Fourier Transform Near Infrared Microspectroscopy, Infrared Chemical Imaging, High-Resolution Nuclear Magnetic Resonance and Fluorescence Microspectroscopy Detection of Single Cancer Cells and Single Viral Particles

Single Cancer Cells from Human tumors are being detected and imaged by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR)Hyperspectral Imaging and Fluorescence Correlation Microspectroscopy. The first FT-NIR chemical, microscopic images of biological systems approaching one micron resolution are here reported. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are also 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 as well as 99% accurate calibrations are also presented here with nanoliter precision. Such high-resolution, 400 MHz H-1 NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. >~20%) compared to the average levels in 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. Tenfold, or hundred-fold, sensitivity increases are predicted for in vivo FT-NIR Fluorescence measurements. Therefore, detailed chemical analyses of oils and phytochemicals are now becoming possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cells. Signalling pathways and key interactions controlling the cell division in normal and transformed cells may be thus monitored in vivo, noninvasively, in human patients with very high sensitivity, accuracy and remarkable speed. In other, agricultural applications, such as the genetic selection and breeding programs, the lower cost, speed and analytical requirements of such genetic selection programs are fully satisfied by FT-NIR spectroscopy and Microspectroscopy, as in the case of whole soybean seeds and soybean embryos that we have investigated extensively and also validated with over 50,000 different soybean samples. 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 (~150) femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD) and 0.25 micron resolution with turbid cell suspensions. Therefore, such powerful techniques allow for ultra-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 sub-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: medical/cancer research, pharmacology, early clinical diagnosis of viral diseases and cancers, agricultural biotechnology and food safety. *Corresponding Author: Professor I.C. Baianu KEYWORDS: FT-NIR and FT-IR Instruments, applications of FCS/NIR; 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 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; Spatial Resolution Test in FT-NIR Micro-Imaging; FT-NIR Images of Soybeans and Embryos; FT-IR Reflectance Chemical Images; Somatic Embryos; 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 somatic soybean embryogenic cultures; 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 Epifluorescence 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.