Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence

Definitions

• the present invention relates to methods and apparatus for determining true fluorescence lifetimes in fluorescent materials, and in particular to a method and apparatus for imaging spatial variations in such fluorescence lifetimes in samples having a composition of varying lifetime species, or species in a multiple of lifetime states, distributed throughout the sample.
• Temporally resolved spectra offer a wealth of information about the state of a fluorescent molecule and its immediate environment.
• the fluorescence lifetime of a molecule is inversely proportional to the sum of all the deactivation pathways out of the excited state. Consequently the fluorescence lifetime of a fiuorophore is sensitive to environmental conditions such as pH, ionic strength and hydrophobicity, and to excited state reactions such as fluorescence resonance energy transfer (FRET), molecular quenching and triplet formation.
• FRET fluorescence resonance energy transfer
• the measurement of fluorescence lifetime is independent of fiuorophore concentration and light path length.
• Fluorescence Lifetime Imaging Microscopy enables the temporal attributes of fluorescence emission to be simultaneously measured in every pixel of a microscope image and has been described in the prior art applied in both the time and frequency domain.
• frequency domain lifetime measurements in general employ a repetitively modulated light source (typically a sinusoidally modulated laser source) to excite the fiuorophore of interest.
• the resulting fluorescence emitted by the sample is also modulated at the same frequency but phase shifted and demodulated relative to the excitation source.
• phase ( ⁇ ⁇ ) and modulation ( ⁇ M ) fluorescence lifetimes can be independently calculated. Only in fluorescent samples composed of a single homogeneous species are these quantities equal to each other and to the true fluorescent lifetime of the fiuorophore. For composite samples the true fluorescence lifetimes can only be determined from phase shift and demodulation measurements acquired at a number of modulation frequencies. A sample composed of N lifetime species requires the phase shift and demodulation to be measured at a minimum of N frequencies. By fitting these to a pair of dispersion relationships the true fluorescence lifetime composition of the sample can be estimated. Ideally, the circular frequencies of the modulations should be chosen so as to span all the reciprocal lifetimes of the sample.
• a fluorescent sample 1 is irradiated with excitation energy 2 which is intensity modulated at a certain frequency by an acousto-optic modulator 3 driven by the amplified voltage output from a frequency synthesizer 4a.
• Fluorescence emission 5 (expressed as F(t) in Figure lb) from the irradiated sample 1 is separated from the excitation light via a dichroic mirror 12 and an emission filter 13 and is focused onto the photocathode 6 of an image intensifier having a microchannel plate device 7.
• Photoelectrons generated by the light image incident upon the photocathode surface 6 are amplified by electron cascade across the microchannel plate 7, maintaining the spatial resolution of the image.
• the amplified electron image 8 exiting the microchannel plate strikes a phosphor screen 9 to generate an amplified light image which can be recorded by imaging onto a CCD camera 10.
• a second frequency synthesiser 4b phase-locked to the first and with controllable phase ⁇ G , is used to modulate the gain of the image intensifier (expressed as G(t) in Figure lb) either by applying the amplified modulated voltage signal 11 across the microchannel plate 7 or (as exemplified in figure lb) at the photocathode 6.
• Frequency mixing of the resulting modulated gain characteristics with the fluorescence signal is thus performed at every pixel of the image output 8 (expressed as D(t) in Figure lb).
• the phosphor screen 9 of the image intensifier behaves as a low pass frequency filter and only the low frequency difference signals are observed by the CCD 10 (expressed as D LP ( ⁇ G ) in Figure lb).
• phase dependent output image D LP ( ⁇ G ) which contains the phase ⁇ F and amplitude M F of the fluorescence signal.
• the phase dependent signal D LP ( ⁇ G ) at every pixel of the image is sampled over a full phase cycle (0° ⁇ ⁇ G ⁇ 360°) by recording an image at each phase setting ⁇ G . From a Fourier analysis of the set of phase dependent images D LP ( ⁇ G ) both the phase shift and demodulation associated with the fluorescence signal can be calculated. The minimum number of phase samples is chosen to satisfy the Nyquist criterion.
• phase shift and demodulation measurements have only been performed at a single modulation frequency for any one experiment, since sequentially collecting lifetime images at every frequency is prohibitively costly, both in terms of data collection times and in terms of the total time the microscopic sample is exposed to the excitation light. Both these points are especially critical in relation to microscopic measurements since the samples under observation are often dynamic in nature (ie. the movement of fluorescently labelled proteins within the spatial environment of a living cell) and far more sensitive to the effects of photobleaching than cuvette samples, where large sample volumes and diffusion ensure that the fluorophores at the point of illumination are continuously replaced. A further consideration is the huge volumes of data to be processed from multiple sets of phase dependent images. It is an object of the present invention to provide a method and apparatus for the simultaneous collection of fluorescence lifetime data and images at multiple frequencies.
• the methods described herein are equally applicable to the simultaneous collection at multiple frequencies of fluorescence lifetime data from cuvette samples using a modulatable point detector ie. by modulating the photocathode voltage of a photo-multiplier tube.
• the present invention provides a method for making fluorescence lifetime measurements comprising the steps of: irradiating a fluorescent material with a beam of excitation energy intensity modulated with a plurality of frequencies in a harmonic series including a fundamental frequency and at least one harmonic thereof; receiving a fluorescence emission from said fluorescent material into a detector having a controllable gain and an output; modulating the output of said detector using said controllable gain, with a modulation function having frequency components corresponding to at least two of said harmonic series of said irradiating beam, and having a controllable phase angle relationship thereto; and sampling said detector output to determine an amplitude for each of a plurality of phase angles.
• the present invention provides a method of imaging spatial variations in fluorescence lifetimes comprising the steps of: irradiating a fluorescent material with a beam of excitation energy intensity modulated with a plurality of frequencies in a harmonic series including a fundamental frequency and at least one harmonic thereof; imaging a fluorescence emission from said fluorescent material onto a detector having a controllable gain and an output; modulating the output of said detector using said controllable gain, with a modulation function having frequency components corresponding to at least two of said harmonic series of said irradiating beam, and having a controllable phase angle relationship thereto; and generating an image of spatial variation in the modulated detector output for each of a plurality of phase angles between said excitation beam and said modulation signal.
• the present invention provides apparatus for making multi-frequency fluorescence lifetime measurements comprising: a source of electromagnetic radiation for irradiating a fluorescent material with a beam of excitation energy intensity modulated with a plurality of frequencies in a harmonic series including a fundamental frequency and at least one harmonic thereof; means for receiving a fluorescence emission from said fluorescent material into a detector having a controllable gain and an output; means for modulating the output of said detector using said controllable gain, with a modulation function having frequency components corresponding to at least two of said harmonic series of said irradiating beam, and having a controllable phase angle relationship thereto; and means for sampling said detector output to determine an amplitude for each of a plurality of phase angles.
• the present invention provides apparatus for imaging spatial variations in fluorescence lifetimes in a sample, comprising: a source of electromagnetic radiation for irradiating a fluorescent material with a beam of excitation energy intensity modulated with a plurality of frequencies in a harmonic series including a fundamental frequency and at least one harmonic thereof; means for imaging a fluorescence emission from said fluorescent material onto a detector having a controllable gain and an output; means for modulating the output of said detector using said controllable gain, with a modulation function having frequency components corresponding to at least two of said harmonic series of said irradiating beam, and having a controllable phase angle relationship thereto; and means for generating an image of spatial variation in the modulated detector output for each of a plurality of phase angles between said excitation beam and said modulation signal.
• Figure 2 shows a graph (Fig 2b) plotting the photoelectron transfer characteristics, in response to constant illumination, of an image intensifier device (Fig. 2a); the effective gain response of the image intensifier to a sinusoidal photocathode voltage (Fig. 2c) is given in Fig. 2d, which can be modelled by a square pulse function with adjustable width D (Fig. 2e).
• Figure 3 shows a series of plots (3a to 3e) illustrating the variation in the relative modulation depth (the ratio of the amplitude in the gain at harmonic n to the average gain, G n /G 0 ) against the fractional pulse width for each of the first five harmonics, calculated from the square pulse wave model gain function shown in Fig. 2e;
• Figure 4 shows an exemplary apparatus for carrying out the method of the present invention
• Figure 5 shows a series of graphs of experimental data plotting the average gain G 0 and relative modulation (G n /G 0 ) for the first four harmonic terms in the gain response of an image intensifier measured as a function of the relative photocathode voltage bias;
• Figure 6 shows the harmonic content of phase sampled reflected excitation light for a set of 'low' and 'high' frequency modulations
• Figure 7 shows the results of processing mfFLIM data in order to separate the fluorescence of two green fluorescent protein mutants co- expressed in a live Hela cell.
• the present invention takes particular advantage of the photoelectron transfer characteristics of an image intensifier device in order to enable control of the higher harmonic content of an image output of the intensifier, by square pulse wave modulation of the gain characteristics.
• each of the harmonics in the gain of the image intensifier can be used in the simultaneous homodyne detection of matching harmonic modulations in the fluorescence.
• the means of introducing a matching harmonic set of modulations in the fluorescent signal will be discussed below.
• excitation field e.g. the excitation energy 2 of Figure la
• excitation energy 2 of Figure la repetitively modulated at frequency /
• Fourier series
• G(t, k) G Q + ⁇ G m - Cos m ⁇ t + ⁇ p m + mkA ⁇ (7)
• m ⁇
• G 0 the average gain amplitude
• G m the gain amplitude for every frequency harmomc with associated phase ⁇ m
• kA ⁇ the adjustable phase setting of the frequency synthesiser, which may be sequentially incremented by ⁇ p .
• the MCP 7 response is proportional to the incident fluorescence intensity multiplied by the gain characteristics of the intensifier.
• the frequency mixing results in a signal 8 composed of a time invariant response, oscillations at the harmonics of the gain and fluorescence modulations, and a combination of oscillations at the sum and difference frequencies of the harmonics.
• the slow response time of the phosphor screen 9 at the output of the imaging device gives an integrated signal image consisting only of the low frequency components of the total MCP response: (G 0 F 0
• the cosine (a coco), sine (bj and dc terms in the Fourier expansion given by equation 10 may be obtained on a pixel by pixel basis from the set of phase dependent images, either by the application of a discrete Fourier transform (DFT) or by fitting to a Fourier expansion 'model' i.e. a band-limited form of equation 10, using a singular value decomposition (SVD) algorithm.
• DFT discrete Fourier transform
• SVD singular value decomposition
• ⁇ £ ⁇ and M E n are the phase and relative modulation of a reflected or scattered excitation light at each harmonic.
• Excitation source Excitation of a sample under analysis by a beam of excitation energy intensity modulated with a plurality of frequencies in a harmonic series can be achieved in several ways.
• a pulsed laser source is used.
• mode- locked lasers typically have pulse repetition rates of approximately 80 MHz. This is ideal for measuring nanosecond fluorescence lifetimes.
• the pulse width from such a laser is typically less than 50 picoseconds which is sufficiently short in comparison to the period (1//) to produce a broad excitation spectrum which will include significant amplitude in many of the higher harmonic terms ie. 160, 240, 320.... MHz.
• a mode locked laser can be employed in combination with a pulse picker (typically performed using a Pockels Cell).
• standing wave acousto-optic modulators provide a means of modulating a continuous wave laser in a sinusoidal manner at high frequencies (e.g. 10' s- 100' s of MHz) to produce a much more limited excitation spectrum.
• high frequencies e.g. 10' s- 100' s of MHz
• Combinations of these AOMs can be employed in series to modulate the excitation light with a combination of frequencies corresponding to the modulation frequencies of all the individual AOMs, their difference frequencies and their sum frequencies.
• AOM modulation frequency Careful choice of AOM modulation frequency is preferable to ensure that the frequency content of the excitation corresponds to an harmonic series.
• This approach is used in a preferred embodiment of the present invention to be described later in connection with Figure 4.
• the number of harmonic components making up the excitation energy spectrum, E(t) is highly constrained. It may then not be necessary to sample the output to satisfy the Nyquist criterion for the highest harmomc possible as determined by the detector bandwidth, merely only to the highest harmonic present in the excitation spectrum.
• problems associated with aliasing when sampling the phase dependent output can be reduced or eliminated.
• the reduction in the sampling requirements has sigmficant benefits with respect to lifetime imaging of microscopic samples, where photobleaching and acquisition times impose significant experimental constraints.
• any voltage applied to the photocathode of an image intensifier acts as a switch to the flow of photoelectrons, where a change in polarity, from negative to positive, switches off this flow.
• a periodic zero-crossing control voltage results in a square wave type modulation of the gain characteristics of the device as the periodic voltage alternates in polarity.
• a sinusoidal control voltage is used.
• By controlling the bias, or DC component, of the sinusoidal control voltage it is possible to control the duration of the 'on' state of the device relative to its 'off state. In this way the width of the square pulse waveform can be used to control the amplitude of the higher harmonic content.
• the sinusoidal control voltage has a frequency which corresponds to the lowest (fundamental) frequency of the harmomc series of the excitation energy modulation.
• Modulation depths of up to 400% are possible from this form of modulation.
• Frequency synthesisers are widely available for providing a highly stable, high frequency sinusoidal voltage source (kHz-GHz) on top of a controllable bias voltage.
• the determination of photoelectron gain as a function of photocathode bias for an exemplary image intensifier unit is shown in Figure 2.
• An experimental configuration (corresponding to the image intensifier configuration in Figure 1) for the measurement is shown in Figure 2a, comprising elements photocathode 6, microchannel plate 7, phosphor screen 9 and CCD camera 10.
• the Hamamatsu C5825 bias voltage is controllable via a ten-turn potentiometer on its power supply module from -50N to +25N, with a resolution of approximately ⁇ 0.01V.
• the response of the image intensifier to the application of a sinusoidal photocathode bias voltage 23 as shown in Figure 2c can be shown as that in Figure 2d.
• This response is obtained from the graphical projection of the sinusoidal photocathode voltage onto the photoelectron transfer plot. To a good approximation, this represents a square pulse wave type response 24, and throughout the present specification, this will be generally referred to as a square pulse wave.
• the width 25 of the square pulse wave modulation can be controlled by changing the photocathode bias 26 ( Figure 2a).
• G(t) is periodic in time ie.
• G(t) G Q + ⁇ a n Cos(n ⁇ t)- b ⁇ in ⁇ n ⁇ t) (11)
• Figure 3 shows the variation in the modulation depth against r for the first five harmomc terms of the gain, where G 0 is the fundamental, and G]...G 5 are the first five harmonics.
• Relative modulation depths up to 400% are possible for sufficiently narrow pulse widths. This is four times greater than the maximal modulation depth possible with pure sine wave modulation. The higher modulation comes at a cost to detection sensitivity.
• detectors can operate satisfactorily with modulated outputs as high as 360 MHz.
• the continuous wave output 42 of an Argon/Krypton laser 41 is passed through a pair of AOMs 43, 44 (either a 40 MHz or 160 MHz in combination with an 80 MHz standing wave AOM 44).
• the output light source excitation beam 45 is thereby sinusoidally modulated at high frequencies at both the fundamental AOM frequencies, and the sum and difference frequencies, to provide a harmonic set of modulation frequencies from 20 MHz to 340 MHz, which are used to excite sample 1 within microscope 71.
• the AOMs 43, 44 are respectively driven by the amplified voltages 50, 51 from two (slave) frequency synthesisers 52, 53 which are phase locked to a third (master) frequency synthesiser 60 using a first output 60a.
• the third frequency synthesiser 60 also has a second, phase-adjustable, output 60b. This is used to derive, via amplifier 61, a gain control signal 62 used to modulate the photocathode voltage of an image intensifier 70, at the fundamental harmonic frequency of the excitation beam 45.
• the photocathode voltage could alternatively be modulated at one of the harmonic frequencies of the excitation beam, but that this would reduce the number of higher harmonics in the gain response G(t) for which lifetimes can be calculated, and would thus be less efficient.
• the relative phase difference between the excitation beam 45 and the gain control signal 62 can be controlled by the phase-adjustable output 60b, enabling phase dependent image collection. This is preferably carried out under the control of a computer system 81 by control bus 82. Iris diaphragms 46, 47 placed approximately 1.5 metres from each of the AOMs 43, 44 select the zero from the higher order diffracted beams (6.4 mrad beam separation) and a variable neutral density wheel 48 of 0-5 OD provides for control of the overall signal intensity.
• the high spatial and temporal coherence properties of the laser beam are removed by passing the light through a rotating ground glass disc 49.
• the scattered radiation from the rotating ground glass disc 49 is collected and collimated with a high numerical aperture lens 58 before being directed into the epi- illumination port of an inverted microscope 71 which results in K ⁇ hler illumination at the sample 1.
• Steady state fluorescence images are capmred using a scientific grade CCD camera 10.
• the sample fluorescence is first imaged onto the photocathode 9 of a C5825 image intensifier ( Figure 2a).
• a telescopic lens 72 optically couples the phase dependent image at the phosphor screen 9 output to the CCD camera 10 and images are downloaded over an AIA bus 80 to an interface card in the computer system 81.
• the computer system 81 also provides positional control and feedback of a motorised stage 83 on which the sample is mounted, by way of a control unit 84 and interface bus 85.
• a series of phase dependent images are taken over the full cycle (0-360°) of the fundamental harmomc, where the number of images satisfies Nyquist' s criterion for the full harmomc content present in the homodyne signal.
• the resulting sampled output is then passed to the computer system 81 where a Fourier analysis can be performed.
• Figure 5 shows exemplary measurements of relative harmonic content present in the gain characteristics of the C5825 image intensifier as function of photocathode relative bias voltage.
• the relative bias voltage is defined as the photocathode offset voltage divided by the amplimde of the applied sinusoidal voltage.
• the average gain in Figure 5a has been normalised to its maximum value and in Figures 5 (b-e), the relative modulation depths in the gain are scaled by half the relative modulation depth of the reflected excitation.
• the data for each figure was acquired with the fundamental frequency of the reflected light and photocathode voltage respectively set to (a, b) 80.236 and 80.236 MHz, (c) 80.236 MHz and 40.118 MHz, (d)80.238 MHz and 26.746 MHz and (e)80.236 MHz and 20.059 MHz.
• FIG. 6 Typical examples of phase sampled excitation light from a reflecting sample and its relative harmonic are shown in Figure 6. Because the sample (a piece of aluminium foil) has no lifetime associated with it these figures essentially show the instrumental response of the detector to the excitation modulations.
• the data shown in Figure 6a designated as the "low" frequencies set, was obtained with the 40MHz and 80MHz AOMs 43, 44 set to modulate the excitation light at frequencies of 42.154 MHz and 63.231 MHz respectively; where frequency mixing gave rise to additional modulations with amplitudes greater than 0.1 at 21.077MHz, 105.385MHz and 126.462MHz.
• the mfFLIM configuration and fitting routines were tested on an equi- Molar (1 ⁇ M) solution of rhodamine 6G and rhodamine B in distilled water.
• the excitation light was modulated by the 40MHz and 80Mhz AOMs tuned 42.154 MHz and 63.231 MHz (including the sum and difference mixing frequencies).
• Homodyne detection of all the excitation frequencies was achieved by modulating the photocathode of the MCP at a fundamental frequency of 21.077 with a relative voltage bias of about 0.9, and 32 phase sampled images were collected in each case (i.e. 11.25° phase steps between images).
• Reference images were obtained by phase sampling reflected light from aluminium foil illuminated with the modulated laser light. All phase dependent images were corrected for dark current and stray light by subtracting an image acquired in the absence of excitation field illumination. No significant photobleaching was observed in the fluorescence signal, due in main to the large diffusional volume of the sample.
• Table 1 lists the results of fitting the phase shift and demodulation images generated from a Fourier analysis of the set of phase dependent images to the dispersion relationships (equations 3 and 4), based on a bi-exponential lifetime model.
• the lifetime values of 1.29 ns and 3.74 ns correspond well with listed literature values of 1.5ns and 4ns for rhodamine B and 6G respectively (Lakowicz & Berndt (1991). Rev. Sci. Instrum. 62, 1727).
• Table 1 Fluorescence lifetime fit parameters of rhodamine B/6G mixture.
• the number of frequencies in the harmomc series present in the excitation and gain modulation spectrum should be at least as many as the number of different lifetime species or states expected in the sample 1 in order to be able to resolve each lifetime species or state by fitting the data to the dispersion relationships.
• the excitation spectra of the detector systems of the present invention are calibrated by using a scattering surface sample 1 having no fluorescence lifetime characteristics. This enables determination of the optical and electrical path lengths of the detection system and the phase and modulation shifts caused thereby. This information is stored by the computer system 81 and used to compensate the output measurements taken.
• FIG 7 shows a practical application of the mfFLIM instrument.
• mfFLIM can be used to disentangle the cellular distributions of two co-expressed green fluorescent protein mutants with differing lifetimes.
• GFP5 Golgi localisation signal N-acetylylglucosaminyltransferase I fused to the green fluorescent protein mutant GFP5 (ZernickaGoetz et al. (1997), Development 124: 1133).
• NA-GFP5 novel green fluorescent protein mutant YFP5 (which will distribute in the cytosol and nucleus).
• YFP5 is described in UK Patent co-pending application entitled "Fluorescent Protein" which has the same filing date as this application. Because of a high degree of spectral overlap it is difficult to efficiently isolate the fluorescence of these two GFP mutants by conventional filtering. In mfFLIM measurements a long pass dichroic and broadband emission filter can be used in order to simultaneously collect the fluorescence from both the GFP mutants.
• Figure 7a shows the dc component of the set of phase dependent images taken of one of the expressing Hela cells.
• Figures 7b and 7c show the isolated fluorescence from the GFP5 and YFP5 respectively, calculated by fitting the phase shift and demodulation images to the dispersion relationships based on a bi- exponential decay model. Improving mfFLIM data analysis
• each pixel in the image can be conceived of as an individual experiment. Global analysis of a multiple of such experiments has a clear advantage over individual analysis of the data at a single point (Beechem (1992) Methods Enzymol. 210, 37). Inter-relationships between decay parameters at each pixel can be encoded in a global fit of the image in order to significantly reduce the fitting errors.
• the lifetimes are pixel invariant and could be linked in the double exponential decay fitting model over the whole image where the amplitudes are left uncoupled.
• mfFLIM data it should be possible to sigmficantly reduce the number of frequency measurements necessary to achieve estimates of fluorescent lifetime parameters with a specified signal to noise ratio. This is critical with microscopic samples where the data acquisition time and total exposure have to be carefully controlled.
• the measurements of the populations of GFP fusion proteins or fluorescence resonance energy transfer (FRET) through donor and acceptor tagged proteins in cells would be examples of systems where the global fitting approach might be expected to bring significant improvements in quantifying the populations or states of molecules.
• FRET fluorescence resonance energy transfer

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