Welcome to Fluorescence-Microscope
The configuration and placement of the three optical filters (excitation filter, dichroic beam splitter, and emission or barrier filter) are shown by the further detail of the fluorescence filter cube. The optical filters are key components enabling the detection of fluorescence light from the sample, thereby enabling the functionality that lies at the heart of a fluorescence microscope. The excitation filter passes a portion of light from the intense light source, which is then reflected by the dichroic beam splitter (also referred to as a mirror) such that it passes through the fluorescence microscope objective and fully illuminates the specimen on the sample slide that is on the microscope stage.
A substantial portion of the fluorescence emission is captured by the microscope objective, and then it is transmitted through the dichroic beam splitter and through the emission filter to the microscope eyepieces. In this so-called epi-fluorescence configuration, the set of three optical filters function together as a complete set to capture fluorescence-emission light and block contaminating excitation light and other potential sources of background light that would otherwise reduce sensitivity or even suppress the fluorescence from the fluorophore. Absorbed by a flourophore is the incoming excitation light at wavelengths in its absorption-spectrum profile. Shown at the optical filter spectral profiles is the superimposed on the FITC absorption and emission spectral profiles.
Here for simplicity, BW refers to the full-width-at-half-maximum transmission bandwidth, but bandwidth may also be specified as the minimum spectral width over which high transmission is guaranteed. A bandpass emission filter in this case (CW = 529 nm and BW = 33 nm) is designed so that it specifically captures fluorescence emission primarily from a single fluorophore (such as green emission). It is worth noting that while the spectra of bandpass excitation and emission filters appear symmetric when the transmission is plotted on a linear scale, suggesting that an excitation filter may be used as an emission filter, or vice versa, so long as the CW and BW are appropriate, in fact the blocking (visible only on a logarithmic scale) is typically not symmetric. The function of the excitation and emission filters is therefore complimentary, ensuring that excitation or other stray light does not spectrally leak, contaminate, and suppress the fluorescence-emission light. Another result of a widening of the BWs of the filters is the increased brightness.
The nonspecific fluorescence and autofluorescence can be minimized by careful sample preparation but not totally eliminated. A balance, therefore, has to be struck between the levels of brightness versus background based on the desired observation criteria for a given application. Dichroic beam splitters and long pass excitation filters are usually specified by a cut-on edge wavelength (EW) above which the filter transmits light. This property enables the clinical observer to see possible counter stains or other fluorescence emission that can be used to enhance contrast between different cells and parts of a biological cell. Optical filters must be impervious to intense light sources that generate ultraviolet (UV) light that could lead to “burnout” (also known as photodarkening or solarization), particularly of the exciter filter as it is subjected to the full intensity of the illumination source.
Absorptive or “colored” substrate glasses are also used in some exciter filters (particularly those optimized for Calcofluor White, DAPI, and other UV-excited fluorophores), and these filters are particularly prone to “burnout” since they contain impurities that lead to photodarkening. These filters are based on a thin-film manufacturing process called Ion-Beam Sputtered (IBS), which enables filters with “hard” oxide thin-film coatings to be constructed, using a single, low-impurity glass substrate the result is the highest possible transmission with exceptional durability and reliability. In fluorescence microscope, the sample you want to study is itself the light source. The technique is used to study specimens, which can be made to fluoresce. The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. The sample can either be fluorescing in its natural form like chlorophyll and some minerals, or treated with fluorescing chemicals.
Call our Sales Hotline at 1-877-384-3931
Click here to view other Fluorescence-Microscope Products.
