Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.

BACKGROUND: Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching. METHODS: A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane. RESULTS: In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field. CONCLUSIONS: Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.

Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching.

Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency. However, in wide-field microscopy, complete acceptor photobleaching is difficult due to the low excitation intensities. In addition, the method is sensitive to inadvertent donor bleaching, autofluorescence and bleed-through of excitation light. In the method introduced in this paper, donor and acceptor intensities are monitored continuously during acceptor photobleaching. Subsequently, curve fitting is used to determine the FRET efficiency. The method was demonstrated on cameleon (YC2.1), a FRET-based Ca(2+) indicator, and on a CFP-YFP fusion protein expressed in HeLa cells. FRET efficiency of cameleon in the presence of 1 mm Ca(2+) was 31 +/- 3%. In the absence of Ca(2+) a FRET efficiency of 15 +/- 2% was found. A FRET efficiency of 28% was found for the CFP-YFP fusion protein in HeLa cells. Advantages of the method are that it does not require complete acceptor photobleaching, it includes correction for spectral cross-talk, donor photobleaching and autofluorescence, and is relatively simple to use on a normal wide-field microscope.

Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.

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, phiFLIM, 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 degrees 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 degrees and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP-transfected HeLa cells were as high as 100%. With phiFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur.

Difference in volume of X- and Y-chromosome-bearing bovine sperm heads matches difference in DNA content.

BACKGROUND: To investigate the possibilities of sperm head volume as a sorting criterion for gender preselection, we determined the magnitude of the difference in volume of X- and Y-chromosome-bearing bull sperm heads. MATERIALS AND METHODS: Bovine sperm heads were sorted on the basis of their DNA content in X- and Y-chromosome-bearing fractions, using an existing flow-cytometric technique. Images of sperm heads of both populations were recorded using Differential Interference Contrast (DIC) microscopy. After reconstructing the DIC images, the area and the optical thickness of sperm heads of both populations were determined. RESULTS: We found a difference in volume of X- and Y-bearing bovine sperm heads matching the difference in DNA content (3.5-4%). CONCLUSIONS: Our findings indicate that volume can be used as a criterion to distinguish X- and Y-chromosome-bearing sperm, making development of a technique to sort X- and Y-chromosome-bearing sperm based on head volume theoretically possible. A strong advantage of such a technique over the existing technique based on DNA content would be that X- and Y-chromosome-bearing sperm cells could thus be sorted without subjecting them to any staining.

Difference in sperm head volume as a theoretical basis for sorting X- and Y-bearing spermatozoa: potentials and limitations.

Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.

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 means.