Abstract
AbstractChemical analysis of soybean seeds, somatic embryos and single cells were carried out by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1 micron (1μ) resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as 2μ3. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision approaching the picogram level. Indeed, detailed chemical analyses of oils and phytochemicals are now becoming possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cells. The cost, speed and analytical requirements of plant breeding and genetic selection programs are fully satisfied by FT-NIR spectroscopy and Microspectroscopy for soybeans and soybean embryos. 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). Therefore, such powerful techniques allow for the most sensitive and reliable quantitative analyses to be carried out both in vitro and in vivo. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allows not only single molecule detection, but also molecular dynamics and high resolution, submicron imaging of femtoliter volumes inside living cells and tissues. These novel, ultra-sensitive and rapid NIR/FCS analyses have numerous applications in important research areas, such as: agricultural biotechnology, food safety, pharmacology, medical research and 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
There are four major fluorescence techniques that are currently employed for the analysis and monitoring of molecular interactions and dynamics: Fluorescence Correlation Spectroscopy (FCS), Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Recovery After Photobleaching (FRAP)
The results reported in this chapter for soybean seeds and embryos were obtained with FT-IR or (FT)- IR and -NIR spectrometers made by the PerkinElmer Co. (Shelton, CT, USA)
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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