1 | Grow transgenic animals expressing the fluorophore in cells or tissues of interest on 60mm plates seeded with OP50, at the standard temperature of 20oC or other appropriate temperature (depends on genetic background and other experimental considerations such as the temperature sensitivity of animals examined). Unless an environmentally controlled chamber or room is available for the epifluorescence, compound light microscope the procedure of fluorescence photobleaching and recovery is performed at ambient temperature. Allow animals to equilibrate at this temperature for 2-3 hours before photobleaching.
2 | The procedure can be performed either directly on a plate (method A) or on a coverslip (method B). When examining worms that express the fluorescent marker in individual cells, photographs of moving worms are hard to analyze. In this case follow method B, below. For worms expressing the fluorescent marker protein globally or in many tissues, the more convenient method A may be applicable. To limit animal mobility, mild anaesthetics that do not interfere with metabolic processes such as levamisole can be used. Avoid the commonly used sodium azide, which blocks the mitochondrial respiratory chain, perturbs energy production and is likely to interfere with the fluorescence recovery process by hindering protein synthesis. An alternative strategy is to use suitable genetic mutants with limited mobility (uncoordinated, paralyzed). Care should be taken when designing the experiment to avoid genetic backgrounds that are likely to have an effect on protein synthesis. It may also be helpful to use the dominant rol-6(su1006) allele as co-transformation marker (plasmid RF4), when constructing transgenic lines. This allele causes animals to roll instead of moving sinusoidally, which confines them in a relatively small area of the plate.
A) Transfer single worms to fresh 35mm plates, seeded with OP50 bacteria. A small bacterial spot in the center of the plate will make localization of the worm easier, while focusing the sample.
B) Spot a drop of 15ul M9 buffer on a microscope slide and place the worm on the drop with the help of an eyelash glued on a pick. Add a cover slip on the top of the drop. The weight of the cover slip is sufficient to keep the worm immobile during the procedure, without damaging it. After photobleaching, recover the worm by adding 100ul of M9 at the edges of the cover slip and sliding off the cover slip. The worm is returned to an OP50-seeded NGM plate for recovery.
3 | Photograph single animals before photobleaching using a camera attached to the microscope (e.g. Axio Cam HR, Carl Zeiss). Images of fluorescent cells or tissues of interest are collected. Imaging parameters such as microscope and camera settings (lens and magnifier used, filters exposure time, resolution, etc.) should be documented.
4 | Perform photobleaching. Use an epifluorescence, compound light microscope (e.g. Axioskop 2 Plus, Carl Zeiss) equipped with a high power light source (HBO 100; 100 Watt mercury arc lamp; Osram, Munich, Germany) and the appropriate excitation/emission filter sets to photobleach the animal. For the applications described here 10 minutes of photobleaching reduce the initial emission intensity adequately (to within 10-30% of pre-bleach levels). The light intensity and the duration of the bleaching period are adjusted accordingly for the specific fluorophore, animal stage and cell or tissue under examination. Investigators should experimentally determine the appropriate duration of irradiation required to reach the extent of photobleaching, appropriate for different specimens.
5 | Photograph each animal immediately after photobleaching. Collect several images of cells or tissues of interest. Move animals to fresh OP50-seeded NGM plates. Recovery timing starts at this point.
6 | Follow fluorescence recovery by photographing animals at defined time points. We use 1 hr intervals between successive photography sessions. A suitable time interval can be determined for each experimental application. Collect several images of cells or tissues of interest. Similar to step 5 above, it is critical that all imaging parameters (microscope and camera settings) are kept identical to those initially set in step 3.
7 | Prepare a stock solution of cycloheximide by diluting in water to a concentration of 10mg/ml. Keep refrigerated. Add cycloheximide on top of OP50-seeded, 35mm NGM plates to 500ug/ml final concentration in the agar volume and allow plates to dry.
8 | Transfer animals on cycloheximide-containing plates and incubate for 2 hours, at the growth temperature. Prepare animals for photobleaching as in step 2 (A) or (B). Return worms in cycloheximide-containing plates during fluorescence recovery.
9 | Process images acquired in steps 3, 5 and 6 with ImageJ to determine the average and maximum pixel intensity for each image of fluorescent cell or tissue of interest in the collected photomicrographs. For each cell, tissue or animal, images should be acquired or converted to a pixel depth of 8 bit (256 shades of grey). To analyze the area of interest manually, use the “freehand selection” tool to enclose the fluorescent area. Select the “measurement” command via the “analyze” drop-down menu to perform pixel intensity analysis.
On occasion (area continuity, high contrast ratios), selection of the fluorescent area can be done automatically. Select “adjust” and then the “threshold” command, within the “image” drop-down menu of ImageJ. Adjust the threshold until the region of interest is marked. Within the “analyze” drop-down menu, select the “analyze particles” command. By selecting “outlines” at the “show” drop-down menu, check whether measurements correspond to the area of interest.
Average and maximum pixel intensity values are collected for each transgenic line and grouped into “Pre-bleach”, “Bleach” and “Recovery(n)”, where n is the time interval after photobleaching. Statistical analysis of data is carried out using the Microsoft Office 2003 Excel software package (Microsoft Corporation, Redmond, USA) and the Prism software package(GraphPad Software Inc., San Diego, USA). The Student’s t test is used for two-way comparisons with a significance cutoff level of p<0.05. Analysis of variance (ANOVA) is used for comparisons of multiple groups of values, followed by Bonferroni-corrected multiple-group comparison t tests.