Hyperspectral fluorescence microfluidic (HFM) microscopy.

We present an imaging system that collects hyperspectral images of cells travelling through a microfluidic channel. Using a single monochrome camera and a linear variable bandpass filter (LVF), the system captures a bright field image and a set of hyperspectral fluorescence images for each cell. While the bandwidth of the LVF is 20 nm, we have demonstrated that we can determine the peak wavelength of a fluorescent object's emission spectrum with an accuracy of below 3 nm. In addition, we have used this system to capture fluorescence spectra of individual spatially resolved cellular organelles and to spectrally resolve multiple fluorophores in individual cells.

[1]  Astrid Magenau,et al.  Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution , 2012, Nature Communications.

[2]  William E. Ortyn,et al.  Cellular image analysis and imaging by flow cytometry. , 2007, Clinics in laboratory medicine.

[3]  Y. Hiraoka,et al.  Multiple-color fluorescence imaging of chromosomes and microtubules in living cells. , 1999, Cell structure and function.

[4]  D. Haussler,et al.  Integration of cytogenetic landmarks into the draft sequence of the human genome , 2001, Nature.

[5]  T. Zimmermann.  Spectral imaging and linear unmixing in light microscopy. , 2005, Advances in biochemical engineering/biotechnology.

[6]  P. Kiesel,et al.  Fluorescence spectrometer-on-a-fluidic-chip. , 2007, Lab on a chip.

[7]  R. Tsien,et al.  Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. , 1989, The Journal of biological chemistry.

[8]  Richard M Levenson,et al.  Spectral imaging perspective on cytomics , 2006, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[9]  P. Chattopadhyay,et al.  Seventeen-colour flow cytometry: unravelling the immune system , 2004, Nature Reviews Immunology.

[10]  S. S. Gorthi,et al.  Microfabricated multiple field of view imaging flow cytometry. , 2012, Lab on a chip.

[11]  S. S. Gorthi,et al.  Phase imaging flow cytometry using a focus-stack collecting microscope. , 2012, Optics letters.

[12]  Liang Gao,et al.  Snapshot Image Mapping Spectrometer (IMS) with high sampling density for hyperspectral microscopy , 2010, Optics express.

[13]  J. Lippincott-Schwartz,et al.  Development and Use of Fluorescent Protein Markers in Living Cells , 2003, Science.

[14]  Hideaki Matsuoka,et al.  Single-cell viability assessment with a novel spectro-imaging system. , 2002, Journal of biotechnology.

[15]  Howland D. T. Jones,et al.  Hyperspectral confocal microscope. , 2006, Applied optics.

[16]  Gregory P. Crawford,et al.  Liquid-crystal materials find a new order in biomedical applications. , 2007, Nature materials.

[17]  A S Verkman,et al.  Green fluorescent protein as a noninvasive intracellular pH indicator. , 1998, Biophysical journal.

[18]  Diane S Lidke,et al.  Hyperspectral Confocal Fluorescence Imaging: Exploring Alternative Multivariate Curve Resolution Approaches , 2009, Applied spectroscopy.

[19]  J. Paul Robinson,et al.  Hyperspectral cytometry at the single‐cell level using a 32‐channel photodetector , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[20]  Victoria J Allan,et al.  Light Microscopy Techniques for Live Cell Imaging , 2003, Science.