Live Imaging of the Drosophila Pupal Eye Using Fluorescence Microscopy

Abstract

Source: Hellerman, M. B., et al. Live-imaging of the Drosophila Pupal Eye. J. Vis. Exp. (2015)

The video demonstrates a live-imaging technique to study eye patterning in a Drosophila melanogaster pupa. Green fluorescent protein (GFP)-tagged markers enable visualization of cellular organization and differentiation during the formation of the developing eye structure.

Protocol

1. Fluorescence Imaging

1. Image the Drosophila pupal preparation using a fluorescent microscope. Every 7 min capture serial sections through the apical domain of the eye neuroepithelium, in the region of the adherens junctions.
   NOTE: a) Appropriate serial sections are usually 4-5 per z-stack, comprised of 0.2 μm z-steps. b) Irregularities in the topology of the epithelium, including mild bumps and folds, may make it challenging to acquire in-focus images of large regions of tissue. While imaging, one may find part of an area of interest to be in focus and a neighboring region to be out of focus. Including a small droplet of distilled water between the pupal eye and coverslip can eliminate some acute tissue folds, but not the mildly uneven topology characteristic of the early stages of pupal eye morphogenesis. In these instances, oversample the tissue by extending the z-stack until in-focus slices are acquired for the entire field. c) Capturing serial sections every 7 min should enable live imaging for 3 to 4 hr without a significant reduction in the intensity of green fluorescent protein (GFP) fluorescence that may compromise image quality. Shorter time intervals may reduce the total time of imaging.
2. Continue imaging for 3 to 4 hr. Do not use an automatic focus and time function that is available on many fluorescent microscopes since pupal growth and pumping of the hemolymph moves the position of the retina and vigilant re-focusing is required every 14-21 min.

NOTE: Imaging beyond 4 hours may slow or stall morphogenesis of the eye.

3. Use appropriate deconvolution software to reduce the background and enhance the contrast of the serial section images. For each z-stack file, perform the following in the Leica application suite advanced fluorescence (LAS AF) software: Navigate to the Tools panel, select 3D Deconvolution, and click Apply.
4. Generate a maximum projection (MP) image for each deconvoluted stack file: Navigate to the Tools panel, select 3D Projection and click Apply.
   NOTE: The MP algorithm may fail to generate a uniformly in-focus image for tissue that has been oversampled.

2. Image Processing

1. Automatic Image Alignment:

NOTE: The live Drosophila tissue will shift and grow throughout the imaging process. Consequently, the centers of images gathered at successive time points may not correspond to the same point in the Drosophila tissue. In addition, the entire retinal disc rotates about 30° between ~21 and 23 hr after puparium formation (APF). To highlight individual cell behaviors and reduce distractions caused by organismal growth, align each MP image. While this can be performed manually, the image editing software used here has built-in algorithms that can expedite the process.

2. From the File tab of the main menu, open Scripts and select Load Files into Stack.
3. Import the MP image files into the Load Layers panel and select OK. Make sure that the Attempt to Align Source Images option is not selected automatically.
4. NOTE: If this option is selected, the software may use an alignment algorithm that distorts the image data.
5. Once all MP files have loaded into the Layers pane, make sure that all images are in chronological order such that the earliest time point is at the top of the layer stack. If the layers are not in the correct order, drag and drop them until they are ordered correctly.
6. Select Auto-Align Layers from the Edit tab of the main menu. Choose Reposition and click OK.
7. If the Auto-Align algorithm fails to orient the frames to a relevant focal point, make minor adjustments using the Move tool.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GMR-GAL4; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP ; + / SM5-TM6b
UAS-dcr-2; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP; + / SM5-TM6b
25 x 75 x 1 mm glass microscope slide	Fisher Scientific	12-550-413
22 x 40 mm glass coverslip	VWR	48393-172
Forceps	Fine Science Tools	91150-20
Whatman 3 mm chromatography paper	Fisher Scientific	05-713-336
Vaseline	Fisher Scientific	19-086-291
30 ml syringe	Fisher Scientific	S7510-30
Adobe Photoshop CS5	Adobe
Leica TCS SP5 DM microscope	Leica Microsystems
LAS AF Version 2.6.0.7266 microscope software	Leica Microsystems