Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Somatic Embryos and Single Cells

Novel methodologies are currently being developed and established 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 one micron 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 two cubic microns. 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. KEYWORDS: Single Cancer Cell Detection by FT-NIR and FT-IR Instruments; applications of FCS/NIR;Single Virus (HIV, HBV, HPV-16) Particle Detection by FCS/FCCS; Agricultural biotechnology; IR Chemical Imaging and NMR, Microspectroscopy; DNA/RNA Micro-array analysis by NIR; High resolution and super-resolution FT-NIR/IR, IR Chemical Imaging by FPAW, Spotlight 300 Microspectrometer; Two photon NIR excitation for FCS, Single Cell and sister; single-molecule dynamics, FCS of molecules, single cells, Soybean oil, protein and moisture analysis, FT-NIR and FT-IR, high-resolution NMR of soybean oil in seeds and somatic embryos; chemical mutagenesis of soybean embryos; picomole FT-NIR and femtomole FCS-NIR analysis of single cells; phytochemicals detection in soybean seeds and cells by FT-NIR; high-power, femtosecond Ti:Sapphire NIR excitation for FCS; FCS/PCR, Nucleic acid hybridization; FT-IR and FT-NIR Images of Soybeans and Embryos, FT-IR and NIR Chemical Imaging Tests; One-Micron Spatial Resolution Tests; FT-NIR Micro-Imaging, FT-NIR Images of Soybeans and Embryos, FT-IR Reflectance Chemical Images, Somatic Embryo; NIR Reflectance Chemical Image of a Red Coat Azuki Red Bean; FT-NIR Chemical Imaging by Difference Spectroscopy (CIDS); High Resolution NMR Analysis of Soybean Oil in Somatic Embryos, HR NMR,H-1 NMR Spectrum of the somatic embryogenic culture of a soybean sample; TEM Micrograph of a Suspension of Soybean Somatic Embryos in Culture; Single Molecule Detection; two-photon excitation, one-photon excitation, three-photon-excitation; Fluorescence Correlation Spectroscopy (FCS); Fluorescence Resonance Energy Transfer (FRET); Fluorescence Lifetime Imaging Microscopy (FLIM); Fluorescence Recovery After Photobleaching (FRAP); Single Photon Confocal Fluorescence Correlation Spectroscopy, Inverted Epi-fluorescence Microscope; FCS auto-correlation, Fluorescence Fluctuations, Fluorescence Intensity; Fluorescence Correlation Spectroscopy and Imaging Experiments in Solutions and Plant Cell Suspensions; Pulsed, Two-Photon NIR Laser Excitation/ Multi-photon (MPE) NIR excitation; FCS Alba Spectrometer Microspectrometer System; FCCS Cross-Correlation with Two Fluorescent Labels; FCCS Applications to DNA Hybridization.