phiFLIM: a new method to avoid aliasing in frequency domain fluorescence lifetime imaging microscopy.

In conventional wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity-modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained. The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation. A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics. Taking more images to avoid this is not always possible owing to phototoxicity or movement. A method is introduced, FLIM, requiring only three recordings that is not susceptible to aliasing. The phase difference between the excitation and the intensifier is scanned over the entire 360? range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image. Three different images are produced following this procedure, each with a different phase profile. Measurements were performed with a conventional wide-field frequency-domain FLIM system based on an acousto-optic modulator for modulation of the excitation and a microchannel-plate image intensifier coupled to a CCD camera for the detection. By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present. Using the conventional method with three recordings, phase errors due to aliasing of up to ? 29? and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP-transfected HeLa cells were as high as 100%. With FLIM, using the same specimen and settings, systematic errors due to aliasing did not occur

Haftwerte von Komposit an Milchzahndentin, nach Präparation mit dem Er:YAG Laser und konventioneller Präparation mit dem Diamanten

Background Photobleaching can lead to significant errors in frequency-domain fluorescence lifetime imaging microscopy (FLIM). Existing correction methods for photobleaching require additional recordings and processing time and can result in additional noise. A method is introduced that suppresses the effects of photobleaching without the need for extra recordings or processing. Methods Existing bleach correction methods and the method introduced in this report whereby the recording order of the phases is permuted were compared using numerical simulations. Results Certain orders were found to make measurements virtually insensitive to photobleaching. At 12 recordings, errors in measured phase and modulation depth decreased by a factor 512 and 393, respectively, compared to recordings using sequential recording order. The optimal order is independent of modulation depth, phase, and extent of photobleaching. Thus, the same order can be used for practically all situations. Application of the method in FLIM measurements of EYFP-transfected HeLa cells was found effectively to suppress photobleaching induced artifacts. Conclusions In view of the ease of implementation, its inherent robustness, and the possibility to still apply existing correction methods afterward, there is no good reason not to use the permuted recording order presented in this report instead of a sequential order

Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge-coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide-field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow-cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal-to-noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide-field DIC microscope becomes possible with this technique, using relatively simple mean

Fluorescence lifetime imaging microscopy (FLIM).

Fluorescence lifetime imaging microscopy (FLIM) is a technique to map the spatial distribution of nanosecond excited state lifetimes within microscopic images. FLIM systems have been implemented both in the frequency domain, using sinusoidally intensity-modulated excitation light and modulated detectors, and in the time domain, using pulsed excitation sources and time-correlated or time-gated detection. In this review we describe the different modes in which both frequency-domain and time-domain FLIM instruments have been constructed in wide-field and in point-scanning (confocal) microscopes. Also, novel additional strategies for constructing FLIM-instruments are discussed. In addition to technical implementation, this chapter gives an overview of the application of FLIM in cell biological en biomedical studies. Especially for in situ protein-protein interaction studies using fluorescence resonance energy transfer (FRET), FLIM has proven to be a robust and established technique in modern cell biology. Other application areas, including usage of lifetime contrast for ion-imaging, quantitative imaging, tissue characterization and medical applications, are discussed