Patent Application: US-58469109-A

Abstract:
described are methods for determining a concentration of a substance in a compartment comprising exciting an endogenous compound , or a functional part , derivative , analogue or precursor thereof , measuring the lifetime of the luminescence and / or transient absorption exhibited by the compound , functional part , derivative , analogue and / or precursor , and correlating the lifetime with the concentration of the substance .

Description:
the spectra of prompt and delayed luminescence were recorded using a ls50b luminescence spectrometer ( perkin - elmer , wellesley , mass ., usa ). prompt fluorescence was measured using the fluorescence mode with excitation source correction . delayed luminescence was recorded in the phosphorescence mode , using varying delay times with respect to the excitation flash and a gate width of 100 μs . the measurements were made at room temperature . excitation and emission wavelengths and slit widths will be specified in the results section . spectra were recorded with either air - saturated samples or samples containing zero oxygen . adding a sufficient amount of ascorbic acid ( 20 μl of 200 mm solution ) to the already ascorbate oxidase - containing samples ( 1 unit ascorbate oxidase per ml , 3 ml total sample volume ) created the zero - oxygen conditions . this method of reducing oxygen levels is explained in more detail below . the amounts of ascorbate oxidase and ascorbic acid used did not interfere with the readings of the spectra . the experiments concerning comparison of triplet - triplet absorption kinetics with delayed fluorescence lifetimes , and the measurement of transient absorption spectra , were performed using an lfdl - 3 / remote flash lamp pumped dye laser ( candela laser corporation , wayland , mass .). this system provided pulses with a duration of approximately 1 μs at 505 nm at a repetition frequency of 10 hz . the output of the laser was directly focused on the sample , consisting of a quartz cuvette containing the ppix solution . the used detector was a r928 ( hamamatsu , hamamatsu city , japan ) photomultiplier tube ( pmt ) with a c1392 - 09 ( hamamatsu , hamamatsu city , japan ) gated socket . the detector was coupled to a monochromator ( oriel 77320 ) in order to select the emission wavelength of interest . the output of the pmt was fed into an oscilloscope ( tektronix 2440 , tektronix inc ., beaverton oreg ., usa ) and transferred to a computer by the serial bus . the wavelength - dependent transient absorption was measured using a white light source and scanning of the monochromator . these experiments were carried out at room temperature ( 20 ° c .). calibration experiments with varying oxygen concentrations were performed with a different set - up . an xecl excimer laser ( lambda physik lpx 110i , göttingen , germany ), operated at 10 hz and producing 50 mj pulses , was used to pump a dye laser ( lambda physik , lpd 3002 ) operating at 405 nm . the output of the dye laser was focused on a quartz optical fiber with a core of 0 . 6 mm ( ensign bickford optics , avon , conn .) using a 3 cm f / 1 . 2 quartz lens . the fiber was coupled to the reaction vessel ( described below ) used for the calibration experiments . the detector was the same r928pmt with c1392 - 09 socked , switched off during 5 μs gate width . the detector was coupled to the reaction vessel by a vis - type liquid light guide with a 5 mm optical core ( oriel , stratford , usa ). instead of the monochromator , three 630 nm long pass glass filters were used for filtering of the emission light . the laser pulse was fired 1 μs after off gating of the pmt ; the repetition rate was 10 hz . per measurement , 64 traces were averaged on a digital oscilloscope ( tektronix tds - 350 , tektronix inc ., beaverton oreg ., usa ). data were transferred to a computer by serial bus and lifetime analysis was performed using labview 5 . 1 graphical programming software ( national instruments , austin , tex ., usa ). mono - exponential fitting was performed using a marquard - levenberg non - linear fit . to perform delayed fluorescence lifetime measurements at varying oxygen concentrations , the oxygen concentration in the ppix solution was varied using the ascorbate oxidase / ascorbic acid enzymatic reaction . calibration experiments , needing precisely controlled oxygen concentrations , were performed using a specially made reaction vessel . the vessel had to be airtight , allow continuous mixing of the ppix solution , temperature control , continuous temperature monitoring and physical access to the content . the latter was necessary to allow injection of aliquots of ascorbic acid solution but should not go at the expense of an interfering oxygen back - diffusion into the sample . it consisted of two glass parts , a bottom part and a top part . both parts were interconnected by screw lock . an airtight connection was assured by a teflon ® ring surrounding the connection site . the bottom of the reaction vessel was flat to allow continuous stirring of the content by a magnetic stirrer . the top part contained three capillary entries : one allowing insertion of a small thermocouple , one for the insertion of the light guide from the excitation source and the latter for injection of ascorbic acid . the capillaries had a length of 2 cm and a lumen of 1 mm diameter . the diffusion barrier was large enough to prevent measurable oxygen back - diffusion within an hour , an adequate time span for calibration experiments . this was checked by oxygen - dependent quenching of phosphorescence of pd - meso - tetra ( 4 )- carboxyphenyl porphine starting at varying oxygen concentrations below 40 μm . the reaction vessel was mounted in a temperature - controlled water jacked on top of a magnetic stirring device . the total content of the reaction vessel , after insertion of the magnetic stirrer , was 30 . 7 ml . injection of 10 μl of a 200 mm solution of ascorbic acid resulted , therefore , in 32 . 5 μm oxygen steps ( po 2 steps of approximately 20 mmhg ). prior to the experiments , the reaction vessel was filled with pre - heated , room - air equilibrated ppix solution . special care was taken to remove all air bubbles from the solution . calibration experiments were performed at 22 ° c . and 37 ° c . pd - meso - tetra ( 4 )- carboxyphenyl porphine was purchased from porphyrin products ( porphyrin products inc ., logan , utah , usa ). protoporphyrin ix disodium salt ( ppix ) was purchased from sigma ( sigma chemical co ., st . louis , mo ., usa ). two regimens of creating ppix solutions were used . in the first regimen , 8 mg / ml ppix was dissolved in distilled water brought at a ph of 8 . 0 by titration with 1 m tris base . from this solution , 0 . 5 ml was added to 50 ml of a human albumin solution ( 40 gr / l ) in phosphate - buffered saline ( pbs ). this mixture was brought to a ph of 7 . 4 by titration with hcl . the ppix is dissolved in an albumin solution to obtain a complex , mimicking the environmental circumstances in cells and tissue ( takemura et al ., 1991 ). the experiments concerning triplet - triplet absorption were performed with ppix solution prepared following this protocol . since dissolving ppix according to the protocol above takes rather long ( ppix is usually not completely dissolved after several hours ), during the course of the study we looked for a more efficient way of preparing the ppix solutions . in the second regimen , 4 . 0 grams of bovine serum albumin ( bsa , sigma chemical co ., st . louis mo ., usa ) was dissolved in 200 ml pbs . to increase the buffer capacity needed to prevent ph changes when adding aliquots of ascorbic acid to the solution , 800 mg hepes was added . ppix was dissolved in methanol ( 6 . 07 mg ppix in 10 ml methanol ) and 2 ml of this ppix solution was immediately added to the albumin solution , resulting in a final concentration of approximately 10 μm ppix . ppix solutions according to the second regimen were used for the recording of the shown spectra and calibration experiments , unless stated otherwise . metallo - porphyrins used for oxygen concentration measurements in vivo can usually be effectively excited at several different wavelengths . for example pd - porphyrin , the most widely used phosphorescent dye for in vivo measurements , can be effectively excited around 400 nm ( the soret maximum ) and 530 nm ( the q - band ). generally , light with a longer wavelength penetrates deeper into tissue , the reason why usually excitation at 530 nm is favored for in vivo measurements , although the excitation efficiency at 400 nm is much higher . fig5 shows the fluorescence emission versus the excitation wavelength of ppix bound to albumin . two peak emissions , one around 400 nm and one around 510 nm , are prominently present . the excitation wavelengths of the used lasers are indicated in the figure for convenience . as will become apparent , both wavelengths are effective for delayed fluorescence measurements . in order to locate an appropriate wavelength for triplet - triplet absorption measurements , the transient transmission spectrum was recorded . fig6 shows the transient transmission spectrum of a 20 μm ppix solution as a function of the transmission wavelength . the maximum at 400 nm is caused by depletion of the ground state by the laser pulse , the minimum at 450 nm is due to population of the t 1 level and absorption to the t 2 level . these results are in good agreement with previous studies ( chantrell et al ., 1977 ; bonnett et al ., 1983 ; sinclair et al ., 1980 ). to identify the type of delayed luminescence that was observed after pulsed excitation of ppix solutions , prompt and delayed luminescence spectra were recorded . fig7 shows the prompt fluorescence spectrum , with its characteristic peak at 636 nm . delayed luminescence spectra , recorded using varying delays after the excitation flash , are shown in fig8 . fig8 , panel a , shows the delayed luminescence in an air - saturated sample . delayed luminescence is hardly detectable 30 μs after the excitation and is totally vanished after a delay of 100 μs . in contrast , fig8 , panel b , shows that under zero oxygen conditions , delayed luminescence can be detected even after a 1 ms delay . from fig8 , panel b , it is also evident that the spectrum of the delayed luminescence is qualitatively the same as the prompt fluorescence spectrum shown in fig7 . the red shift , characteristic for phosphorescence , is especially absent . we , therefore , identify the delayed luminescence as delayed fluorescence . to be useful for quantitative oxygen measurements , the delayed fluorescence lifetime should be an appropriate representative of the t 1 lifetime . to test this , the delayed fluorescence lifetime was compared to the lifetime of transient triplet - triplet absorption in a deoxygenated sample . fig9 shows the decay of the triplet state measured with both delayed fluorescence and triplet - triplet absorption . panel a displays the decay curve measured by delayed fluorescence at 636 nm after pulsed excitation at 505 nm . the fast decaying first part of the curve is an artifact introduced by the excitation source . panel b contains the corresponding decay trace as measured by triplet - triplet absorption at 470 nm . from fig8 , it is already noticeable that the lifetimes of the delayed fluorescence are highly dependable upon the oxygen concentration in the solution . a quantitative relationship between the lifetime and the oxygen concentration is mandatory if delayed fluorescence lifetimes are to be used for oxygen concentration measurements . to test the applicability of the stern - volmer relationship , we measured delayed fluorescence lifetimes at varying oxygen concentrations . these experiments were performed using the described reaction vessel . starting at a high oxygen concentration ( the sample was equilibrated with room air ), the oxygen concentration was lowered in steps of 32 . 5 μm as described in the materials and methods section . the stern - volmer relationship predicts a linear relationship between the reciprocal lifetime ( 1 / τ ) and the oxygen concentration . fig1 shows the measured values of the reciprocal lifetime versus the oxygen concentration at 22 ° c . and 37 ° c . by performing a linear fit procedure on these data , the quenching constant k q was determined . the best - fit results are also shown in fig1 . at 22 ° c ., k q was found to be 243 ± 5 m − 1 μs − 1 and at 37 ° c ., this value was 471 ± 7 m − 1 μs − 1 . measured values for the decay time at zero oxygen conditions , τ 0 , were 1 . 4 ± 0 . 1 ms and 1 . 0 ± 0 . 1 ms for 22 ° c . and 37 ° c ., respectively . from fig1 , it is clear that a good linearity between the reciprocal values of the lifetimes and the oxygen concentration exists , as is confirmed by correlation coefficients of 0 . 9882 and 0 . 9924 for 22 ° c . and 37 ° c ., respectively . moreover , no significant departure from linearity could be detected by a runs test , providing p - values of 0 . 07 and 0 . 79 for 22 ° c . and 37 ° c ., respectively . these results show that , at least over the tested oxygen concentration range , the stern - volmer is accurate in quantifying the relationship between the delayed fluorescence lifetimes and oxygen concentrations . as a check , a calibration experiment with a ppix solution prepared according to the first regimen was run . the result was comparable to the calibrations performed with solutions according to the second regimen ( data not shown ), indicating that the reported phenomena are independent of the followed preparation procedure . the main findings of this study can be summarized as follows : 1 ) ppix shows delayed luminescence besides the already known prompt fluorescence . 2 ) the emission spectrum of the delayed luminescence overlaps the spectrum of the prompt fluorescence and a red shift is absent , therefore , the delayed luminescence is classified as delayed fluorescence . 3 ) the lifetime of this delayed fluorescence is a representative of the lifetime of the first triplet state . 4 ) oxygen is a known quencher of the triplet state of ppix and this study shows that the delayed fluorescence lifetime is also oxygen dependent . 5 ) moreover , it is shown that the stern - volmer relationship quantitatively describes the dependence of the delayed fluorescence lifetime on the oxygen concentration . these findings show that oxygen - dependent quenching of delayed fluorescence provides an exciting new method to measure oxygen concentrations , since it allows non - invasive tissue - and intracellular oxygen concentration measurements by an endogenous compound , such as , a porphyrin . in this example , we demonstrate the feasibility of the proposed method for measuring intramitochondrial oxygen levels in living cells . in this example , a method of the invention is , for instance , performed in the time domain using pulsed excitation from an experimental high - power tunable laser . the laser of this example consists of a doubled flash - lamp pumped nd - yag laser pumping an optical parametric oscillator ( opo ). this results in a tunable laser providing 10 mj pulses of 6 ns duration . the laser is coupled to a quart cuvette containing the studied samples using a glass fiber . perpendicular to the laser beam is a detector consisting of coupling lens , monochromator and photomultiplier tube ( pmt ). the photomultiplier ( hamamatsu r928 ) is working in photon - counting mode and is gated during laser excitation by reversing the polarities of the second and third dynode . the current from the pmt is voltage converted using a fast - switching integrator ( integration time 3 . 5 μs and reset time 0 . 5 μs ). the voltage is digitized at a sample rate of 250 khz using a data - acquisition board in a pc . the signal of 64 pulses is averaged before applying a mono - exponential fit procedure to the measured decay curves . the lifetime typically varies from 20 ms at high oxygen levels to 700 ms at zero - oxygen conditions . first , the intracellular distribution of protoporphyrin ix as a function of time after the administration of 5 - aminolevulinic acid ( ala ) was investigated . therefore , neuroblastoma cells were incubated with ala during varying periods of time . cells were observed using a leica fluorescence microscope with appropriate filterset . fig1 shows the distribution of the ppix fluorescence at three different time points ( two , four , and eight hours for panels a , b and c , respectively ). at least until four hours , the ppix fluorescence shows a spotty appearance corresponding to a mitochondrial pattern . at eight hours , a more diffuse fluorescence is observed located in the cytosol . to demonstrate the ability to measure intramitochondrial oxygen levels , calibration experiments were performed in suspensions of neuroblastoma cells ( 4 × 10 6 cells / ml ) after four hours of incubation with ala . extracellular oxygen levels were controlled using a rotational cell oxygenator and gas flow controllers . intramitochondrial oxygen measurements were performed before and after administration of rotenone . rotenone is a blocker of complex 1 of the mitochondrial respiratory chain and , therefore , inhibits mitochondrial oxygen consumption . if the measurement is indeed mitochondrial of nature , adding rotenone will cause a decrease of intracellular oxygen gradients until ultimately the intramitochondrial oxygen level is the same as the extracellular oxygen level . for the measurement , this implies that adding rotenone will cause an increase in the measured intramitochondrial oxygen concentration . fig1 shows the results of such a measurement . it is clear that adding rotenone causes the predicted effect , thus , the ppix signal is mitochondrial in nature . moreover , the signal can be calibrated , making quantitative measurements possible . from this example , it is concluded that after administration of ala , a time window exists in which ppix accumulates inside the mitochondria . moreover , it is concluded that quantitative intramitochondrial oxygen measurements are possible in living cells . an example of an experimental two - photon set - up is given in fig1 . in this example , excitation is achieved using a q - switched laser operating at 1064 nm ( laser 1 - 2 - 3 , schartz electro - optics inc ., orlando , fla ., usa ). the laser provides pulses of approximately 10 ns duration and an energy ranging from 10 mj per pulse for in vitro experiments to 100 mj per pulse in in vivo experiments . the bundle diameter of the laser beam is slightly expanded to a final diameter of 5 mm by a beam expander , before being directed to the focusing lens by an optical mirror with an enhanced silver reflection surface ( opto sigma , santa anna , calif ., usa ). the focusing lens is a single plan - convex lens with a focal length of 2 . 0 cm . based on gaussian beam optics , the bundle diameter of 5 mm combined with a lens with a focal length of 2 . 0 cm results in a focal spot size of 8 μm and a focus length of 94 μm ( in air ). assuming a refractive index in tissue of 1 . 4 , the measurement volume is approximately a cylinder with diameter of 10 μm and a length of 130 μm . the focusing lens is connected to a micrometer - screw for manual adjustment of the focal plane , thereby allowing longitudinal measurements to be made . for in vivo application , the reading of the micrometer screw is multiplied by the refractive index of tissue , assumed to be 1 . 4 . emission light is collected by the same lens and directed towards the photo detector by two mirrors . selection of the phosphorescence light is achieved by two 700 ± 20 nm bandpass filters ( oriel , stratford , conn ., usa ), positioned in series before the cathode of the photomultiplier tube ( pmt , type r928 , hamamatsu , hamamatsu city , japan ). the output of the pmt is voltage - converted by a current - to - voltage converter with subsequent wide - band amplifier ( 30 mhz ) and fed into a digital oscilloscope ( tektronix 2440 , tektronix inc ., beaverton , oreg ., usa ). to increase signal - to - noise ratio , luminescent traces are averaged on the oscilloscope . for instance , an average of 32 traces is used . the resulting averaged traces are transferred to a computer by serial bus for data - collection and analysis using software , for instance , written in labview ( national instruments , austin , tex ., usa ). luminescence lifetimes can be measured both in the time domain as well as in the frequency domain . in the time domain , the real decay curve is measured after photo excitation with a short pulse of light . in the frequency domain , the ( continuous ) excitation light is modulated with a known frequency and the lifetime can be determined from the phase shift between excitation and emission light . both methods have their specific advantages and disadvantages : a monochromator is preferably used instead of filters in order to avoid possible disturbance of the delayed fluorescence signal as a result of fluorescence and / or phosphorescence of the filters themselves . unfortunately , monochromators have low transmission efficiency and a gain in performance is achieved by using a different optical system . a cost - effective solution is a use of bandpass filters combined with an optical system that at least partly prevents a high amount of excitation light to reach the filters . an example of this embodiment is shown in fig1 . considering the low signal levels , pmts are a good . choice . due to the high energetic laser pulse and the resulting high amount of prompt fluorescence , the detector and electronics are preferably protected against damage . in one embodiment , gating of the pmt is performed by switching the voltages of the second and third dynodes during the laser pulse . this causes distortion of the first 20 to 30 μs of the signal , diminishing adequate measurement of short lifetimes . a dedicated microchannel plate pmt is , therefore , a preferred option . an alternative is using a fast shutter in front of a standard pmt , e . g ., a pockels cell . an even cheaper alternative is the use of semiconductor devices like avalanche - 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