Real-time, label-free detection and identification of bacteria through non-invasive optical imaging.

Currently, traditional and newer molecular and mass spectrometry techniques of identifying bacteria from biological samples requires lengthy sample preparation, growth and labelling/staining assays. Thus, there is a pressing clinical need for an adjunct method that accurately identifies bacteria in real time. Here we report on the evaluation of confocal microscopy for the identification of clinically important and multi-drug resistant (MDR) bacteria in real time, using their intrinsic fluorescence features, i.e., emission spectra and fluorescence lifetime. The results demonstrate that difference in emission spectra and fluorescence lifetimes can be used as a fingerprint for identification of 12 bacterial species and MDR strains in real-time. Photostability or time-traces of bacteria demonstrated that these parameters could be used for tracking and recording without a need for labelling. Further, dilution experiments demonstrated that using intrinsic fluorescence S. aureus, Klebsiella pneumoniae and Escherichia coli bacteria can be detected and identified at clinically relevant concentrations as low as 2 × 102 CFU/mL. This non-invasive, non-labelling optical methodology may serve as the basis for development of a device that would quickly and accurately identify bacteria in biological samples. Thus, this intrinsic fluorescence technique would provide clinicians information, within minutes from sampling, to base accurate and specific treatments for patients.

Detection and identification of amino acids and proteins using their intrinsic fluorescence in the visible light spectrum.

The detection and identification of biomolecules are essential in the modern era of medical diagnostics. Several approaches have been established, but they have significant limitations such as laborious and time-consuming sample preparation, analysis, and the need to use external probes which provide adequate but not desired levels of accuracy and sensitivity. Herein, we have explored successfully a non-invasive technique to detect and identifybiomolecules such as amino acids and proteins by utilizing their intrinsic fluorescence. The developed confocal microscopy method revealed high and photostable emission counts of these biomolecules including amino acids (tryptophan, phenylalanine, tyrosine, proline, histidine, cysteine, aspartic acid, asparagine, isoleucine, lysine, glutamic acid, arginine) and proteins (HSA, BSA) when they are excited with a green laser. The fluorescence lifetime of the samples enabled the identification and distinction of known and blind samples of biomolecules from each other. The developed optical technique is straightforward, non-destructive and does not require laborious labeling to identify specific proteins, and may serve as the basis for the development of a device that would quickly and accurately identify proteins at an amino acid level. Therefore, this approach would open an avenue for precise detection in imaging and at the same time increases our understanding of chemical dynamics at the molecular level.