Three dimensional image restoration in fluorescence lifetime imaging microscopy

A microscope set‐up and numerical methods are described which enable the measurement and reconstruction of three‐dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high‐frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase‐dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase‐dependent data gives three‐dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov–Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.

[1]  Robert M. Clegg,et al.  Fluorescence Lifetime-Resolved Imaging Microscopy: A General Description of Lifetime-Resolved Imaging Measurements , 1996 .

[2]  W. Reeves.  Royal Microscopical Society , 1873, Nature.

[3]  T M Jovin,et al.  Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation , 1995, The Journal of cell biology.

[4]  Kjell Carlsson,et al.  Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation , 1997 .

[5]  S. Hell,et al.  Lens Aberrations in Confocal Fluorescence Microscopy , 1995 .

[6]  W. Richardson,et al.  The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen , 1988, The Journal of cell biology.

[7]  Robert M. Clegg,et al.  Wide-band acousto-optic light modulator for frequency domain fluorometry and phosphorimetry , 1989 .

[8]  Y. K. Levine,et al.  Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy. , 1995, Analytical biochemistry.

[9]  A. C. Mitchell,et al.  Photon correlation system for fluorescence lifetime measurements , 1995 .

[10]  Aggelos K. Katsaggelos,et al.  A regularized iterative image restoration algorithm , 1991, IEEE Trans. Signal Process..

[11]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data , 1994 .

[12]  Jeffrey A. Squier,et al.  Double‐pulse fluorescence lifetime measurements , 1997 .

[13]  Mario Bertero,et al.  III Super-Resolution by Data Inversion , 1996 .

[14]  T M Jovin,et al.  Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Thomas M. Jovin,et al.  Time-resolved imaging fluorescence microscopy , 1992, Photonics West - Lasers and Applications in Science and Engineering.

[16]  J N Turner,et al.  Blind deconvolution of fluorescence micrographs by maximum-likelihood estimation. , 1995, Applied optics.

[17]  R. D. Spencer,et al.  MEASUREMENTS OF SUBNANOSECOND FLUORESCENCE LIFETIMES WITH A CROSS‐CORRELATION PHASE FLUOROMETER * , 1969 .

[18]  Joseph R. Lakowicz,et al.  Lifetime‐selective fluorescence imaging using an rf phase‐sensitive camera , 1991 .

[19]  Enrico Gratton,et al.  Time-resolved fluorescence microscopy using two-photon excitation , 1995 .

[20]  H Szmacinski,et al.  Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2. , 1994, Cell calcium.

[21]  J. Pines,et al.  Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus. , 1995, The EMBO journal.

[22]  B. Maliwal,et al.  Construction and performance of a variable-frequency phase-modulation fluorometer. , 1985, Biophysical chemistry.

[23]  G. M. P. VAN KEMPEN,et al.  A quantitative comparison of image restoration methods for confocal microscopy , 1997 .

[24]  S. Suzuki,et al.  Ultrafast microchannel plate photomultipliers. , 1988, Applied optics.

[25]  R. Aikens,et al.  Chapter 16 Solid-State Imagers for Microscopy , 1988 .

[26]  D. Rawlins,et al.  The point‐spread function of a confocal microscope: its measurement and use in deconvolution of 3‐D data , 1991 .

[27]  E Gratton,et al.  Fluorescence lifetime imaging by asynchronous pump-probe microscopy. , 1995, Biophysical journal.

[28]  F. A. Seiler,et al.  Numerical Recipes in C: The Art of Scientific Computing , 1989 .

[29]  Toshimitsu Asakura,et al.  Reduction of coherent noise using various artificial incoherent sources , 1985 .

[30]  Mario Bertero,et al.  Super-resolution by data inversion , 1996 .

[31]  Peter J. Verveer,et al.  Efficient superresolution restoration algorithms using maximum a posteriori estimations with application to fluorescence microscopy , 1997 .

[32]  Jordi Vitrià,et al.  Reconstructing 3D light microscopic images using the EM algorithm , 1996, Pattern Recognit. Lett..

[33]  A. N. Tikhonov,et al.  Solutions of ill-posed problems , 1977 .

[34]  R. Clegg,et al.  Fluorescence Lifetime-Resolved Imaging Microscopy (FLIM) , 1996 .

[35]  G. J. Brakenhoff,et al.  3‐D image formation in high‐aperture fluorescence confocal microscopy: a numerical analysis , 1990 .

[36]  H. M. Voort,et al.  Restoration of confocal images for quantitative image analysis , 1995 .

[37]  Aggelos K. Katsaggelos,et al.  A class of iterative signal restoration algorithms , 1990, IEEE Trans. Acoust. Speech Signal Process..

[38]  J. Lakowicz,et al.  Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing. , 1995, Cell calcium.

[39]  Aggelos K. Katsaggelos,et al.  Maximum likelihood blur identification and image restoration using the EM algorithm , 1991, IEEE Trans. Signal Process..

[40]  Nikolas P. Galatsanos,et al.  Methods for choosing the regularization parameter and estimating the noise variance in image restoration and their relation , 1992, IEEE Trans. Image Process..

[41]  Thomas Cremer,et al.  Volume ratios of painted chromosome territories 5, 7 and X in female human cell nuclei studied with confocal laser microscopy and the Cavalieri estimator , 1995 .

[42]  M. Zernicka-Goetz,et al.  Following cell fate in the living mouse embryo. , 1997, Development.

[43]  R. Aikens,et al.  Solid-state imagers for microscopy. , 1989, Methods in cell biology.

[44]  F S Fay,et al.  Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. , 1995, Science.

[45]  E. Gratton,et al.  A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. , 1983, Biophysical journal.

[46]  S. Nagata,et al.  pEF-BOS, a powerful mammalian expression vector. , 1990, Nucleic acids research.

[47]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale , 1993 .

[48]  Robert M. Clegg,et al.  Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications , 1997 .