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For many analytical and life science applications the optical properties of the membrane are important because a constituent of the filtration stream is captured on the filter and then processed to allow visualization. These applications include particle monitoring and counting microorganisms. Most filters are opaque when dry. Although the polymer used in the filter’s manufacture may be transparent in another form, the porous nature of the filter results in a high degree of diffraction and light scattering. A notable exception is track-etched membranes where the transparency of the film used as the starting material is typically retained when the membrane is produced.
Some membrane filters become semitransparent or translucent when wet in aqueous fluids. In fact, the uniformity of membrane wetting can be quickly assessed by looking for the uniformity with which the membrane’s appearance changes from opaque to semitransparent or translucent. Not all filters exhibit this property. Depth filters typically remain opaque when wet as do some membrane filters, for example membranes made from polyethersulfone.
In many instances, the filter is encased in an opaque housing and is invisible to the user. Devices used for filtration of smaller volumes, generally less than one liter, sometimes employ transparent plastics for the housing. The plastic is chosen primarily for compatibility with the filter during device fabrication and not for visibility of the filter.
The optical properties of the membrane become important in the context of these visualization techniques. For some types of microscopy, it is necessary to clear the membrane. In other instances, this is unnecessary. For optical readers, a signal on the membrane can be assayed via:
For reflectance measurements light at a defined wavelength is shined onto the membrane surface and the light reflected back to the detector is measured. The consistency of the readings depends in part on the consistency of the membrane surface. While the membrane’s surface does not need to be glossy, it should have a uniform roughness so that the background reflection is uniform. Relative to the scale at which membranes are manufactured (thousands of square meters), a single reflectance measurement may be made on a tiny surface area (<1 mm2).
In some configurations, particles or optical signals on the membrane are measured using transmitted light. For maximum sensitivity, the membrane needs to be made transparent. This can be done by choosing a clearing agent with the same refractive index as the membrane. This is called the pore filling technique. Filling the pores with a liquid that has the same refractive index as the membrane allows light to pass through the filter, rendering it transparent. This technique can be used for most membrane filters with a single refractive index. The refractive index is 1.42 for PVDF membranes and 1.50 for nitrocellulose membranes. This technique does not work for filters having more than one refractive index, including polycarbonate membranes (refractive indices of 1.62 and 1.58) and composite membranes. Fluids used for clearing the membrane are typically oils or combinations of organic solvents.
In molecular biology applications, proteins or nucleic acids are immobilized on the surface of the membrane. Minute quantities of the molecules can be assayed using molecular probes such as antibodies and oligonucleotides. The final probe molecule in the detection scheme is conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase. A substrate molecule specific to the enzyme is then applied to the membrane. When the substrate is acted upon by the enzyme, one product of the reaction is photons of light, which can be detected on film or by camera. The directionality of the photons is random. Only those photons that come into contact with the detection system will be measured.
Fluorescent probes can be used on membrane filters. For cell-based assays, this may be the direct measurement of cellular constituents or cellular responses to various stimuli. For molecular assays, this may involve the indirect assay of immobilized proteins or nucleic acids. There are many different fluorophores available for different applications. One of the criteria for choosing a fluorophore is its optical compatibility with the membrane. Depending on the excitation wavelength, the membrane may exhibit autofluorescence, which will reduce the signal-to-noise ratio. Autofluorescence of the membrane may be caused by the polymer used in the manufacture of the membrane or contaminants introduced onto the membrane during its manufacture. If the membrane is in a device, the housing may also contribute autofluorescence. Autofluorescence of the membrane and the device, if present, should be measured before assaying samples.