Fluorescence spectral imaging microscopy

Fluorescence spectral imaging microscopy (FSPIM)—a method that uses a spectroscopic approach to quantify changes in the acceptor intensity at donor excitation wavelengths [24, 27, 51, 52] (see Chapter 8) ... 

Hanley, Q. S., Arndt-Jovin, D. J. and Jovin, T. M. (2002). Spectrally resolved fluorescence lifetime imaging microscopy. Appl. Spectrosc. 56,155-66. 

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

Ecker, R. C., de Martin, R., Steiner, G. E. and Schmid, J. A. (2004). Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis. Cytometry A 59, 172-81. 

Tinnefeld, R, Herten, D.P., and Sauer, M. 2001. Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence hfetime imaging microscopy (SFLIM). J. Phys. Chem. A 105 7989. 

Knemeyer, JP, Herten, DP, and Sauer, M, Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy. Analytical Chemistry 75 (2003) 2147-2153. 

Our previous approaches to detect endogenous complexes of dynamin and auxilin using co-immunoprecipitation approaches were unsuccessful, so we turned to fluorescence lifetime imaging microscopy (FLIM). While fluorescence microscopy provides two- or three-dimensional information about fiuorophore concentration, FLIM can reveal spatial differences in fluorophore population lifetimes that are independent of concentration. Besides being useful in fiuorophore identification, which transcends issues of spectral overlap, FLIM inherently observes lifetime truncations on a pixel by pixel basis that are induced by fluorescence resonance energy... 

Dumas, D. Stoltz, J. F. New tool to monitor membrane potential by FRET voltage sensitive dye (FRET-VSD) using spectral and fluorescence lifetime imaging microscopy. Clin. Hemorheol Microcirc. 2005, 33, 293-302. 

Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31, 1272-8. 

Spectral imaging fluorescence microscopy. Genes Cells 7, 881-7. 

Dickinson, M. E., Bearman, G., Tille, S., Lansford, R., and Eraser, S. E. 2001. Multi-spectral imaging and hnear unmixing add a whole new dimension to laser scaiming fluorescence microscopy. Biotechniques 31(6) 1272-8. 

B. Rajwa, T. Bemas, H. Acker, J. Dobracki and J.P. Robinson, Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes, Microscopy Research and Technique, 70(10), 869-879 (2007). 

In this chapter we explore several aspects of interferometric nonlinear microscopy. Our discussion is limited to methods that employ narrowband laser excitation i.e., interferences in the spectral domain are beyond the scope of this chapter. Phase-controlled spectral interferometry has been used extensively in broadband CARS microspectroscopy (Cui et al. 2006 Dudovich et al. 2002 Kee et al. 2006 Lim et al. 2005 Marks and Boppart 2004 Oron et al. 2003 Vacano et al. 2006), in addition to several applications in SHG (Tang et al. 2006) and two-photon excited fluorescence microscopy (Ando et al. 2002 Chuntonov et al. 2008 Dudovich et al. 2001 Tang et al. 2006). Here, we focus on interferences in the temporal and spatial domains for the purpose of generating new contrast mechanisms in the nonlinear imaging microscope. Special emphasis is given to the CARS technique, because it is sensitive to the phase response of the sample caused by the presence of spectroscopic resonances. 

We demonstrate the Mil method, which couples the sensitivity of multiphoton excitation on the spectral phase of the laser pulses to probe microscopic chemical environment-induced changes in the multiphoton excitation spectrum of sensitive reporter molecules. We carry out the optimization of the required phase functions in solution and provide theoretical simulations. We show experimental images whereby pH-selective two-photon microscopy is achieved and demonstrate how selective excitation can be used to enhance contrast and, consequently, to achieve functional imaging, using fluorescent probes sensitive to changes in their local environment. 

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