Patent Application
20170059488
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The, Applicant has no objection to the facsimile reroduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. Further, no references to third party patents or articles made herein is to be construed as an admission that the present invention is not entitled to antedate such material by virtue of prior invention.
The present invention is related to the field of sample identification, in particular to a method for identifying or discriminating biological species from non-biological species, whether they are individual particles or components of a composition. The invention lies in the field of pump-probe fluorescence spectroscopy for time-resolved imaging. It uses the differences in UV induced fluorescence of biological samples vs. non-biological samples when they are exposed to radiation, in particular the fact that the UV fluorescence of bio particles changes, in particular is depleted, by the addition of visible radiation, whereas the UV fluorescence of non-bio particles does not change, in particular is not depleted, under the same condition.
Similar effects were reported using quantum coherence effects in pump-probe femtosecond experiments as described e.g. in “Pump-Probe Depletion Spectroscopy Discriminates Organic and Biological Molecules” by F. Courvoisier, J.-P. Wolf et al. in App. Phys. Lett. 87(6) 063901 (2005) and some subsequent publications by the same group. These experiments use a femtosecond 266 nm pump laser and a femtosecond 800 nm probe pulse.
This coherent control experiment requires rather complex and expensive equipment and thus limits its use to laboratories or to experimental setups. A main disadvantage of prior art solutions and applications is the necessity to use lasers with extremely short pulse durations, usually in the femtosecond range. Such lasers are sensitive, usually rather expensive, unrugged instruments requiring precise adjustment and cautious handling. Also, they often produce non-linear perturbations and unwanted dispersion in attached fibers and probe media to be investigated, thus complicating measurements and leading to unexact results.
It would thus be advantageous if one could use compact, cost effective and rugged lasers producing longer pulses instead of such sensitive femtosecond devices.
On the other hand it was found that biological organic material like amino-acids or material containing aromatic amino-acids, like peptides, proteins, bacteria, viruses, pollens, spores, as well as other biological species like flavins or NAD/NADH (nicotinamide adenine dinucleotide, a coenzyme), exhibits fluorescence depletion in pump-probe arrangements longer than one picosecond, while non-biological organic material like aromatic (AH) or polyaromatic hydrocarbons (PAR) or containing such hydrocarbons, carbonaceous aerosols, or soot, do not exhibit such fluorescence depletion.
This finding allows to discriminate biological material or particles from non-biological particles by using the pump-probe method/arrangement with a laser producing longer pulses, here meaning pulses longer than one picosecond, preferably in the nanosecond range. Even the use of continuous wave (CW) lasers seems reasonable.
The optical identication of bioaerosols in the atmosphere and their discrimination against combustion related particles is a major issue for real-time, field-compatible instruments. The present invention aims at embedding advanced pump-probe depletion spectroscopy accordingly in such portable equipment.
To summarize, contrary to the known experiments and methods, the present invention makes use of two long, here meaning beyond the vibronic coherence time, overlapping nanosecond laser pulses, one in the UV and one in the visible, to distinguish between biological and non-biological material or particles. The advantages are obvious: simpler, portable equipment, less error sources, more economical, mostly even faster. Even continuous wave (CW) or quasi-CW lasers may be used. The possible use of standard, long pulse, typically nanosecond or more, or even CW lasers opens a wide field of applications.
Advanced discrimination methods are needed since the UV fluorescence spectra of bio- and non-bio hydrocarbons strongly overlap. The difference in radiative lifetimes between some PAE and aromatic amino acids can be used to a certain extent, but not with all PAHs. Furthermore, precise lifetime measurements of individual particles containing a mixture of PAH require sensitive and expensive analyzers as described in “Stroboscopic technique for measurement of fluorescence lifetimes of bacteria and biological interferents” by M. Wlodarwski et al. in Proc. SPIE 6398, 69380L (2006) or in “On-the-Fly Fluorescence Lifetime Determination with Total Emission Detection in HPLC” by M. A. Dvorak et al. in Anal. Chem. 69, 3458-3464 (1997).
As compared to chemical or mass spectrometric methods, the present invention offers the advantages of real-time analysis, it is maintenance-free, needs no consumables, and requires no sampling. In particular, large volumes (of air, clean room atmospheres, or water, etc.) can be analyzed instead of checking randomized probes that should be representative of ensembles.
The invention discriminates the fluorescence signals of bio and non-bio particles using a differential approach, namely the comparison of the total fluorescence recorded with and without the additional visible radiation. This allows to assess individual particles, like in flow cytometry, microscopy, imaging, or single aerosol particle analysis, as well as for ensembles of different particles. In this latter case, the outcome of the measurement provides an estimate of the ratio between aromatic amino acids and non-bio hydrocarbons.
The underlying mechanism for the observed fluorescence depletion in the case of amino acids and the absence of fluorescence depletion in the case of other organic compounds relies the difference in the excited state absorption (ESA) characteristics. From the intermediate excited state S1 (populated by the UV laser), the visible photon re-excite the molecules in some upper lying states Sn, which are likely to auto-ionize or dissociate, so that fluorescence is depleted. This is the case of AA, but not for AH and PAH, in which either
It should be understood however that the present invention is not limited to the above applications, but may be used in any field where a variation of fluorescence, whether depletion or increase, can be observed as discriminating feature of particles or materials.
In the appended drawings show:
The sequence of laser pulses after mirror 10 then has the following time structure:
In this particular case, each laser pulse has a duration of about 5 ns. This sequence is schematically indicated on
Through focusing lens 11, this laser pulse sequence is focused onto the sample in measuring chamber 12 and produces the desired fluorescence. The sample consists of individual aerosol particles described below.
The produced fluorescence of the sample 12 is collected by a pair of lenses 13, a spectral filter set 14 centered at 340 nm (20 nm FWHM) and rejecting 263 nm and 527 nm, photomultiplier tube 15 and a fast, typical 300-500 MHz, digital oscilloscope interfaced to or integrated into a computer 16.
As mentioned above, this embodiment serves to discriminate bioaerosols from non-bioaerosols. The sample is constituted by an aerodynamically focused flow of aerosol particles, as described, e.g. in L. Bonacina, J.-P. Wolf et al, European Patent Application EP 12167800,7 “Measurement Device and Method for Detection of Airborne Particles”, equivalent to U.S. Patent Application 2013/0301047A1 (2013).
The presence of aerosol particles is detected by Mie scattering from additional laser diodes (not shown), and the scatter signals trigger the laser 1. Notice that the sample can also be constituted by a liquid flow cytometer, a liquid (e.g. water) containing biological material and aromatic hydrocarbons, or contaminated surfaces, contaminated food etc.
The discrimination is obtained by comparing the fluorescence signals measured for the combined UV+visible excitation and for the reference UV only excitation, both recorded within the same transient. More precisely, the depletion ratio D (0<D<1) is defined as:
Where FUV+VIS is the depleted fluorescence intensity and FUV is the undepleted fluorescence. FUV+VIS clearly depends on the intensity of the visible laser IVIS and has to be calibrated beforehand. In the cases of water droplets containing tryptophan and other bioaerosols like bacteria, pollen gains etc., at the used intensities (iUV=107W/cm2 and IVIS=108W/cm2), DBio=0.3 was measured. In the case of AH and PAH (naphtalene, liquid diesel mixture, soot particles), DNonBio=0 was measured.
This large difference clearly evidences the power of the method for discriminating between bioaerosol particles and non-bioaerosol particles. Notice also that this method is insensitive to the pulse-to-pulse fluctuations of the UV laser. Moreover, FUV/IUV provides information about the tryptophan content in the sample.
More generally, when e is a predefined threshold value between 0 and 1, it can be determined that, if D<e, the investigated species or sample is non-biological, or, if D>e, that the investigated species is biological or that the sample contains biological particles.
On the basis of
Several types of aerosols were analyzed with this instrument to assess the discriminability offered by the fluorescence spectrum/lifetime/pump-probe depletion approach. An common aerosol generator (Model 3076 from TSI Inc) and a nebulizer were used to inject different aerosols, like Enterococcus bacteria, diesel droplets, tryptophan particles and humic particles, while the exhausts of two different Diesel cars were used for analyzing non-bio particles emitted by combustion in actual conditions.
In
The approach shown in
The same approach as in
The approach shown in
A different example of an application of the invention is the remote identification of pathogen aerosols released in the atmosphere (e.g. terrorist attacks) using a depleted-fluorescence LiDAR. (light detection and ranging) instrument.
Similar to the first embodiment shown in
An electro-optic (or acousto-optic) modulator 29 is synchronized with the laser 21 in such a way that it blocks the visible green laser pulse every other pulse. In other words, it blocks the 532 nm visible radiation at half the repetition rate f of the laser 21.
The UV at 266 nm, after being reflected by mirror 27, is then recombined with the chopped visible radiation of 532 nm by a second dichroic mirror 28. The thus produced combined laser pulse sequence contains UV (266 nm) and contains alternating in one case also the 532 nm visible (called “ON”) and in the other case no 532 nm visible (called “OFF”). The little diagram in the upper right corner of
The laser pulses sequence after mirror 28 then has the following time structure for a laser f=1 kHz repetition rate:
This laser pulse sequence is focused through a collimating/focusing telescope or other transmitter optics 30 onto the sample 31 and produces the desired fluorescence. Sample 31 here is a “suspect” aerosol cloud, potentially of a mixture of biological and non-biological materials as mentioned above, standing in the atmosphere at a distance R from the LiDAR.
The backwards emitted fluorescent light is collected by a Newtonian telescope 32, focused on an iris for adapting the field of view, expanded and collected, respectively, by lenses 33 to allow splitting into two channels by a dichroic mirror 34 which reflects the UV of 266 nm and transmits the fluorescence at 350 nm. The UV channel, consisting of a filter 36 centered at 266 nm, another lens 33, and a first PMT 39 records the backscattered UV signal EUV (in number of photons) as a function of time (i.e, distance R=ct with c the light velocity). The fluorescence channel, consisting of a filter 35 centered at 340 nm, another lens, 33, and a second PMT 37, records the fluorescence E(R) in number of photons as a function of distance R also for the ON and OFF laser pulses separately. Both signals from PMT 37 and PMT 39 are recorded as a function of time in the data acquisition system 38. From the inversion of the three signals EON(R), EOFFR), and EUV(R), one derives an assessment of the probability that the suspect cloud is mainly formed by biological substances or “harmless” AH. This could be applied, e.g., when checking a typical bioterrorisin threat consisting of sprays of pathogen bacteria, like anthrax, yersina pestis, or liquid drops containing viruses.
Here, a flow of 1 m/s of liquid in a tube and a sapphire nozzle 47 provided as a liquid jet 48 is subjected to a measurement spot with a size of 50 μm. A typical bacterium is about 1 μm in diameter, so very small in comparison.
From measurements conducted, it was found that laser powers of 100 kW/cm2 for the pump section and 1 MW/cm2 forhe probe section leads to a detectable depletion of only about 3%. These are, however, single shot measurements, i,e, a single fluorescence depletion cycle/particle.
In the present example with a CW laser, each particle would experience an illumination time of 50 μm/1 m/s=50 μs. Assuming 50 ns for the fluorescence cycle, which is a conservative value, this leads to an improvement in signal-to-noise ratio (SNR) of roughly the square root of 1000, i.e. a factor of about 30 in SNR. The intensities could therefore be reduced by about 30, resulting in approximately 3 sW/cm2 for the pump section and 30 kW/cm2 for the probe section. On a spot size of 50 μm, this corresponds to reasonable CW laser powers of 60 mW and 600 mW, respectively. To be on the safe side, standard CW lasers providing 1 W of UV and 10 W of visible (green) appear to be a suitable choice.
The 266 nm UV beam is chopped by an electro-optic modulator 43 which is synchronized with the laser 40, blocking the 532 nm UV laser pulse every half of the time within the measuring cycle of 50 μs. In other words, it provides a square modulated signal with a 50%/50% duty cycle. Lock-in detection might be implemented to improve the signal-to-noise ratio (SNR).
The chopped 532 nm laser beam is recombined with the 266 nm radiation by a second dichroic mirror 45. The recombined beam is focused by lenses 46 onto the 50 μm measurement spot of the liquid jet 48.
Fluorescence reading is done through lenses 49 with an intermediate spectral filter 50 transmitting the fluorescence at 340 nm to a photomultiplier (PMT) detector 51, connected to the acquisition computer 52.
The embodiment shown in
The above detailed description of the function and of various embodiments of the present invention permit a person skilled in the art to devise further implementations without departing from spirit and scope of the present invention.
This application claims the benefit of U.S. Provisional Application 62/209,454, filed 25 Aug. 2015, the contents of which are incorporated herein by reference thereto.
| Number | Date | Country | |
|---|---|---|---|
| 62209454 | Aug 2015 | US |
