Sample Preparation

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ZEISS Lightsheet Z.1 Sample Preparation

White Paper

ZEISS Lightsheet Z.1 Sample Preparation Author: Flood P.M., Kelly R., Gutiérrez-Heredia L. and E.G. Reynaud School of Biology and Environmental Science, University College Dublin, Ireland Date:

Sept 2013

This paper describes theoretical and practical aspects of sample preparation for Light Sheet Fluorescence Microscopy (LSFM). We present general rules for sample handling and mounting, as well as guidelines with respect to the best preparative technique to use, taking into account sample type, structure and properties. Step by step protocols and recommended materials for ZEISS Lightsheet Z.1 samples are included. These protocols cover sample preparation ranging from micrometer-sized fluorescent beads to millimeter-sized insects, providing detailed information relating to preparation and observation techniques. Finally, this paper identifies the main artifacts and problems that can result from the preparation techniques. CONTENTS (click text to go to page): 1 INTRODUCTION







3

3

SPECIFIC EXAMPLES OF SAMPLE

PREPARATION

22 22

2

SAMPLE MOUNTING FOR LSFM

5

3.1

Preparation of Fluorescent Beads

2.1

The perfect Sample for LSFM

5

3.2

Preparation of a Medaka Fish Embryo

2.2

Holding the Sample

6

(Oryza latypes)

2.2.1

Embedded Samples

8

3.3

2.2.2

Hanging Samples

12

(Drosophila melanogaster)

2.2.3

Enclosed Samples

13

3.4

2.2.4

FEP Tubing

15

(Arabidopsis thaliana)

24

2.3

Materials and Equipment

15

3.5

Imaging Cell Cysts in an Extracellular Matrix Gel

25

2.3.1

Sample Chambers

15

3.6

Immunostaining and Preparation of

2.3.2

Molding and Mounting Supports

16



MDCK Cell Cysts

2.3.3

Sample Holder

18

3.7

Preparation of a Whole Mount of a Mosquito

2.3.4

Gels and Polymers

18

(Anopheles gambiae)

2.3.5

Hydrogel Preparation

19

2.4

Fixation and Fixatives

20

4

TIPS, TROUBLESHOOTING AND

2.5

Stains and Staining

20



ADDITIONAL INFORMATION

2.5.1

Choosing a Fluorescent Label

20

4.1 Tips

28

2.6

Antifading Agents

21

4.2 Troubleshooting

28

2.7

Cleaning, Labelling and Storing Samples

21

4.3

Suggested Additional Sources of Information

30

4.4

References and Further Reading

31

23

Preparation of a Fly Pupa 24

Preparation of a Plant Root

5 Index

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