Dual Channel Detection of Ultra Low Concentration of Bacteria in Real Time and via Scanning Fcs

Altamore, Ilaria; Lanzanò, Luca; Gratton, Enrico

doi:10.1016/j.bpj.2012.11.1929

Cited by 8 publications

(5 citation statements)

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“…We propose that cuvette FCS may be adapted to perform experiments such as scanning FCS on samples held inside regular glass or quartz test tubes. Scanning FCS has been used to detect fluorescently dim but large objects such as bacteria (15). We think that the high sensitivity of the cuvette FCS would enable detection and characterization of much smaller hazardous pathogens such as viruses.…”

Section: Discussionmentioning

confidence: 99%

“…For example, Garai et al performed FCS measurements using a single-mode optical fiber with NA ¼ 0.13 for detection of amyloid aggregates inside a centrifuge tube (14). Altamore et al used a low NA (NA ¼ 0.4), long working distance objective to perform scanning FCS measurements inside glass test tubes for single-particle detection and characterization of bacteria (15). However, these experiments are applicable only for characterization of highly bright and large particles because of the poor S/N of the FCS data.…”

Section: Introductionmentioning

confidence: 99%

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A Fluorescence Correlation Spectrometer for Measurements in Cuvettes

Sahoo

Sil

Karmakar

et al. 2018

Biophysical Journal

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We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single-molecule measurements on samples inside regular cuvettes. The cuvette FCS uses a horizontally mounted extra-long working distance, 0.7 NA, air objective with a working distance of >1.8 mm instead of a high NA water or oil immersion objective. The performance of the cuvette FCS is found to be highly sensitive to the quality and alignment of the cuvette. The radial resolution and effective observation volume obtained using the optimized setup are $340 nm and 1.8 fL, respectively. The highest molecular brightness and the signal/noise ratio in the autocorrelation data achieved using an aqueous solution of rhodamine B are greater than 44 kHz and 110, respectively. Here, we demonstrate two major advantages of cuvette FCS. For example, the cuvette FCS can be used for measurements over a wide range of temperatures that is beyond the range permitted in the microscope-based FCS. Furthermore, cuvette FCS can be coupled to automatic titrators to study urea-dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette FCS in the experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Fluorescence Correlation Spectrometer for Measurements in Cuvettes

Sahoo

Sil

Karmakar

et al. 2018

Biophysical Journal

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“…In this type of particle counting device, the fluid sample is transferred into a cuvette that is rotated and translated in front of a lens focusing the illumination beam into the cuvette. The signal from a small observation volume is detected in a particular wavelength band with a fast detector such as a photomultiplier [2,3]. Because the sample volume is explored by moving the container rather than passing the fluid through a tube or channel the specimen is not subjected to turbulence or shear stress and a large volume can be processed in a short amount of time.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence lifetime detection with particle counting devices

Hedde

Abram²,

et al. 2019

Biomed. Opt. Express

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Fluorescence-based single particle counting devices have become very powerful tools for human health-related applications such as the detection of blood-borne pathogens. Instead of passing the sample fluid through a thin tube or microfluidic chip, as it is commonly practiced in flow cytometers and sorter devices, single particle counters scan the fluid volume by rotation and translation of the sample container. Hence, single particle counters are not limited by the fluid flow friction and can scan a large volume in a short timeframe while maintaining high sensitivity. A single particle can be detected in a milliliter of the fluid sample within minutes, and diagnostics are being developed using this principle. Until now, signal detection with particle counters has been based on signal intensity and signal separation into multiple wavelength bands coupled with multiple detectors, which limits the number of species that can be resolved. In this paper, we applied fluorescence lifetime detection to single particle counting to increase specificity and enable multiplexing with a single detector. We demonstrate how this principle can be used for diagnostic assays based on fluorescence quenching.

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“…For instance, a potential issue of the cuvette-FCS setup is represented by the huge difference between the small observation volume required to detect single-molecule fluctuations ($1.8 fL) and the total cuvette volume ($3.5 mL). In this respect, mechanical scanning of the cuvette could be useful to explore a larger portion of the cuvette, especially in the case of very dilute samples (10,11). This idea has been demonstrated only with large, bright particles such as fluorescently labeled bacteria and yeast cells (10,11).…”

mentioning

confidence: 99%

“…In this respect, mechanical scanning of the cuvette could be useful to explore a larger portion of the cuvette, especially in the case of very dilute samples (10,11). This idea has been demonstrated only with large, bright particles such as fluorescently labeled bacteria and yeast cells (10,11). The introduction of a cuvette-FCS setup sensitive to single-molecule fluctuations opens interesting perspectives toward detection of very low concentrations of toxic agents.…”

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confidence: 99%

Back to the Future: Fluorescence Correlation Spectroscopy Moves Back in the Cuvette

Lanzanò

2018

Biophysical Journal

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Dual Channel Detection of Ultra Low Concentration of Bacteria in Real Time and via Scanning Fcs

Cited by 8 publications

References 0 publications

A Fluorescence Correlation Spectrometer for Measurements in Cuvettes

A Fluorescence Correlation Spectrometer for Measurements in Cuvettes

Fluorescence lifetime detection with particle counting devices

Back to the Future: Fluorescence Correlation Spectroscopy Moves Back in the Cuvette

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