Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Abstract

Intrinsically fluorescent sterols, like dehydroergosterol (DHE), mimic cholesterol closely and are therefore suitable to determine cholesterol transport by fluorescence microscopy. Disadvantages of DHE are its low quantum yield, rapid bleaching, and the fact that its excitation and emission is in the UV region of the spectrum. Thus, one has to deal with chromatic aberration and low signal-to-noise ratio. We developed a method to correct for chromatic aberration between the UV channel and the red/green channel in multicolor imaging of DHE compared with the lipid droplet marker Nile Red in living macrophage foam cells and in adipocytes. We used deconvolution microscopy and developed image segmentation techniques to assess the DHE content of lipid droplets in both cell types in an automated manner. Pulse-chase studies and colocalization analysis were performed to monitor the redistribution of DHE upon adipocyte differentiation. DHE is targeted to transferrin-positive recycling endosomes in preadipocytes but associates with droplets in mature adipocytes. Only in adipocytes but not in foam cells fluorescent sterol was confined to the droplet-limiting membrane. We developed an approach to visualize and quantify sterol content of lipid droplets in living cells with potential for automated high content screening of cellular sterol transport. ' 2008 International Society for Advancement of Cytometry Key terms cholesterol; fluorescence; deconvolution; Nile Red; adipocyte; macrophage CHOLESTEROL is an essential lipid constituent of cellular membranes. It plays an important role in membrane trafficking and signal transduction (1). These functions depend on the tightly controlled intracellular distribution of cholesterol. Determination of cholesterol's intracellular distribution and transport dynamics rely either on labeling cells with radioactive cholesterol isotopes and membrane fractionation or on imaging-based approaches. While radioactive cholesterol isotopes monitor the behavior of cholesterol in cells, membrane fractionation is a crude and time-consuming method during which the sterol can redistribute between isolated organelles. Fluorescence microscopy provides deepened insight into cellular architecture and function but requires accurate reporter molecules. Fluorescent analogs of cholesterol have been designed bearing a chemically linked extrinsic reporter moiety whose properties are optimized for detection of the molecule by fluorescence spectroscopy or microscopy (2). A disadvantage of those analogs is the fact that the fluorophore has a large impact on the physico-chemical properties of the labeled sterol due to its charge, polarity and size. A way to circumvent the labeling problem is to use sterols with intrinsic fluorescence like the natural sterol dehydroergosterol (DHE). DHE differs from cholesterol in having three additional double bounds and an extra methyl group in the sterol side chain; it is closely related to ergosterol, a sterol found in yeast cells and other fungi. DHE emits light in the UV ...

Potential of ultraviolet wide‐field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

“…For comparison, we labeled HepG2 cells with DHE exactly as described for the MP microscopy experiment and acquired ten planes along the optical axis using our UV-WF microscope followed by bleaching correction and image restoration using a maximum likelihood deconvolution algorithm implemented in Huygens software ( Fig. 3G; Wü stner, 2007b;Wü stner and Faergeman, 2008a (2002)]. Thus, MP microscopy is better suited to get high-resolution 3D images of DHE-containing endosomes in a crowded cellular environment.…”

“…4C and 4D). Note that the FWHM of 0.1-lm fluorescent beads determined in the UV channel of our UV-WF microscope as a measure of lateral resolution of the microscope was $710 nm prior to and 390 nm after deconvolution (Wü stner and Faergeman, 2008a). Thus, the very small DHE-containing patches shown in Figure 4 can probably not be resolved with this precision by UV-WF microscopy, even after applying deconvolution (Wü stner, 2007b).…”

“…Colocalization in UV-WF imaging modus requires correction for chromatic aberration between the UV and green or red channel providing good-quality images of colocalizing species (Figs. 7D-7H; Wü stner and Faergeman, 2008a). Together, colocalization of DHE with EGFP-NPC1L1 can be detected by both methods.…”

“…For staining cells, a DHE labeling solution containing DHE/MCD was generated as described Wü stner et al, 2002). Cells were pulse labeled for 1-2 min with DHE/MCD, washed with buffer medium, and either imaged directly or chased for another 30 min at 378C (Wü stner, 2007b;Wü stner and Faergeman, 2008a).…”

“…We tried previously to circumvent this problem in two steps: (1) acquiring a bleach stack by repeated acquisition of DHE-stained specimen and fitting a monoexponential decay function to the fading DHE intensity over the whole image field followed by (2) multiplying the images in a postprocessing step by the reciprocal of this decay function, eventually followed by frame averaging (Markham and Conchello, 2001;Wü stner, 2005;Wü stner and Faergeman, 2008a). This method allowed for some gain in SNR and for applying deconvolution algorithms to small z-stacks of DHEstained cells acquired on a UV-WF microscope system (see Fig.…”