Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Summary

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Abstract

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Introduction

Fluorescence microscopy is one of the most essential imaging techniques in the life sciences, especially in cell and developmental biology. In confocal fluorescence microscopes¹, which are state-of-the-art for three-dimensional fluorescence imaging since the mid-1990s, the same lens is used for fluorophore excitation and emission light detection. The illumination laser beam excites all fluorophores along the illumination/detection axis and the respective out-of-focus signal is discriminated prior to detection by a pinhole. Hence, for each two-dimensional image, the entire specimen is illuminated. Consequently, for each three-dimensional im....

Protocol

1. Preparatory work

1. Choose an illumination lens/detection lens/camera combination for the LSFM that suits the scientific question and set up the microscope. The size of the field of view is the quotient of the camera chip size and the magnification of the detection lens. The illumination lens should be chosen so that the entire field of view is covered by a roughly planar light sheet³⁴. Three recommended combinations are listed in Table 1.
2. To prepare agarose .......

Discussion

One of the exclusive application areas of LSFMs is developmental biology. In this discipline, it is of importance to look at living specimens, otherwise morphogenetic processes cannot be described in a dynamic manner. An experimental framework for the simultaneous live imaging in sample chamber-based LSFMs, as described here, is convenient for two major reasons.

Ambient variance, which is unavoidable in sequential live imaging, can be addressed pro-actively. In insect embryo-associated live im.......

Materials

Name	Company	Catalog Number	Comments
6-well plate	Orange Scientific	4430500
24-well plate	Orange Scientific	4430300	Only for live imaging involving Tribolium
35-mm Ø Petri dish	Fisher Scientific	153066	Only for live imaging involving Drosophila.
90-mm Ø Petri dish	Fisher Scientific	L9004575
100-µm mesh size cell strainer	BD Biosciences	352360
250-µm mesh size sieve	VWR International	200.025.222-038	Only for live imaging involving Tribolium
300-µm mesh size sieve	VWR International	200.025.222-040	Only for live imaging involving Tribolium
710-µm mesh size sieve	VWR International	200.025.222-050	Only for live imaging involving Tribolium
800-µm mesh size sieve	VWR International	200.025.222-051	Only for live imaging involving Tribolium
405 fine wheat flour	Demeter e.V.	SP061006	Only for live imaging involving Tribolium
commercially available Drosophila medium	Genesee Scientific	66-115	Only for live imaging involving Drosophila / Custom-made Drosophila medium may also be used
fluorescent microspheres, 1.0 µm Ø	Thermo Fisher Scientific	T7282
inactive dry yeast	Genesee Scientific	62-108	Only for live imaging involving Tribolium
low-melt agarose	Carl Roth	6351.2
narrow vials	Genesee Scientific	32-109	Only for live imaging involving Drosophila
small paint brush	VWR International	149-2121
sodium hypochlorite (NaOCl), ~12% active Cl	Carl Roth	9062.3	Caution: sodium hypochlorite is corrosive
whole wheat flour	Demeter e.V.	SP061036	Only for live imaging involving Tribolium / United Kingdom: wholemeal flour
wide vials	Genesee Scientific	32-110	Only for live imaging involving Drosophila