ABSTRACT
Novel methodologies are currently being evaluated for the chemical analysis of soybean seeds, embryos, and single cells 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 μm 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 2 μm3. 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 with nonmutagenized 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). Multiphoton (MP), pulsed femtosecond laser NIR fluorescence excitation techniques were shown to be capable of single molecule detection. Therefore, such powerful techniques allow highly sensitive and reliable quantitative analyses to be carried out both in vitro and in vivo. Thus, MP NIR 242excitation 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, ultrasensitive, 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.
