Dynamic Fluorimetry
Fluorimetry
Fluorescence continues to be a powerful tool for life science research due to its sensitivity, selectivity and general usability. The safety benefits over radioactivity-based assays have also been recognized. Typically, the presence and amount of a fluorescently-tagged analyte is correlated to the intensity of the fluorescence measured. This can be done in many formats including bulk biochemical assays, spot arrays, bead and cell-based assays. Multiple emission colors can be used to multiplex multiple analytes within the same assay.
Laser Scanning Fluorimetry
The majority of fluorescence-based assays are performed in bulk solution as relatively well-controlled biochemical assays. Laser scanning offers a two-dimensional image of fluorescence emitting from objects (spot, beads, cells, etc.) on the surface of slides or microplate well bottoms. Traditionally this has been a method of choice for reading arrays for nucleic acids, proteins, and recently adherent cells. In laser scanning methodology, a small micron sized laser spot is rastered along a surface on which the array elements, beads, or cells are located. At every position that the spot covers, the emission intensity is recorded and a two-dimensional intensity map can be reconstructed. The reconstructed map is essentially an image of the surface containing the fluorescent objects.
Fluorescence Polarization
The use of fluorescence polarization (FP) builds on these techniques by using a reporter fluorophore's fluorescence properties to indicate binding state and microenvironment. Polarization is a measurement of a molecule's rotation from its excitation until it's emission. Polarization (P) is defined as:
Where I|| is the emission intensity parallel to the excitation plane and I⊥ is the intensity perpendicular to the excitation plane.
If the emission is completely maintained in the parallel direction then P=+1. If the emitted light is totally polarized in the perpendicular direction then P=-1. In the actual measurement of polarization there is usually an angle between the excitation dipole and the emission dipole within a molecule. For example, even a fixed, non-rotating fluorophore, will have some "intrinsic" depolarization due to this angle.
Anisotropy
Another term representing the degree of polarized emission is anisotropy (r) which is defined as:
The difference in the way polarization and anisotropy is defined is the presence of a second perpendicular intensity term in the denominator. The definition of anisotropy takes into account the second possible perpendicular emission plane, which is oriented along the axis of propagation. Anisotropy is a more accurate representation of the physical phenomena because it takes into consideration the contribution of all degrees of rotational freedom and reflects orientations of constrained molecules that can be detected through two-dimensional mapping of polarized fluorescence emission.
Time Resolved Fluorescence
Time-resolved fluorescence (TRF) is another aspect of fluorimetry that has been adapted for increased information content or sensitivity in the assay readout. Fluorescent molecules have a fluorescent lifetime associated with the emission process. Typically lifetimes for small fluors such as fluorescein and rhodamine are in the 2-10 nanosecond range. These lifetimes can be influenced by interactions with their environment. Energy transfer, quenching, conformational changes, binding, and hydrophilic or lipophilic environments can dramatically effect lifetime.
Traditionally, TRF assays use a long-lived (>1000 ns) fluorophores to discriminate assay signal from short-lived interference such as autofluorescence of the matrix or fluorescent samples which almost always have lifetimes much less than 10 ns. Instruments for TRF simply delay the measurement of the emission until after the short-lived fluorescence has died out and the long-lived reporter fluorescence still persists. One of the latest versions of this technique utilizes a proximity sensitive resonant energy transfer between two fluorophores, one of which is long lived and hence shifts the readout away from the short-lived (background) interference. This can be described as time gated fluorescence since no determination of the absolute lifetime is performed. The ability to assemble an image in which each pixel reflects a true lifetime is a much more powerful technique.
Fluorescence lifetime can be determined in two fundamental ways. The time domain technique uses very short pulses (picosecond) of excitation and then monitors the emission in real time over the nanosecond lifetime. Fitting the decay curve to an exponential yields the lifetime. The frequency domain technique modulates the excitation at megahertz frequencies and then watches the emission intensity fluctuate in response. The phase delay and amplitude modulation can then be used to determine lifetime. Blueshift uses the frequency technique for fast and economical lifetime imaging.
Rotational Mobility and Lifetime
Mobility and lifetime are the two factors that determine anisotropy. No matter how fast a molecule is rotating, if its lifetime is short enough, there will be a high anisotropy, i.e. very little perpendicular emission. Likewise, if a large, slowly rotating molecule has a very long lifetime, there will also be a high anisotropy. Hence, both size (rotational mobility) and fluorescence lifetime can influence anisotropy. A molecule rigidly bound to a surface or cellular organelle is essentially an infinitely large molecule, and binding assays based on anisotropy can offer a sensitive, powerful approach. Lifetime and Energy Transfer
When Forster Resonant Energy Transfer (FRET) occurs between a donor and acceptor fluorophore, the lifetime of the donor's fluorescence is reduced by as much as 50%. Quenching is another energy transfer mechanism which produces the same effect. These proximity sensitive phenomenon can be exploited with the appropriate assay components and instrumentation. Co-localization of less than 200 nanometers cannot be determined by light microscopy alone, FRET allows the detection of co-localization in time and space, without the need for high optical resolution.
Object-Based Dynamic Fluorimetry
One of the many technologies that Blueshift Biotechnologies is providing makes use of laser-scanning technology with object-based fluorescence anisotropy readouts. Object-based fluorimetry refers to discriminating an object's signal from that of the bulk signal. Sub-populations of objects within a sample, such as cells in mitosis or those successfully transfected, can be evaluated while ignoring others. Many have likened this to flow cytometry without the flow. Multiplexed and more homogenous microarray, bead binding, and cell-based assays become easy to develop and run as primary screens. For the first time, high throughput, two-dimensional anisotropy can provide novel screening information on the interactions of relevant biomolecules and structures. It has been shown that even fluorescent proteins have anisotropy changes upon conformational changes or translocating between cellular environments.