ph.D., Director of Purdue University Cytometry Laboratory, Purdue University, West Lafayette, IN, USA. 

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