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Figure 3. The NMSU team: (left to right in front) Wanqin Yu, JoAnne M. Dupre, (behind) John E. Gustafson and Jesus A. Cuaron


E. coli strain DH5cx and four strains of Staphylococcus aureus (LP9, MM61, MM66, and MM66-4) were chosen for the study. All the bacterial cell samples were grown, harvested, and lyophilised by researchers at New Mexico State University. In total, 15 unidentified samples in disposable cuvettes were presented to the ARA team and they were told that the first five samples were unique and the remaining ten samples were replicates, two each, of the first five. LIBS spectra were collected by focusing pulses from a Q-switched Nd:YAG laser (1064 nm, 60 mJ/pulse, 10 Hz) into the open end of a cuvette contained within a Class II biological safety cabinet and sparking the pathogenic sample within.


Each spectra was the accumulation of ten spectra from the laser-induced plasma plumes, with the collection of each spectrum accurately delayed by 1 microsecond from the laser pulse and integrated on a 20 microsecond temporal scale. Because the sample identities were unknown and to mimic an analysis situation in which data are collected and not controlled for quality, all collected spectra were used in the analysis with no screening for spectral quality and no data preprocessing. A total of 1050 accumulated spectra (70 spectra datasets for each sample) over the entire spectral range of 205.42 to 1000 nm from individual samples were collected. The LIBS classification spectra from Sample B (E. coli) is shown in Figure 5. The units of the ordinate axis is detector counts.


By using chemometric analysis of the LIBS spectra in combination with an identification algorithm, the ten blind samples were correctly matched to the unique samples thus demonstrating the spectral identification of all five bacterial samples with 100% accuracy.


The iStar Intensified CCD camera is perfectly adapted to cope with these challenging LIBS measurements, equipped with a fully integrated, software- controlled digital delay and ultrafast and ultralow jitter electronics for sub-2ns to ms optical shuttering capabilities. Coupled with the Echelle spectrograph, the camera delivers the highest spectral and time resolution across a very large bandwidth.


A new generation of rapid, automated biosensor detection systems?


This study demonstrates that LIBS can be used to differentiate the common hospital-borne bacterial pathogens, E. coli and S. aureus in pure form in less than a minute with 100% accuracy using only the raw LIBS spectra in an automated system. Furthermore, in combination with appropriately constructed chemometric models and defined testing flows, LIBS could be used to successfully classify an unknown pathogen in pure form, both species and strain, provided the unknown pathogen is within a defined set of pathogens. In medical treatment applications, by classifying pathogens in matrices of interest (such as blood or tissue), this capability could possibly be used to create testing algorithms to assist in rapid pathogen identification, thereby speeding the initiation of an appropriate antimicrobial-therapeutic regimen.


Figure 5. LIBS classification spectra from E. coli


Figure 4. LIBS setup at ARA: (A) Nd:YAG laser, (B) laser focusing optics, (C) biological target in Class II safety cabinet, (D) collection fibre optics, (E) Echelle spectrometer, (F) Andor iStar ICCD


As a ‘proof-of-principle’ for a LIBS-based instrument for rapid pathogen detection, these are important results since LIBS has many advantages as a biosensing method which include: little or no sample preparation; simplicity of use (focus the laser pulse on the material and collect the light); and rapid in situ analysis with results in less than a minute with automated analysis.


The potential for saving lives through the development of rapid diagnostic instrumentation that can be operated by personnel without any specific technical or LIBS expertise should not be underestimated.


References


1. Baudelet, M, Yu, J, Bossu, M, Jovelet, J. and Wolf, J. P. ’Discrimination of Microbiological Samples using Femtosecond Laser-Induced Breakdown Spectroscopy’, Applied Physics Letters 89, 163903 (2006)


2. Multari, R. A, Cremers, D.A, Dupre, J. M. and Gustafson, J. E. ‘The Use of Laser-Induced Breakdown Spectroscopy for Distinguishing Between Bacterial Pathogen Species and Strains’, Applied Spectroscopy 64 (7), 750-759 (2010)


Authors


a) Applied Research Associates, Southwest Division, 4300 San Mateo Blvd, NE, Suite A-220, Albuquerque, NM 87110, USA b) NetDyaLog Limited, The Annexe, Crispin Way, Farnham Common, Bucks, SL2 3UE, UK


Speeding up High Content Screening


TTP LabTech’s Acumen eX3 is the fastest imaging system available for cell-based screening. It collects and simultaneously analyses over 40 images/second, covering every cell in the entire well area, without the trade-off of having to use lower resolution. Acumen is well established for high-content screening, but researchers have recently applied its large field of view to rapidly analyse complex cellular or animal models, such as angiogenic tube formation, C. elegans or drosophila larvae.


In addition to its built in software, Acumen offers the flexibility of simultaneously exporting whole well 8- or 16- bit TIFF images. These images are open source files and can be used for batch processing by a large range of third party image analysis software. Acumen can be used in cytometry mode to provide a rapid primary screen of compounds or RNAis whilst exporting TIFF files for subsequent secondary analysis/hit confirmation studies using image analysis packages, without the requirement to have to prepare a new set of plates.


This new screening paradigm represents a major breakthrough in how microplate cytometers can be applied to complex cellular models since rapid cytometric analysis can now be combined with image-processing methodology.


Circle no. 218


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