Abstract:
A quantitative fluorescence technique is presented as &#34;differential real-time fluorescence spectroscopy&#34; (DRFS), pursuant to which one can simultaneously make time-resolved measurement of fluorescence changes in two cuvette samples (e.g., experimental and control), wherein the fluorescence is induced by excitation light, and wherein both samples have been identically prepared with a selected ratiometric dye. The ratiometric dye may either be of excitation-shifted variety or of emission-shifted variety. With DRFS, it is possible to monitor the response of specimen cells to a chemical, biological or physical agent, in the absence and in the presence of a selected experimental variable, and to determine both of these responses in real time, i.e., at the same time as the measurements are being made. The invention is therefore ideally suited to monitor response from experimental stimuli that are expected to induce only relatively small cellular changes during the typical time course of a fluorescence measurement, namely, 20 minutes or less. The reason for this capability is that DRFS eliminates problems attributable to inherent biological variability associated with preparations of living cells, such as known and unknown (e.g., time-dependent) changes in base-line biological activity during the storage or handling of prepared cells, prior to and awaiting a particular experiment.

Description:
BACKGROUND OF THE INVENTION 
     The invention relates to fluorescence spectrophotometric equipment and methods, particularly for making high-precision measurements in the determination of specimen response to a chemical, biological or physical agent wherein evaluation is made for a &#34;control&#34; sample of the specimen in relation to an &#34;experimental&#34; sample of the specimen. 
     U.S. Pat. Nos. 4,795,256 and 5,039,219 are illustrative of state-of-the-art precision fluorescence spectrophotometric equipment in which instrument sensitivity (e.g., as defined by signal/noise ratio) is high and cuvette-based fluorescence experiments are conventionally made, using one sample at a time. However, high-precision measurements do not necessarily yield meaningful and accurate results, particularly when one is concerned with the accuracy with which weak external influences on real-time kinetics of samples of biological cells are to be determined by fluorimetry. The accuracy of any experimental evaluation is limited by repeatability, that is, the extent to which a measurement with the same initial conditions (e.g., including the same biological state of the sample) will produce the same result. For example, when data from two or more consecutive fluorescence recordings (i.e., &#34;experimental&#34; and &#34;control&#34;) are compared, it is generally assumed that the quantitative biological response patterns (i.e., kinetics) are the same. However, this may not always be the case, in that the false assumption of a constant cell response will be reflected in erroneously interpreted results. In other words, two separate recordings will differ, not because of imprecise measurements, but because the response kinetics may have changed in one or more significant ways. If a change in response kinetics is not accounted for, the change will factor into a measured fluorescence change, consequently prompting an inaccurate interpretation of results. 
     BRIEF STATEMENT OF THE INVENTION 
     It is an object of the invention to provide an improved method and apparatus for fluorescent spectroscopic examination of a specimen, wherein the above-noted inaccuracy can be eliminated or at least very substantially reduced. 
     It is a specific object to meet the above object with methodology whereby two or more samples of virtually identical biological response patterns can be analyzed simultaneously, whereby to achieve results that can be compared on a real-time basis. 
     Another specific object is to meet the above objects for the case of a dual-fluorescence analysis. 
     A further specific object is to provide an improved method and apparatus for fluorescent spectroscopic analysis whereby to achieve substantially greater accuracy and reproducibility of observations, particularly for cases in which the experimental specimen is subjected to a relatively weak chemical, biological or physical agent, such as a weak electromagnetic field. 
     It is also a specific object in a dual-beam dual-wavelength fluorescence system to make instrumental time resolution independent of any switching of light paths that may be involved in the measurement process. 
     A general object is to meet the above objects with apparatus which involves relatively little departure from what is commercially available, and which is inherently capable of performing a vastly greater number of experiments in a given time, as compared to prior techniques. 
     The invention in a preferred mode meets the above objects with what I believe to be a new quantitative fluorescence technique which I term differential real-time fluorescence spectroscopy (DRFS), pursuant to which one can simultaneously make time-resolved measurement of fluorescence changes in two cuvette samples (e.g., experimental and control), wherein the fluorescence is induced by excitation light, and wherein both samples have been identically prepared with a selected ratiometric dye. The ratiometric dye may either be of excitation-shifted variety or of emission-shifted variety. With DRFS, it is possible to monitor the response of specimen cells to a chemical, biological or physical agent, in the absence and in the presence of a selected experimental variable, and to determine both of these responses in real time, i.e., at the same time as the measurements are being made. The invention is therefore ideally suited to monitor response from experimental stimuli that are expected to induce only relatively small cellular changes during the typical time course of a fluorescence measurement, namely, 20 minutes or less. The reason for this capability is that DRFS eliminates problems attributable to inherent biological variability associated with preparations of living cells, such as known and unknown (e.g., time-dependent) changes in base-line biological activity during the storage or handling of prepared cells, prior to and awaiting a particular experiment. 
     By way of illustration, actual use of the invention will be described for an investigation of the dose-dependent acute effects of an inhibiting drug on stimulus-induced increases in intracellular calcium levels [Ca 2+  ] i , in human leukemic T-lymphocyte cells. 
    
    
     DETAILED DESCRIPTION 
     Preferred and other embodiments of the invention will be described in detail, in conjunction with the accompanying drawings, in which: 
     FIG. 1 is a diagram schematically showing optical and electrical components of a preferred embodiment of the invention, for the case of using an excitation-shifted ratiometric dye in dual-wavelength excitation measurements; 
     FIG. 2A is graphical recording made in use of the embodiment of FIG. 1, showing, in real time, measured fluorescence for identical &#34;control&#34; and &#34;experimental&#34; specimens as a function of the same time scale, and for the illustrative case of subjecting the experimental specimen to a relatively weak concentration of an inhibitory drug; 
     FIG. 2B is a recording similar to that of FIG. 2A, for case of a doubled concentration of the inhibitory drug; 
     FIG. 3 is a diagram similar to FIG. 1, to illustrate application of the invention to real-time dual-wavelength fluorescence observation of a greater plurality of specimens; 
     FIG. 4 is a diagram similar to FIG. 1 for another embodiment of the invention, for the case of using an emission-shifted ratiometric dye for dual-wavelength emission measurements; 
     FIG. 5 is a diagram similar to FIG. 1 for a modification of FIG. 1, for making dual-wavelength excitation measurements using an excitation-shifted dye; and 
     FIG. 6 is a diagram similar to FIG. 5 for a modification wherein dual-wavelength emission measurements are made using an emission-shifted ratiometric dye. 
    
    
     Referring initially to FIG. 1, the invention is shown as a dual-wavelength fluorescence spectrophotometric system for concurrently comparatively analyzing two identical specimens, respectively contained and supported in identical cuvettes 10, 11 in separate chambers 12, 13. Preparation of the specimens will be understood to have included a ratiometric fluorescent dye that will have been selected for the purposes of a particular experiment. In the present illustrative situation, changes in intracellular calcium concentration [Ca 2+  ] i  will be assumed to be important to the experiment, and the dye Fura-2 will be assumed to have been selected for the purpose. Fura-2 is a well-known excitation-shifted dye suitable for detection of calcium; it is commercially available from Molecular Probes, Inc., Eugene, Oreg. Fura-2 is one of a variety of excitation-shifted dyes; it has the property of responding to excitation in one or both of two spaced relatively narrow bandwidths that are respectively centered at 340-nm and 380-nm, with fluorescence emission at 510-nm. 
     The chambers 12 (13) are identical, each having an inlet-porting alignment for exposing its cuvette 10 (11) to excitation light from an optical-fiber cable 14 (15), and an outlet-porting alignment for delivery of resultant fluorescent light to a photon detection device 16 (17) to measure fluorescent output. The designations PMT at 16 (17) will be understood to apply for devices which measure fluorescent output in the exiting beam from each of the respective chambers. Further symbolism within chambers 12 (13) will be understood illustratively to include identical coils 18 (19) for generating magnetic fields, identical thermostatically controlled cooling/heating systems 20 (21), and identical optical systems such as concave mirrors 22 (23) for more efficient direction of fluorescent-light output to the respective PMT&#39;s at 16 (17). In the labeling of the PMT&#39;s, parenthetical reference at 16 to &#34;control&#34; and at 17 to &#34;EXP.&#34; (meaning &#34;experimental&#34;) will be understood to designate the respective functional purposes of the identically prepared specimens at cuvettes 10, 11. 
     In accordance with an important feature of the invention, the lines 14, 15 of optical-fiber connection to chambers 12, 13 are identical and are supplied with like shares of light at 340-nm and at 380-nm, in equally shared time-interlaced relation. As shown, such light originates with a source 25, which may be a Xenon-arc lamp, producing an output beam on an optical axis to a continuously rotated mirror chopper 26, for splitting the lamp beam into two beams 27, 28, in equally shared time-interlaced alternation. The chopped and unreflected light of beam 27 is shown restricted by a first filter F(340) to the Fura-2 excitation-wavelength band, centered at 340-nm, for delivery of the same via a first optical-fiber cable 29 to an optical device 30; and the chopped and reflected light of beam 28 is restricted by a second filter F(380) to the second Fura-2 excitation-wavelength band, centered at 380-nm, for delivery of the same via a second optical-fiber cable 31 to the optical device 30. Cables 29, 31 are preferably of equal length and contain identical quantities of like optical fibers; cables 29, 31 may be the two branches of a single bifurcated fiber-optics bundled assembly. Similarly, the optical-fiber cables 14, 15 which deliver excitation light to the respective specimens 10, 11 may be the two branches of another and identical bifurcated fiber-optics bundled assembly, in turn assembled back-to-back at 30 with the assembly for cables 29, 31. The function at optical connection 30 is to assure in each of the cables 14, 15 an equally divided share of excitation light from each of the cables 29, 31. Description of items related to excitation light in FIG. 1 is completed by identifying a light beam 32 between a local source and a photocell 33, with interruption by chopper 26, whereby, with suitable amplification at 34, a signal is available in a synchronizing line 35, for purposes to be described. And a shutter 25&#39; is suggested at the exit of light from source 25 to prevent incidence of excitation light on specimens at 10, 11, unless and until an experiment is being run. 
     The &#34;control&#34; PMT 16 will be seen to respond, for a specific calcium concentration, with a greater signal level for the chopped intervals of 340-nm exposure, in time-interlaced relation with a lesser signal level for the intervening chopped intervals of 380-nm exposure; in this connection, signal-to-noise at the excitation-shifted fluorescence wavelength 510-nm is enhanced by a suitable filter F(510). These PMT signals are then supplied to signal-processing means, collectively designated 36 for the &#34;control&#34; side of the system. Identical processing will be understood to exist for identical excitation on the &#34;exp.&#34; side of the system, for which identical, duplicate signal-processing means is collectively designated 36&#39;. The only difference between signals detected at PMT 16, as compared with those detected at PMT 17, is the fact of a selected one or more chemical, biological, or physical stimuli for the experimental-cuvette contents of chamber 13, as compared with the control-cuvette contents of chamber 12. 
     As shown, &#34;control&#34; signal-processing means 36 comprises an electronic switch 37 operating from the chopper-synchronizing signal in line 35. Switch 37 separates the time-interlaced signals, supplying to a first processing circuit 38 the segregated 510-nm fluorescent response to 340-nm excitation, and supplying to a second processing circuit 39 the segregated 510-nm fluorescent response to 380-nm excitation; suitably and preferably, the processing at 38, 39 includes analog/digital conversion, so that ensuing functions can be digitally processed. Output of the respective circuits 38, 39 is connected to a quotient-determining circuit 40, whereby the ratio of the signal which reflects response to 340-nm excitation, in relation to the signal which reflects response to 380-nm excitation is available for recording of the indicated &#34;control&#34; ratio, as a function of time in the on-going course of a given experiment. Identical signal-processing components for the &#34;experimental&#34; signals detected by PMT 17 need no further description and are therefore given the same reference numbers, with primed notation; the net result is that an &#34;experimental&#34;-signal ratio output from circuit 40&#39; which also reflects response to 340-nm excitation, in relation to the signal which reflects response to 380-nm excitation, is available for recording of the indicated &#34;experimental&#34; ratio, as a function of the same time scale as the &#34;control&#34; ratio, in the same on-going course of the experiment. In FIG. 1, a single display means 41 is shown connected for simultaneous display and/or storage of involved &#34;control&#34; and &#34;experimental&#34; ratio determinations in real time. 
     FIGS. 2A and 2B are illustrative recordings made simultaneously and on a real-time basis, for a &#34;control&#34; signal ratio (CTR) and an &#34;experimental&#34; signal ratio (EXP), wherein Fura-2 was the excitation-shifted dye used in the apparatus of FIG. 1, and wherein the traces shown in these FIGS. 2A and 2B are the raw-processed data available at the control/experimental display 41 of FIG. 1. 
     FIGS. 2A and 2B represent the time-evolution of the intracellular calcium concentration in Fura-2-loaded human leukemic T-lymphocyte cells (JURKAT-clone E6-1), wherein the displayed evolution proceeds in the time period 0 to 900 seconds, and wherein an increase in the ratio F340/F380 signifies an increase in intracellular calcium concentration in the T-lymphocyte cells, the same being suspended in a physiological buffer solution. 
     More specifically, in both FIGS. 2A and 2B, the time period 0 to 300 seconds reflects the base-line calcium levels in both the &#34;experimental&#34; and the &#34;control&#34; T-lymphocyte cell suspensions at cuvettes 10, 11, respectively. At time 300 seconds, a dose of a calcium-flux inhibitory drug, ECONAZOLE (0.25 μM in FIG. 2A, and 0.50 μM in FIG. 2B) was added, exclusively to the &#34;experimental&#34; cuvette sample 11; such doses will be observed not to lead to baseline deviations, as between the &#34;experimental&#34; and the &#34;control&#34; samples at 10 and 11. Then, at time 420 seconds, a &#34;stimulus&#34; applied equally to both the cell suspensions at 10, 11, was administered; the stimulus was &#34;THAPSIGARGIN&#34; at a concentration of 0.1 μM. This stimulus induced a rapid increase in intracellular calcium concentration in the involved cells, characterized by a peak at approximately 500 seconds, followed by a decline to essentially a plateau at about 900 seconds. FIGS. 2A and 2B illustrate the very noticeable difference in this peak attributable to the inhibitory drug, not only in the comparisons against the &#34;control&#34; response but also as a function of having doubled the drug concentration in the experiment of FIG. 2B. It is notable that even the influence of relatively weak drug administration on the stimulus-induced calcium-concentration rise (FIG. 2A) can be clearly discerned, by the dual-beam, dual-wavelength fluorescence spectrophotometric technique involved in a single experimental run with the apparatus of FIG. 1. 
     The system of FIG. 3 will be seen as having great similarity to FIG. 1, with the exception that FIG. 3 illustrates that the invention is equally applicable to simultaneous real-time observation and measurement of plural specimens when the plurality exceeds two. The only limitation to the number of simultaneously observed specimens is that sufficient excitation-light flux shall be available from source 25, after spectral restriction to F(340) and F(380) distribution, with equally shared fractions of F(340) and F(380) light, divided at optical connection 30&#39; into each of a plurality of optical-fiber cable branches 14&#39;, 15&#39; and 15&#34;, serving plural prepared specimens at cuvettes 10, 11&#39; and 11&#34; in chambers 12, 13&#39; and 13&#34;. Legends at the single display means 41 indicate the capability of concurrently responding to the ratio output of signal-processing means 36 for the fluorescent output of the control specimen at 10, as well as (i) the ratio output of signal-processing means 36&#39; for the fluorescent output of a first or Experiment-A specimen at 11&#39;, and (ii) the ratio output of signal-processing means 36&#34; for the fluorescent output of a second or Experiment-B specimen at 11&#34;. In each of these three cases, the prepared specimens may illustratively have been identical, and the only difference in the course of a given run may, for example, be in the concentration of an added chemical agent, or the strength of an ambient electrical or magnetic field, as between Experiment A and Experiment B. A further reference numeral 15 n  applied to a fragmentary phantom optical-fiber cable branch will be understood to indicate further equal sharing (at 30&#39;) of the time-interlaced dual-wavelength excitation light, to serve up to N experiments at a time, with processing identical to what has been described for Experiments A and B. And a legend applicable to an arrow in connection with display 41 is indicative of the fact that all processed experiments (A to N), plus the processed control signal can be presented comparatively, in real time and to the same time scale. As noted above, the limiting number of such further simultaneous experiments or measurements will depend upon whether the equal-sharing operation at 30&#39; will deliver to each specimen sufficient light flux (at both of wavelengths 340-nm and 380-nm) to stimulate meaningful fluorescence at 510-nm (for each of the excitation wavelengths, 340-nm and 380-nm). 
     The system of FIG. 4 illustrates use of the invention in a mode wherein dual-wavelength fluorescence results from use of an emission-shifted dye, which may be Indo-1, for observation of intracellular calcium [Ca 2+  ] i  in a biological specimen. In an emission-shifted dye for dual-wavelength emission measurements, and for the case of Indo-1 in particular, a single narrow band of excitation light (centered at 350-nm) will develop fluorescent emission at two spaced narrow bands (centered at 425-nm and at 490-nm, respectively). Functional components in FIG. 4 therefore utilize these wavelength numbers for Indo-1 to designate functional relation to one or more of these wavelengths in a typical use of a ratiometric emission-shifted dye for dual-wavelength emission measurements, which in the case of FIG. 4 (as in FIG. 1) result in real-time development and display and/or storage of &#34;control&#34; and &#34;experimental&#34; ratio signals. 
     In the emission-shifted situation of FIG. 4, source 25 is required, in connection with a single filter F(350), to deliver only a single narrow band of excitation light to a single bifurcated optical-fiber bundle 45, with provision for equally shared delivery via branches 46, 47 to &#34;control&#34; and to &#34;experimental&#34; specimens at cuvettes 10, 11, within chambers 12, 13 as described in connection with FIG. 1. This may be done by direct and continuous delivery of light from source 25, via filter F(350), to the inlet end of the bifurcated fiber bundle 45; but in the form shown, preference is indicated that this delivery be made via chopper 26, with which other switching means as at 26&#39;, are synchronized, for signal-to-noise enhancement purposes; in FIG. 4, the legends &#34;sync.&#34; applied at 26 and 26&#39; will be understood to symbolize such synchronism. 
     In view of the dual-wavelength emission nature of Indo-1 dye used in specimens at 10, 11 in FIG. 4, the exiting fluorescent beams 48, 49 which issue from chambers 12 and 13 are each characterized by the two narrow bands centered at 425-nm and at 490-nm, respectively. Beams 48, 49 are shown folded at mirrors 43, 44 and split at 50, 50&#39; into two spaced axes for narrow-band filtering at F(425) and at F(490), prior to separate measurement at 51 (PMT 425) and 52 (PMT 490) of &#34;control&#34; specimen fluorescence, and prior to separate measurement at 51&#39; (PMT 425) and 52&#39; (PMT 490) of &#34;experimental&#34; specimen fluorescence. Gate-26&#39; controlled signal processing at 53, 54 of the respective PMT 425 and PMT 490 detected &#34;Control&#34; outputs at 51, 52 sets the stage for development at 55 of a ratio signal reflecting the instantaneous relative magnitude of &#34;control&#34; specimen fluorescence at the dual wavelengths attributable to use of Indo-1, and the ratio signal for the &#34;control&#34; specimen is supplied directly and continuously to display/storage means 56. Concurrently, gate-26&#39;  controlled signal processing at 53&#39;, 54&#39; of the respective PMT 425 and PMT 490 detected &#34;experimental&#34; outputs at 51&#39;, 52&#39; sets the stage for development at 55&#39; of a ratio signal reflecting the instantaneous relative magnitude of &#34;experimental&#34; specimen fluorescence at the dual wavelengths attributable to use of Indo-1, and the ratio signal for the &#34;experimental&#34; specimen is supplied directly and continuously to display/storage means 56, for display with the &#34;control&#34; ratio signal and to the same time scale. The plotted display at 56 will be like that of FIGS. 2A and 2B, all other conditions being analogous, except for the fact of using an emission-shifted dye in FIG. 4, in place of an excitation-shifted dye in FIG. 1. 
     The system of FIG. 5 will be recognized for its correspondence to that of FIG. 1, except for use in FIG. 5 of a single chamber 60 to contain two identical cuvette specimens at 61, 62, for concurrent exposure to time-interlaced delivery of 340-nm excitation light in alternation with 380-nm excitation light, all as described for FIG. 1. For this reason, the same reference numbers are adopted in FIG. 5 as in FIG. 1 and no further separate discussion is needed, beyond pointing out that in chamber 60 of FIG. 5 there are two spaced inlet ports and two spaced outlet ports, with a light barrier 63 between cuvette specimens, for assurance against internally scattered &#34;cross-talk&#34; between &#34;control&#34; specimen fluorescence and &#34;experimental&#34; specimen fluorescence. Finally, the rectangular outline 64 around both cuvette specimens will be understood to indicate provision for thermostatically controlled uniform thermal environmental conditions for both specimens. 
     The system of FIG. 6 illustrates that the single chamber 60 of FIG. 5, containing plural cuvette specimens at 61, 62, is equally adaptable to use with an emission-shifted ratiometric dye (such as Indo-1) as such a single chamber was described in FIG. 5 for use with an excitation-shifted dye. This being the case, it is suitable in FIG. 6 to borrow from FIG. 4 most of the components described and shown in connection with involved emission-shifted fluorescence. Accordingly, where appropriate, the same reference numbers are used in FIG. 6 for components and functions as in FIG. 4, and further discussion is unnecessary. 
     It will be seen that the presently described techniques and apparatus meet the above-stated objects and provide substantial advantages over past and present techniques; these advantages include but are not limited to the following: 
     1. Problems associated with sequential measurements on a given specimen are eliminated by the simultaneous nature and real-time analysis provided by the invention. 
     2. The invention significantly reduces the time required for analysis of the effect of a given agent on a specific specimen, thus vastly improving efficiency of the measurement process. 
     3. For many applications, e.g., not involving magnetic-field experiments, the invention can be fully served by plural cuvette samples in a single chamber; and the use of separate chambers, i.e., one cuvette sample in each chamber, enables the efficient real-time analysis and measurement of the effect of a magnetic field on a given sample, in real-time comparison to a &#34;control&#34; sample which is not exposed to a magnetic field.