Wide field imaging

Ratio imaging nicely cancels out some of the main complications in the interpretation of wide-field images in that it normalizes fluorescence intensity differences caused by for example, cell height (Fig. 7.T1) as well as possible slow drift in excitation intensity. Light sources invariably are much less stable than detectors. Incidentally, for these reasons emission ratio imaging has been applied for over 3 decades by the Ca2+ imaging community. 

These differences add up to one major distinction on wide-field imaging setups, it suffices to calibrate the setup just once for a given set of filters and fluorophores, and then use it for weeks or months without bothering about it. In contrast, for confocal filterFRET imaging, calibrations must be made every time a gain setting or laser line is adjusted, and preferably, for every image. 

An endoscope, coupled with video rate wide field imager, operating in either the time or frequency domain [92] could be an invaluable tool for early detection of cancer or possibly other diseases whose AF signatures are yet to be studied. 

Fig. 7 3D orbital tracking of a polyplex inside a cell(a) The 3D trajectory (blue) of a polyplex was tracked in HuH7 cells with EGFP labeled tubulin (green structures in the image). Overlayed onto the 3D trajectory are two wide-field images taken at different z-positions during the measurement. 

Fig. 10 PL from single PF molecules containing on-chain fluorenone defects, a Chemical structure of the dioctylfluorene-fluorenone copolymer investigated, b Room-temperature single molecule fluorescence wide-field images of the copolymer dispersed in a Zeonex matrix. Note the PL intensity encoded in a negative scale with respect to Fig. 2b. Excitation was performed at 400 nm in the tail of the backbone absorption. The PL was recorded in 5-s exposure windows. Spectral selection was performed by means of blue (left column) and green (centre column) band-pass filters centred at 460 and 550 nm, respectively. Adapted from [26]...

A few comments should be made about the differences between the TCSPC scanning technique and TCSPC wide-field imaging. The obvious difference is that wide-field imaging by position-sensitive TCSPC imaging does not yield any depth resolution or out-of-focus suppression. Moreover, two-photon excitation cannot be used. Wide-field TCSPC therefore lacks the contrast of the TCSPC scanning technique and is not useful for deep tissue imaging. 

The benefit of TCSPC wide-field imaging is that it can be easily adapted to almost any microscope or other optical system. It may also be a solution for samples that preclude, for whatever reason, scanning by a laser spot of high power density. 

An additional push can be expected from new technical developments in TCSPC itself. The largest potential is probably in the development of new detectors. The introduction of direct (wide-field) imaging techniques is clearly hampered by the limited availability of position-sensitive detectors. In addition the selection of multianode PMTs is still very limited, especially for NIR-sensitive versions. Large-area detectors with 64 or more channels may result in considerable improvements in DOT techniques. Single photon APDs with improved timing stability are urgently required for single-molecule spectroscopy and time-resolved microscopy. 

Using the application software, direct the light path to the camera and select the 488 filter cube for wide field image acquisition. 

Leisawitz et al. (2003) proposed an extension of the FOV by using a focal plane detector array for optical wavelengths, technique called Wide-Field Imaging Interferometry. However, direct detector arrays in the Far Infrared are still expensive and a similar approach is not straightforward. 

D. T. Leisawitz, B.J. Frey, D.B. Leviton, A.J. Martino, W.L. Maynard, L.G. Mundy, S.A. Rinehart, S.H. Teng, X. Zhang, in Wide-field imaging interferometry testbed I purpose, testbed design, data, and synthesis algorithms, ed. by M. Shao. Society of Photo-Optical Instiumentation Engineers (SPIE) Conference Series, vol. 4852 of Society of Photo-Optical Instrumentation Engineers (SPIE) Conference Series (2003), pp. 255-267. doi 10.1117/12.460704... 

Wide-Field Imaging Interferometry Testbed (WIIT)... 

In this chapter the two Spectro-Spatial Interferometry Testbeds in which I have been involved during my programme have been presented the FIRI laboratory testbed and WIIT, the Wide-field Imaging Interferometry Testbed. Both testbeds demonstrate the theory of Double Fourier Spatio-Spectral Interferometry or the application of a Fourier-transform spectrometer (FTS) to aperture synthesis interferometry presented in the previous chapter, at far infrared wavelengths and optical wavelengths, respectively. 

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