Abstract
AbstractNovel techniques are currently being developed and established for the accurate chemical analysis and detection of single cancer cells, single embryos and single seeds by Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fourier Transform Infrared (FT-IR), Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1micron resolution are here reported. 400 and 500 MHz, H-1 NMR analyses were carried out that allowed the selection of mutagenized embryos. Detailed chemical analyses are being demonstrated to be also possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cancer cells. FT-NIR Microspectroscopy and Chemical Imaging are also shown to be potentially important in Functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allowed not only single molecule detection, but also molecular dynamics observations and high resolution, submicron imaging of sub-femtoliter volumes inside living cells with 0.25 micron spatial resolution, in both normal and cancer cells, as well as neoplastic tissues. These novel, ultra-sensitive and rapid FT-NIR/FCS analyses have, therefore, substantial potential for numerous applications in important research areas, such as: medicine, medical/cancer research, pharmacology, agricultural biotechnology, food safety, as well as clinical diagnosis of viral diseases and cancers.
Highlights
Infrared (IR) and Near Infrared (NIR) commercial spectrometers employ, respectively, electromagnetic radiation in the range from to ~150 to 4,000 cm-1, and from 4,000 to ~14,000 cm-1
Detailed chemical analyses of oils and phytochemicals are becoming possible by Fourier Transform Near Infrared (FT-NIR) Chemical Imaging/ Microspectroscopy of single cells
FT-NIR Microspectroscopy and Chemical Imaging are shown to be potentially important in functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA)
Summary
Infrared (IR) and Near Infrared (NIR) commercial spectrometers employ, respectively, electromagnetic radiation in the range from to ~150 to 4,000 cm-1, and from 4,000 to ~14,000 cm-1. The employment of high-power, pulsed NIR lasers for visible fluorescence excitation has resulted in a remarkable increase of spatial resolution in microscopic images of live cells, well beyond that available with the best commercial FT-NIR/IR microspectrometers, allowing even for the detection of single molecules. This happens because fluorescent molecules can absorb two NIR photons simultaneously before emitting visible light, a process referred to as "two-photon excitation." Using two-photon NIR excitation (2PE) in a conventional microscope provides several great advantages for studying biological samples.
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