Abstract
A method for detecting the presence of or quantification of carbon black and/or black carbon in a sample or carrier medium. The method includes providing the sample or carrier medium without labelling or pre-treatment of the carbon black and/or black carbon particles; illuminating the sample or carrier medium at a temperature below 90° C. by a pulsed light with a pulse duration below 500 femtoseconds, a repetition rate above 1 MHz with an average power below 20 mW, and a wavelength of a femtosecond laser pulse ranging from 700 to 1200 nm, to generate non-incandescence related light emission from the carbon black and/or black carbon particles; and analysis of the light emission.
Claims
1. A method for quantification of carbon black and/or black carbon in a sample or carrier medium, the method comprising: providing the sample or carrier medium without labelling of the carbon black and/or black carbon particles and without pre-treatment of the carbon black and/or black carbon particles; illuminating the sample or carrier medium at a temperature below 90° C. by a pulsed light with a pulse duration below 500 femtoseconds, a repetition rate above 1 MHz with an average power below 20 mW, and a wavelength of a femtosecond laser pulse ranging from 700 to 1200 nm, to generate non-incandescence related light emission from the carbon black and/or black carbon particles; and analysis of the light emission to quantify the carbon black and/or black carbon in the sample or carrier medium.
2. The method according to claim 1, wherein the illumination is performed with a femtosecond laser focused on the sample using a stationary or scanning beam delivery.
3. The method according to claim 1, wherein the average power of the pulsed light is between 4 and 5 mW.
4. The method according to claim 1, wherein the sample or carrier medium comprises a material or compound with autofluorescence properties.
5. The method according to claim 1, wherein the sample or carrier medium comprises a compound selected from the group consisting of mitochondria, lysosomes, flavins, extracellular matrix, collagen, elastin, NAD(P)H, chlorophyll, retinol, cholecalciferol, folic acid, pyridoxine, tyrosine, dityrosine, excimer-like aggregate, glycation adduct, indolamine, lipofuscin, polyphenol, tryptophan, and melanin, or a combination thereof.
6. The method according to claim 1, wherein the intensity and/or wavelength of the pulsed light is varied until emission from the carbon black and/or black carbon particles is distinguishable from emission noise from the sample.
7. The method according to claim 1, wherein the pulsed light intensity and/or wavelength is modulated to reach near infrared emission.
8. The method according to claim 7, wherein shorter emission wavelengths than the near infrared emission are filtered out by a wave length filter to distinguish carbon black and/or black carbon particles emission from background noise emission.
9. The method according to claim 1, wherein the sample or carrier medium is illuminated at a temperature between 1° C. and 80° C.
10. The method according to claim 1 wherein the sample or carrier medium is illuminated at a temperature between 10° C. and 50° C.
11. The method according to claim 1, wherein the sample is blood or urine.
12. The method according to claim 1, wherein the sample is a liquid sample, cell sample or tissue sample of biological origin, wherein the cells in the cell sample or tissue sample have an intracellular water content greater than 40%.
13. The method according to claim 1, wherein the method comprises quantification of black carbon particles without labelling of the black carbon particles and without pre-treatment of the black carbon particles.
14. The method according to claim 13, wherein the black carbon particles are aggregated black carbon particles.
15. The method according to claim 1, wherein the sample or carrier medium comprises carbon black or black carbon particles from environmental or industrial pollution.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:
(2) FIG. 1A. Schematic schematic representation of the illumination and emission process of CB particles for the presented detection method. FIG. 1B. TEM image of an ufPL aggregate. Scale bar: 300 nm. FIG. 1C. CCB (600 μg/mL) imaging in ultrapure water, ethanol and glycerol at room temperature upon illumination with 5 or 10 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bars: 15 μm. Emission band: 450-650 nm.
(3) FIG. 2A. Extinction spectra of aqueous CB suspensions. FIG. 2B. Two-dimensional excitation-emission plot of ufPL particles in water under single photon excitation with a false black-white map based on the emission intensity in arbitrary units. The arrow points towards the Raman line of water.
(4) FIG. 3A. Normalized WL emission spectra of aqueous CB particle suspensions using femtosecond 810 nm laser illumination (8 mW, 150 fs, 80 MHz). FIG. 3B. Normalized WL emission spectra of aqueous ufP90 suspensions recorded at different femtosecond illumination wavelengths (from 780 (1) to 900 nm (7), with an interval of 20 nm). FIG. 3C. Temporal response of aqueous carbon suspension measured by femtosecond photoluminescence up-conversion experiments. Also shown is the instrument response function, (dashed line).
(5) FIG. 4. Imaging of cellular compartments of fixed MRC-5 cells stained with commonly utilized fluorophores and in combination with the detection of CCB particles (4 h incubation of 5 μg/cm.sup.2CCB at 37° C. prior to imaging). Emission of the carbonaceous particles can be probed at different wavelengths, here shown (FIG. 4A) 400-410 nm in the non-descanned mode and (FIG. 4B) 650-710 nm in descanned mode (4 mW average laser power at the stage). From left to right: CCB particles, tubulin cytoskeleton (Ex/Em 495/519 nm, ˜3 μW radiant power at the sample), vimentin which is an intermediate filament protein of the cytoskeleton (Ex/Em 555/565 nm, ˜3 μW radiant power at the sample), paxillin expressed at focal adhesions (Ex/Em 650/665 nm, ˜3 μW radiant power at the sample), and overlay image. Scale bars: 25 μm.
(6) FIG. 5. Tubulin cytoskeleton (green, Ex/Em 495/519 nm, ˜3 μW radiant power at the sample) of normal human lung fibroblasts incubated with 5 μg/cm.sup.2 CCB particles (red, 4 mW average laser power at the sample, emission detection: 400-410 nm in non-descanned mode) at 37° C. FIG. 5A. Control cells. FIG. 5B. 4 h incubation. FIG. 5C. 8 h incubation. FIG. 5D. 24 h incubation. Scale bars: 30 μm. Arrow heads: some locations of very small, engulfed CCB particles.
(7) FIG. 6 is a perspective view explaining one embodiment of an apparatus for analysing carbonaceous particles in fluids, cells or tissues of biological and carrier media of non-biological origin according to the present invention.
(8) FIGS. 7A, 7B and 7C are drawings showing an outline of a functional composition of a specimen distribution section of the apparatus for detecting and analysing particles in fluids.
(9) FIG. 8 is a drawing showing a composition of the optical detection section for detecting and analysing carbonaceous particles in air, fluids, cells or tissues of biological and carrier media of non-biological origin.
(10) FIG. 9 is a block diagram showing a whole composition of the apparatus for analyzing particles in fluids, cells or tissues of biological and carrier media of non-biological origin shown in FIG. 6.
(11) FIG. 10 is a drawing showing a composition of the battery and temperature controller section of the apparatus for detecting and analyzing carbonaceous particles.
(12) FIG. 11 is a flowchart (first half) showing urine analysis procedures using the apparatus for analysing particles in urine relating to one embodiment according to the present invention.
(13) FIG. 12 is a flowchart (second half) showing urine analysis procedures using the apparatus for analysing particles in urine relating to one embodiment according to the present invention.
(14) FIG. 13 is an exemplary result of ultrapure water linearly spiked with carbonaceous particles and measured using the presented invented method of measuring carbonaceous particles. There is a linear relation between the added concentration of carbonaceous particles in ultrapure water and the amount of particles detected per mL ultrapure water. The data (N=3) are plotted linearly (R.sup.2=0.99).
(15) FIG. 14 is an example of carbonaceous particle (600 μg/mL) imaging in ultrapure water, ethanol and glycerol (room temperature) upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bars: 15 μm. Emission band: 450-650 nm.
(16) FIG. 15 is an exemplary result obtained by imaging carbonaceous particles in the body fluid urine at room temperature upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bar: 50 μm. Emission band: 450-650 nm.
(17) FIG. 16 is an exemplary result obtained by imaging carbonaceous particles in non-biological carrier medium polydimethylsiloxane at room temperature upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bar: 50 μm. Emission band: 450-650 nm.
(18) FIG. 17 is an exemplary result obtained by imaging cellular compartments of fixed human lung fibroblast cell (MRC-5 cell line) which had engulfed carbonaceous particles when exposed to 5 μg/cm.sup.2 particles at 37° C. prior to imaging. Emission of the carbonaceous particles has been probed at 400-410 nm upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bar: 20 μm.
(19) FIG. 18 is an exemplary result obtained by imaging living human lung fibroblast cell (MRC-5 cell line) exposed to 5 μg/cm.sup.2 particles at 37° C. Emission of the carbonaceous particles has been probed at 400-410 nm upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bar: 20 μm.
(20) FIG. 19 is an exemplary result obtained by imaging carbonaceous particles in the biological tissue placenta at room temperature upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bar: 25 μm. Emission band: 450-650 nm.
(21) FIG. 20 is an exemplary result obtained by imaging carbonaceous particles in the biological plant tissue ivy leafs at room temperature upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bar: 50 μm. Emission band: 450-650 nm.
(22) FIG. 21 is a schematic depiction of the flowchart of an optimized experimental protocol for BC detection in urine.
(23) FIG. 22 is an exemplary result obtained by imaging CB and/or BC particles in urine at room temperature upon illumination with 9.7 mW average laser power at the sample (excitation 810 nm) (using the procedure as depicted in FIG. 25). Scale bar: 20 μm. Emission band: 400-410 nm.
(24) FIG. 23. TEM images of the four different types of carbon black particles. Upper Left (ufPL), Upper Right (ufP90), Lower Left (CCB), and Lower Right (fCB). Scale bar: 200 nm.
(25) FIG. 24. CCB imaging in glycerol and immersion oil using two different illumination powers of 5 and 10 mW at the sample (810 nm, 150 fs, 80 MHz, MaiTai laser, Spectra Physics, USA). Scale bars: 50 μm. Emission band: 450-650 nm.
(26) FIG. 25. CCB imaging in PDMS at room temperature upon illumination with 5 mW average laser power at the sample (excitation 810 nm, 80 MHz). Scale bars: 50 μm. Emission band: 450-650 nm.
(27) FIG. 26. Two-dimensional excitation-emission plot (similar to FIG. 2B) of fCB particles in water under single photon excitation. Each vertical slice corresponds to an emission spectrum at the excitation wavelength.
(28) FIG. 27. Raman data of ufPL superposed on a reasonable luminescence background. Insert shows data typical for amorphous carbon (blue), triple Lorentzian line fit (red), baseline correction (blue, dashed line), spectral components (black, dash dot) fit results for D- and G-bands. For clarity two components are displayed shifted vertically.
(29) FIG. 28. Normalized white light spectra of various dry carbonaceous particles deposited on a cover glass. Droplets of CB suspensions were dried on cover glasses and illuminated using a femtosecond laser (810 nm, 150 fs, 80 MHz repetition rate, MaiTai, Spectra Physics, USA).
(30) FIG. 29. Raw data of FIG. 3B. WL emission spectra of aqueous ufP90 suspensions recorded at different femtosecond illumination wavelengths (from 780 (1) to 900 nm (7), with an interval of 20 nm).
(31) FIG. 30. Temporal response of CCB particles dried on glass (1) and in aqueous suspension (2) measured by time correlated single photon counting timing. The instrument response function (IRF) is overlaid (3). The relative strength of the signal cannot be deduced from the relative peak values of the curves as different particle concentrations were used when performing the experiment in dry and aqueous state.
(32) FIG. 31. Comparison of femtosecond and picosecond illumination of CB particles at 810 nm. 10 mW average laser power at the sample was applied to fCB similar to the experimental conditions above. The same area was consecutively imaged with a seven picosecond laser system (Levante OPO, APE, Berlin pumped by a 532 nm pulse train from a Picotrain laser, HighQ, Austria) and a femtosecond laser (810 nm, 150 fs, 80 MHz, MaiTai, Spectra Physics, USA). The lasers were switched between individual frames. Scale bars: 5 μm.
(33) FIG. 32. Emission power dependence of a sample of immersed ufPL particles. Power spectra were recorded by using a multiphoton microscope with 800 nm excitation by a femtosecond laser (810 nm, 150 fs, 80 MHz repetition rate, MaiTai, Spectra Physics, USA) and a 1.05 NA Objective (Olympus, Japan). As the photomultiplier tubes were quickly saturated on CB emission, the effective number of pixels visible in a scan was measured. Therefore a constant threshold was set and the number of pixels calculated by means of a MATLAB routine.
(34) FIG. 33. Tubulin cytoskeleton (ex/em 495/519 nm) of fixed normal human lung fibroblasts (MRC-5 cell line) incubated at 37° C. with 5 μg/cm.sup.2 ufP90 particles (red, 4 mW average illumination power at the samples at 810 nm, emission band detection: 400-410 nm in non-descanned mode). (A) Control cells. (B) 4 h incubation. (C) 8 h incubation. (D) 24 h incubation. Scale bar: 20 μm.
REFERENCES TO THE APPLICATION
(35) ASTM, American Society for Testing Materials D3053-13a, Standard on Terminology Relating to Carbon Black. ASTM International: West Conshohocken, Pa., 2013. Castro, L.; Pio, C.; Harrison, R. M.; Smith, D. Atmos. Environ. 1999, 33, (17), 2771-2781. Arnal, C.; Alzueta, M.; Millera, A.; Bilbao, R. Combust. Sci. Technol. 2012, 184, (7-8), 1191-1206. Wang, R., Global Emission Inventory and Atmospheric Transport of Black Carbon: Evaluation of the Associated Exposure. Springer Theses: Beijing, China, 2015. J. C. G. Esteves da Silva and H. M. R. Goncalves, Trends Anal. Chem., 2011, 30, 1327 Chow, J. C.; Watson, J. G.; Doraiswamy, P.; Chen, L.-W. A.; Sodeman, D. A.; Lowenthal, D. H.; Park, K.; Arnott, W. P.; Motallebi, N. Atmos. Res. 2009, 93, (4), 874-887. Nemmar, A.; Hoet, P. M.; Vanquickenborne, B.; Dinsdale, D.; Thomeer, M.; Hoylaerts, M.; Vanbilloen, H.; Mortelmans, L.; Nemery, B. Circulation 2002, 105, (4), 411-414. Ferrari, A.; Meyer, J.; Scardaci, V.; Casiraghi, C.; Lazzeri, M.; Mauri, F.; Piscanec, S.; Jiang, D.; Novoselov, K.; Roth, S. Phys. Rev. Lett. 2006, 97, (18), 187401. Belousova, I.; Mironova, N.; Scobelev, A.; Yur'ev, M. Opt. Commun. 2004, 235, (4), 445-452. Zelensky, S. Semicond. Phys. Quantum Electron. Optoelectron. 2004, 7, (2), 190-194. Rulik, J. J.; Mikhailenko, N.; Zelensky, S.; Kolesnik, A. Semicond. Phys. Quantum Electron. Optoelectron. 2007, 10, (2), 6-10. Strek, W.; Cichy, B.; Radosinski, L.; Gluchowski, P.; Marciniak, L.; Lukaszewicz, M.; Hreniak, D. Light Sci. Appl. 2015, 4, (1), e237. Hamilton, B.; Rimmer, J.; Anderson, M.; Leigh, D. Adv. Mater. 1993, 5, (7-8), 583-585. Imholt, T.; Dyke, C. A.; Hasslacher, B.; Perez, J. M.; Price, D.; Roberts, J. A.; Scott, J.; Wadhawan, A.; Ye, Z.; Tour, J. M. Chem. Mater. 2003, 15, (21), 3969-3970. Fougeanet, F.; Fabre, J.-C. In Nonlinear mechanisms in carbon-black suspension in a limiting geometry, MRS Proceedings, 1997; Cambridge Univ. Press: p 293. Li, H.; Kang, Z.; Liu, Y.; Lee, S.-T. J. Mater. Chem. 2012, 22, (46), 24230-24253. Ghosh, S.; Chizhik, A. M.; Karedla, N.; Dekaliuk, M. O.; Gregor, I.; Schuhmann, H.; Seibt, M.; Bodensiek, K.; Schaap, I. A.; Schulz, O. Nano Lett. 2014, 14, (10), 5656-5661. Usman, A.; Chiang, W. Y.; Masuhara, H. Sci. Prog. 2013, 96, (Pt 1), 1-18. Usman, A.; Chiang, W.-Y.; Masuhara, H. In Femtosecond trapping efficiency enhanced for nano-sized silica spheres, 2012; pp 845833-845833-7.
