Principles and applications in biomedical diagnostics. The endogenous fluorescence of human skin has been investigated in vivo on normal human volunteers as well as on patients with psoriasis and it was found that characteristic bands can be identified in the fluorescence spectra that are associated with specific skin fluorophores. Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fiberoptic probe article pdf available in biomedical optics express 52. This article presents an overview of timeresolved lifetime fluorescence techniques used in biomedical diagnostics. For our analysis we employ the principle of fluorescence lifetime crosscorrelation spectroscopy flccs. In this paper, we used in vivo fluorescence lifetime imaging with her2targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17dmag an hsp90 inhibitor on tumors with. From the optical diffusion equation describing the propagation and generation of fluorescence within solutions of 0. In vivo imaging of cerebral energy metabolism with twophoton. Dimensional imaging, lifetime measurements and fluorescence spectroscopy a. Content fluorescence lifetime and its measurement use in spectroscopy and microscopy applications 1. A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole. Fluorescence based methods like twocolor fluorescence crosscorrelation spectroscopy can provide this information but measurement precision is.
Reporters, dyes, and instrumentation proceedings of spie, vol. Design and construction of an intravital depthresolved fluorescence lifetime spectrometer 67 5. Fluorescence imaging is a type of noninvasive imaging technique that can help visualize biological processes taking place in a living organism. Conference proceedings laboratory for fluorescence dynamics. The major components are a tunable pulse laser tsunami, spectra physics, mountain view, ca with a pulse width of 100 fs and repetition rate of 80 mhz, a time. Fluorescence lifetime imaging of cancer in vivo springerlink. Fluorescence based methods like twocolor fluorescence crosscorrelation spectroscopy can provide this information but measurement precision. It provides the reader with an excellent source for a detailed discussion of the techniques, their implementation in innovative instrumentation. Fluorescence lifetime imaging with a confocal endomicroscope.
Under this flim condition, the temporal decay of fluorescence from deep tissue can be directly used to recover both the in vivo lifetimes and their corresponding. Tomographic fluorescence lifetime imaging fluorescence lifetime imaging based on exogenous probes 464 real part of fr. Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. The ability to quantify protein concentrations and to measure protein interactions. Krasieva, a chiara stringari, b feng liu, c chungho sun, a yu kong, d mihaela balu, a frank l. Fluorescence lifetime cross correlation spectroscopy. Biophysical journal volume72 june 1997 24052412 multiphoton excitation fluorescence microscopyandspectroscopyof in vivo humanskin barry r. We recently used a fluorescence lifetime sensor as a readout of cytosolic glucose levels in resting and stimulated neurons diaz.
Fluorescence resonance energy transfer between fluorescent proteins as powerful toolkits for in vivo studies a l rusanov and a p savitsky. Fluorescence lifetime cross correlation spectroscopy resolves. As shown in figure \\pageindex1\, in absorption, wavelength. In this paper, we used invivo fluorescence lifetime imaging with her2 targeted fluorescent probes as an alternative imaging method to. Folding is far more complicated in eukaryotic cells for example. Fluorescence spectroscopy of human skin has the potential to provide useful morphologic and biochemical information.
Therefore, fluorescence lifetime spectroscopy and imaging are among the most promising techniques to unravel the elusive nature of lipid domains in vivo. Jun 29, 2010 fluorescence correlation or crosscorrelation spectroscopy fcs or fccs, a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. Jan 25, 2012 this article presents an overview of timeresolved lifetime fluorescence techniques used in biomedical diagnostics. Osa in vivo imaging of cerebral energy metabolism with two. In vivo fluorescence spectroscopy and imaging for oncological. By combining diffuse fluorescence tomography with fluorescence lifetime imaging flim, implemented using widefield timegated detection of fluorescence excited by ultrashort laser pulses in a. Fiberoptic multiphoton fluorescence spectroscopy for biosensing and in vivo flow cytometry by yuchung chang a dissertation submitted in partial fulfillment of the requirements for the degree of doctor of philosophy electrical engineering in the university of michigan 2009 doctoral committee. In vivo imaging of cerebral energy metabolism with twophoton fluorescence lifetime microscopy of nadh the harvard community has made this article openly available.
In vivo use of fret is challenging because of the scattering properties of bulk tissue. The impact of visible cwlaser irradiation on skin autofluorescence lifetimes was investigated in spectral range from 450 nm to 600 nm. Timedomain wholefield fluorescence lifetime imaging with optical. View the article pdf and any associated supplements and figures for a period of 48 hours. Yaseen,1 sava sakadzic,1 weicheng wu,1 wolfgang becker,2 karl a. In addition, the use of these techniques for dosimetry in photodynamic therapy pdt or to guide tissue this paper is dedicated to the former editor of photochemistry and photobiology. Fluorescence lifetime spectroscopy for ph sensing in.
The goal of this work is to develop methods based on twophotonexcitedfluorescence tpef for measuring relative concentrations of these compounds. The observed fluorescence lifetime, is the average time the molecule spends in the excited state, and it is 1. Pdf fluorescence lifetime spectroscopy of glioblastoma. Fluorescence lifetime flt properties of organic molecules provide a new reporting strategy for molecular imaging in the near infrared nir spectral region. Theemission spectra and fluorescence lifetime images wereobtained at.
Photoluminescence, phosphorescence, and fluorescence. Fluorescence lifetime spectroscopy in multiplescattering environments. Multiphoton excitation fluorescence microscopy and. The fluorescence decay amplitudes corresponding to the irfp720 lifetime, recovered from the basis function approach, as in fig. In comparing the in vivo fluorescence spectral shape of bronchial tissue collected by this system with the intrinsic one obtained in vitro, we found no obvious distortion in the in vivo spectra. Invivo fluorescence microscopy laser diode illumination. In vivo fluorescence lifetime imaging monitors binding of specific. This genetically encoded sensor harnesses the glucose. Twophoton excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo article pdf available in journal of biomedical optics 183. Fluorescence lifetime imaging flim in confocal microscopy. For accurate in vivo fluorescence observation on freely moving animal, we strongly recomend you choice doric rotary joint and optical fiber systems using with below products. Fluorescence lifetime techniques in medical applications. Molecular brightness characterization of egfp in vivo by fluorescence fluctuation spectroscopy.
Book chapters laboratory for fluorescence dynamics. Pdf fluorescence lifetime imaging microscopy of nanodiamonds. Osa in vivo imaging of cerebral energy metabolism with. Images can be produced from a variety of methods including. Thus, flim is a fluorescence imaging technique where the contrast is based on the lifetime of individual fluorophores rather than their emission spectra. In vivo imaging of cerebral energy metabolism with twophoton fluorescence lifetime microscopy of nadh mohammad a. Singlecolor fluorescence lifetime crosscorrelation. Fluorescence lifetime spectroscopy in the presence of tissuelike scattering is demonstrated from measurements of phase and modulation ratio as a function of modulation frequency using a phsensitive dye, carboxy seminaphthofluorescein1 csnafl1. University of cyprus biomedical imaging and applied.
Dec 12, 2012 twophoton excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo tatiana b. We found the molecular brightness of egfp to be a very robust parameter, and anticipate the use of pch analysis for the study of oligomerization processes in vivo. Pdf imaging proteins in vitro using fluorescence lifetime. Methods and protocols serves as a valuable reference for researchers from numerous fields who wish to become more familiar with in vivo fluorescence imaging techniques. Protein folding in cells in vivo is extremely complicated and differs depending on the organism.
We attached nearinfrared nir fluorescent probes to human epidermal growth factor 2 her2neuspecific affibody molecules and used our. In vivo fluorescence lifetime tomography spie digital library. We describe a customized twophoton imaging system with time correlated single photon counting detection and. Fluorescence spectroscopy in vivo nirmala ramanujam university of wisconsin, madison, usa 1 fluorescence spectroscopy 1 1.
Functional and morphological optical biopsy based on three. Characteristics of lifetime p id dditi l di i f i f ti i i i. Twophoton excited fluorescence emission spectra of melanins in vitro were obtained using the 32channel meta detector of the zeiss lsm 510 meta nlo microscopy system. Trfs and flim detect biochemical changes in various biological tissues, both in vitro and in vivo fite et al. Flccs is a modification of fccs, whereby a pulsed laser is used for fluorophore excitation and where each detected. In addition, the use of these techniques for dosimetry in photodynamic therapy pdt or to guide tissue this paper is dedicated to the former editor of photochemistry and photobiology, dr. Fluorescence excitation spectroscopy provides information. Multiphoton excitation microscopy of in vivo human skin. Fluorescence spectroscopyfluorescence spectroscopy. Monitoring intracellular nanomolar calcium using fluorescence. Unfortunately, most of the nir fluorescent dyes have short flt typically clustered below 1. The smallest fluorescence microscope body ever was built that easily snaps into chronically implanted imaging cannula via selfcentering latching mechanism.
Fluorescence lifetime imaging flim produces an image based on the differences in the excited state decay rate from a fluorescent sample. One approach to address this issue for in vivo applications is to combine raman spectroscopy with imaging methodologies able to provide an initial faster assessment of tissue biochemical or morphological features, as for instance optical coherence tomography oct, fluorescence spectroscopy, fluorescence lifetime imaging flim, ultrasound or. Canti, timegated fluorescence spectroscopy of porphyrin derivatives and aluminum. Here, we present highresolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. For example, fluorescence lifetime imaging microscopy. The excitation wavelength used was 900 or nm from a chameleonultra femtosecond pulsed tunable from 690 to 1040 nm laser coherent inc. Combined fiber probe for fluorescence lifetime and raman. In vivo imaging of cerebral energy metabolism with two. Molecular brightness characterization of egfp in vivo by. Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy.
In this study, we demonstrate that a new class of nir fluorescent dyes, pyrrolopyrrole cyanine dyes, have exceptionally long flts ranging. Fluorescence and phosphorescence come at lower energy than absorption the excitation energy. We have developed a fluorescence lifetime diffuse optical tomography dot system for in vivo preclinical imaging. Autofluorescence lifetimes were measured before and immediately after the optical provocation. However, the inherent drawback of fcs is that species with similar molecular weight could not be differentiated.
To study molecular processes in vivo, we have developed a laserscanning flim endomicroscope built around a. To study molecular processes in vivo, we have developed a laserscanning flim endomicroscope built around a commercial mauna kea. By dj bornhop, ch contag, and em sevickmuraca editors. Fluorescence correlation or crosscorrelation spectroscopy fcs or fccs, a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. Using in vivo fluorescence lifetime imaging to detect her2. Authoritative and practical, in vivo fluorescence imaging. Jun 10, 2016 akers w, lesage f, holten d, achilefu s 2007 in vivo resolution of multiexponential decays of multiple nearinfrared molecular probes by fluorescence lifetimegated wholebody timeresolved diffuse optical imaging. Assessing molecular biomarkers in living mice using. Specifically the chapter focuses on timeresolved fluorescence spectroscopy trfs and. Importance of studies in vitro to design and rationalize studies in vivo 2.
Fluorescence lifetime imaging system for in vivo studies ncbi nih. Akers w, lesage f, holten d, achilefu s 2007 in vivo resolution of multiexponential decays of multiple nearinfrared molecular probes by fluorescence lifetimegated wholebody timeresolved diffuse optical imaging. Fluorescence lifetime imaging microscopy in life sciences. Heterogeneity in tumor chromatindoxorubicin binding. Canti, time gated fluorescence spectroscopy of porphyrin derivatives and aluminum.
In vivo fluorescence lifetime imaging for monitoring the. The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Martinos center for biomedical imaging, massachusetts general hospitalharvard medical school, charlestown, massachusetts. Fluorescence itself, is a form of luminescence that results from matter emitting light of a certain wavelength after absorbing electromagnetic. Jan 25, 2020 the ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Singlecolor fluorescence lifetime crosscorrelation spectroscopy in vivo. Fluorescence lifetime spectroscopy and imaging techniques. Invivo fluorescence lifetime imaging for monitoring the efficacy of. Study lipid domains away from the complexity and dynamics of a living cell. Fluorescence lifetime based bioassays franzjosef meyeralmessinglemolecule techniques in biophysics. Twophoton excited fluorescence lifetime imaging and.
The fluorescence lifetime is defined as the average. Jul 04, 2003 fluorescence lifetime spectroscopy in the presence of tissuelike scattering is demonstrated from measurements of phase and modulation ratio as a function of modulation frequency using a phsensitive dye, carboxy seminaphthofluorescein1 csnafl1. Twophoton excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo tatiana b. Osa in vivo fluorescence lifetime tomography of a fret. Applications of fluorescence lifetime spectroscopy and. Skin optical provocations were performed during 1 min by 405 nm low power cw laser with power density up to 20 mwcm 2. After absorption, the vibrationally excited s 1 molecule relaxes back to. Fluorescence lifetime spectroscopy and imaging is a valuable modern compendium of this exciting field of research that extends innovative fluorescence techniques to clinical diagnosis and treatment. The book also includes chapters on the emerging fields of cell tracking, imageguided treatment, and fluorescence imaging in the second nir window, as well as protocols for evaluation methods before and after in vivo imaging. Pdf twophoton excited fluorescence lifetime imaging and. Yan chen, joachim d muller, qiaoqiao ruan, and enrico gratton department of physics, university of illinois at urbana champaign, urbana, illinois 61801, usa. Forster resonance energy transfer fret is a powerful biological tool for reading out cell signaling processes. Pdf fluorescence lifetime techniques in medical applications.