Leica Microsystems M165 FC Application Note

WORK MORE EFFICIENTLY IN DEVELOPMENTAL

BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

AUTHORS

James DeRose

Scientiﬁc Writer, Stereo & Digital Microscopy Marketing,

Leica Microsystems AG, Switzerland

Adam Cliffe

Sales & Application Specialist, Leica Microsystems, Singapore

Heinrich Buergers*

Product Manager, Life Science Research Stereo Microscopy, Leica

Microsystems AG, Switzerland

*corresponding author: heinrich.buergers@leica-microsystems.com

LIFE SCIENCE RESEARCH TECHNICAL REPORT

From Eye to Insight

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

Introduction

For scientists and technicians working with fruit ﬂies, most often

genus

Drosophila

, this report is intended to give useful information

to help improve daily laboratory work by making the steps of ﬂy

pushing, ﬂuorescent screening, dissection, and documentation/

imaging more efﬁcient. It also details various possibilities for

properly equipping or stocking a ﬂy lab.

Drosophila melanogaster

has been a model organism for genetic

studies for more than 100 years and continues to be widely

used today for developmental biological research in the areas

of genetics, physiology, pathogenesis, and evolution [1,2]. It is

relatively easy and inexpensive to care for in large numbers. It

breeds quickly, lays many eggs, and has a short life cycle of just

2 weeks [1,2]. The fruit ﬂy has only 4 pairs of chromosomes [3],

many of which are large, making it easy to locate mutations [4]

and deletions [5]. Its entire genome [6] has been sequenced and

many mutations are available for use. The homology of cancer

and disease related genes found in both the genomes of

melanogaster

and humans is signiﬁcant [7,8].

There are several common steps when doing routine work with

fruit ﬂies, often

D. melanogaster

1. “ﬂy pushing”, i.e., the basic sorting of ﬂies by stereo

microscopic observation of visible markers, normally done day

to day;

2. ﬂuorescent screening, similar to ﬂy pushing, but normally

involves observing the embryos or larvae with ﬂuorescence

stereo microscopy to separate successfully transfected, gene-

expressing ones from those which are non-expressing;

3. dissection performed via a stereo microscope;

4. documentation or imaging, often done with confocal

or compound microscopy, but high performance stereo

microscopy is also possible.

For clarity, in this text the sequence of steps mentioned above for

routine fruit ﬂy work will be referred to as “workﬂow” (refer to Fig.1).

In many cases, the workﬂow is not a linear “ﬂow” of work steps.

Workow for Fruit Fly

(D. melanogaster)

Fly Pushing

Fluorescent

Screening

Dissection

Documentation/

Imaging

Work Steps

Figure 1: Workﬂow for describing a non-linear sequence of work steps typically done in fruit ﬂy laboratories.

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

Key Considerations for Optimizing Workow Efciency with Fruit Flies

There are several important points which must be considered when attempting to develop a more efﬁcient workﬂow:

Identiﬁcation of the critical tasks for a speciﬁc study, for example, sorting fruit ﬂies for selective breeding using visible markers or using

ﬂuorescent protein markers;

Time spent for each step and the overall number of steps: generally the workﬂow is a daily repetitive, tedious, time-consuming work, which

must be done reliably and accurately in order to maintain the required pedigree of the fruit ﬂy;

Ordinary stereo microscopy: better efﬁciency with good illumination, resolution, and ergonomic approach;

Specimen characteristics: in general ﬂies and other insects are largely composed of the polysaccharide chitin and various protein molecules

which can contribute to autoﬂuorescence (unwanted background ﬂuorescence signals from the specimen) [9] and decrease the signal-to-noise

(S/N) ratio during observation with ﬂuorescence microscopy;

Fluorescent stereo microscopy: faster workﬂow with quick switching between ﬁlters and between ﬂuorescence and bright ﬁeld, as well as

rapid zooming in and out;

Dissection: ﬂexible illumination and microscope stand options provide an ergonomic workﬂow; and

Documentation/imaging: recording mutant phenotypes, ﬁlming behavioral changes, measuring anatomical features, such as wing or leg sizes,

and time lapse imaging of cell migration (frequently done with confocal microscopy). Documentation with stereo microscopy often requires

different setups of the digital color camera.

Stereo Microscope Systems for Fly Pushing and

Fluorescent Screening

Fly Pushing

Fly pushing refers to the daily sorting of ﬂies by anatomical

characteristics and phenotype [10]. Flies are generally

anesthetized with CO

and placed on a CO

perfused pad for

sorting. Normally, sorting is done with stereo microscopy.

There are several types of stereo microscopes available from

Leica Microsystems which can be used with fruit ﬂies, usually

melanogaster

, for ﬂy pushing, notably the Leica M50, M60, and

M80 CMO (common main objective) stereo microscopes [11].

Some of the advantages that these microscopes offer:

> Optics with extremely high light transmission, enabling many

details to be seen at a glance with no need to use extra bright

illumination that can potentially create shadows or bright

spots;

Figure 2: At lowest magniﬁcation, the Leica M60 offers a FOV diameter (via the

eyepieces) of 36.5 mm (blue circle), compared to 30.7 mm (red circle) for a Leica

M80. The larger FOV of the Leica M60 can be an advantage for more efﬁcient ﬂy

pushing. However, the Leica M80 offers higher magniﬁcation and better resolution.

The Image of fruit ﬂies was taken with a Leica M60 having a 0.5x objective and

0.63x zoom setting. The original image shows a larger ﬁeld of view (FOV) and was

cropped afterwards.

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

> Eyepieces with a ﬁeld number (FN) of 23 mm, which can provide a

larger ﬁeld of view (FOV)/object ﬁeld (OF), giving the user a quick

overview of the ﬂies over a large area of the pad for fast, efﬁcient

ﬂy pushing;

N.B. the Leica M80 has a smaller FOV than the M50 and M60

(refer to Fig. 2 on page 3);

> The Leica M80 achieves better resolution (maximum numerical

aperture [NA] of 0.206) than the M60 and M50 (maximum NA

of 0.15) - higher resolution facilitates the identiﬁcation of subtle

phenotypes, such as bristle morphology;

> For the wider FOV of the Leica M50 and M60, depending on

the application, plan achromatic objective lenses are normally

recommended, while achromatic ones are sufﬁcient for the M80

[12,13];

> The Leica M50, M60, and M80 can be equipped with the Leica

LED2000 or LED2500 all-in-one microscope stand and illumination

system which keeps the table top working space free of cables and

external light sources

Figure 3: Typical fruit ﬂy lab where Leica M60 and M80 stereo microscopes

with incident lighting (spotlight illumination) are used for ﬂy pushing (sorting

of the ﬂies).

Figure 4: Fly pushing station showing a Leica M80 stereo microscope with

gooseneck spotlight illumination, tube with CO2 to anesthetize ﬂies inside

vials (1), ﬂypad with CO

to keep ﬂies anesthetized during sorting (2), rubber

bands to bundle tubes together, and stickers or tape to label tubes.

N.B. normally fruit ﬂies are kept in hundreds of plastic vials and

these ﬁll up the working space, occasionally cables from equip-

ment can get bumped by users and accidentally knock some vials

onto the ground;

> Ergonomic accessories for stereo microscopes, such as Leica

ErgoWedges, ease the strain of repetitive work;

N.B. when microscope users in the laboratory feel more

comfortable while ﬂy pushing all day long, they will be more

productive, and ergonomic accessories can be retroﬁt economically

to older model Leica microscopes;

> Compact LED light sources, which are attached to the stand, allow

the stereo microscopes to be easily moved for cleaning and give

the users more free bench space.

The photos below show a typical

Drosophila

lab with stereo microscopes for ﬂy pushing (courtesy of T. Hummel, Dept. of Neurobiology,

University of Vienna, Austria).

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

Useful Information (Hints and Tips)

Size of Microscope Base Plate

Microscope footprint is a key consideration for every laboratory.

Fly pads for CO

anesthetization are available in different sizes, but

all tend to fall off the microscope base during ﬂy pushing, as the base

can be too small.

Solutions to this potential problem are:

> Using medium size bases; or

> Alternatively one can use small swingarm (boom) stands and work

directly on the bench (refer to Fig. 5). Leica’s LED3000 spotlight

illumination (SLI) with goosenecks can be mounted directly on the

swingarm stand and all lighting controls are directly accessible on

the goosenecks, so they do not crowd the bench.

Figure 5: Photo of Leica stereo microscope mounted on a swingarm (boom)

stand with ﬂypad underneath. Courtesy of B. Reversade, IMB, A*STAR,

Singapore.

Advantages versus Disadvantages of Greenough and CMO (Common Main Objective) Stereo Microscopes

for Fly Pushing

Greenough Advantages:

> Normally longer working distances than CMO stereo microscopes; and

> An economic solution.

Greenough Disadvantages:

> Limited modularity; and

> Optical axes of the lens systems (for each eye) converges to a center point where the specimen is placed. The two separate focal planes for

each eye does not allow clear focus for both eyes on a specimen positioned away from the center (refer to Fig. 6 on page 6).

CMO Advantages:

All specimens are in the same focal plane for both eyes, which leads to a larger useable ﬁeld of view at a speciﬁc magniﬁcation value compared

to Greenough stereo microscopes [14,15] and allows:

> larger overview with less need to refocus and move the ﬂy pad around;

> faster identiﬁcation of fruit ﬂies; and

> smoother workﬂow, making ﬂy pushing less tedious.

Figure 6 compares the optics of a Greenough and a CMO stereo microscope.

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

Figure 6: Two ﬂies observed by a stereo microscope: A) and C) Greenough optics where specimens in the center at the point of convergence of the two

objective light beam paths are in focus and give a good 3D impression, but specimens away from the center cannot be in sharp focus simultaneously for both

eyes; and B) and D) CMO (common main objective) optics where the ﬂies are in the same focal plane over the entire ﬁeld of view and in sharp focus for both

eyes, permitting more efﬁcient ﬂy pushing.

A: Greenough Stereo B: CMO Stereo

Top view specimen plane via eyepiece

C: Center area ﬁeld of view:

sharp focus both eyes

D: Entire ﬁeld of view:

sharp focus both eyes

blurry for one

or both eyes

Greenough

objectives

specimen

observation

plane

objective focal

planes left (red),

right (green)

objective focal

and specimen

observation

plane

CMO

(common main

objective)

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

Fluorescent Screening

In many laboratories, special strains of fruit ﬂies, which have

modiﬁed genotypes [16] to express markers such as Green (GFP),

Red (RFP), Cyan (CFP), and Yellow (YFP) Fluorescent Protein, are used

[17,18]. Typically, the ﬂy larvae or embryos are observed and analyzed

with ﬂuorescence stereo microscopy and then selected based on

the presence or absence of ﬂuorescent markers, hopefully in the

expected organ. Using ﬂuorescent protein markers, like GFP, make

the job of sorting non-mutated from mutated larvae much easier.

However, in order to gather sufﬁcient data for analysis, many fruit ﬂy

larvae must be screened and analyzed.

There are several types of stereo microscopes available from Leica

Microsystems which can be used for ﬂuorescent screening of fruit

ﬂies: the Leica M165 FC, M205 FA, and MZ10 F (see example images

in Fig. 7). Some of the advantages that each microscope can offer

users are the following:

Leica M165 FC, M205 FA, or MZ10 F uorescence stereo

microscopes offer y screeners:

Best signal-to-noise (S/N) ratio for ﬂuorescence microscopy due

to the unique triple beam design;

The Leica M205 FA has the best resolution (maximum NA of

0,349), followed by the M165 FC (maximum NA of 0,3), and then

the MZ10 F (maximum NA of 0.25);

The Leica M165 FC and M205 FA use plan apochromatic objective

lenses, while achromatic ones are typical for the MZ10 F;

The Leica M165 FC and M205 FA have encoded optics for reliable

measurements of ﬂy anatomy, e.g., dimensions and surface area of

legs, wings, eyes, etc.;

The Leica M205 FA has the FusionOptics technology [19] for a

visual 3D perception through the eyepieces with the highest

resolution and depth of ﬁeld possible, simultaneously and even

at high magniﬁcation, which is very practical for ﬂuorescent

screening;

The Leica M205 FA is a fully automated ﬂuorescence stereo

microscope and, as a result, also can be used for complex imaging

like z-stacking and multichannel acquisition; and

Ergonomic accessories, such as Leica ErgoTubes, ease the strain of

repetitive work by allowing the height and angle of the eyepieces

to be adjusted for the comfort of the individual user, leading to

higher productivity.

Figure 7:

Fluorescence stereo

microscope image

of anesthetized

Mediterranean fruit

ﬂies recorded with a

Leica M205 FA. The

ﬂies are expressing

RFP (red ﬂuorescent

protein).

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

Dissection

Dissection of fruit ﬂies and their larvae is done to allow more detailed

studies of the organs and tissues [20]. Generally, a high performance

stereo microscope is used for dissection.

For dissection of fruit ﬂies and larvae, the Leica M80 stereo

microscope is often sufﬁcient, but the M125 has higher magniﬁcation

and resolution and a good ﬁeld of view which makes it more practical

for preparation of ﬂy organs, e.g., brains (refer to the example in Fig.

8). Normally, the organs are later imaged using higher resolution

confocal, compound, or stereo microscopy.

Drosophila brain

Figure 8: Image of 3 brains dissected from fruit ﬂies using a Leica M125

stereo microscope. The ﬂy brains are expressing RFP (red ﬂuorescent

protein). The image was taken with a Leica ﬂuorescence stereo microscope.

The brains were oriented as seen for later confocal imaging. Courtesy of L.

Geid and T. Hummel, Dept. of Neurobiology, University of Vienna, Austria.

Documentation/Imaging

Phenotypic characterization of mutations in the eyes, legs, body, and wings of the fruit ﬂy often are documented using encoded stereo

microscopy, normally a higher performance system, such as the Leica M205 FA, M165 FC, M205 A, M205 C, M165 FC, or M165 C.

To take videos for the observation of fruit ﬂy behavior, a digital macroscope like the Leica DMS1000 or DMS300 can be useful, allowing the

capture of high frame rates and large overviews on a relatively small setup. In addition, the Leica Application Suite X (LAS X) software offers

features like extended depth of ﬁeld (EDOF) or image z-stacking for quick, routine ﬂy characterization in 3D. Using encoded Leica stereo

microscopes in combination with LAS X software, all images will be correctly calibrated and thus guarantee accurate measurements of wing

size, leg length, eye surface, and other relevant changes in phenotype.

For many studies, characterization of the fruit ﬂy phenotype must be performed at the subcellular level [21]. Documentation of dissected fruit

ﬂy parts to acquire details at the subcellular level is normally done with a confocal or compound microscope, such as the Leica TCS SP8 or SPE

(confocal) or DM6 (compound).

The Leica M205 FA is fully encoded and automated to allow complex imaging tasks to be done more easily. The encoding and automation

provide reliable measurements at the cellular and subcellular level. The Leica M205 FA uses FusionOptics technology [19] and ergonomic

accessories are available.

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

Conclusion

The fruit ﬂy,

Drosophila melanogaster

, has been a model organism for developmental biology research for more than a century [1,2]. Its genome

is fully sequenced and the disease related genes show appreciable homology to those of humans [7].

The routine workﬂow for fruit ﬂies, usually

D. melanogaster

, involves multiple steps using stereo microscopy:

> ﬂy pushing, daily basic sorting of ﬂies;

> ﬂuorescent screening, observing the embryos or larvae; and

> dissection for later documentation or imaging.

Documentation or imaging of the anatomy or whole organs of fruit ﬂies is usually done with stereo microscopy and parts dissected from them

with confocal or compound microscopy to obtain subcellular details.

This report refers to examples of scientists and technicians working with fruit ﬂies, such as

D. melanogaster

, and shows different possible

setups with a large range of microscopes and accessories. Because the demands of each laboratory can vary widely, a large range of

conﬁgurations and instruments are available to address speciﬁc tasks in the workﬂow or even enable more work steps to be performed by one

instrument. This short report presents recommended workﬂows based upon the experiences of different well-established fruit ﬂy labs and can

be a very useful reference or guidelines when setting up or expanding a ﬂy lab.

Acknowledgements

We would like to thank Laura Geid and Prof. Thomas Hummel, Dept. of Neurobiology, University of Vienna in Austria for supplying photos of his

Drosophila laboratory, as well as, Prof. Bruno Reversade, IMB, A*STAR, Singapore.

WORK MORE EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO MICROSCOPY:

FRUIT FLIES

(DROSOPHILA)

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T +41 71 726 34 34 · F +41 71 726 34 44

www.leica-microsystems.com

LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH. FusionOptics is a trademark of Leica Microsystems (Schweiz) AG registered in Europe.

References / Additional Reading

G. Manning, A quick and simple introduction to

Drosophila melanogaster, Drosophila

Virtual Library

Thomas H. Morgan – Biographical, The Nobel Prize in Physiology or Medicine 1933, The Nobel Foundation, Stockholm, Sweden

Chromosomes, National Human Genome Research Institute (NHGRI), National Institute of Health (NIH), Bethesda, Maryland, USA

Mutation, Talking Glossary of Genetic Terms, National Human Genome Research Institute (NHGRI), National Institute of Health

(NIH), Bethesda, Maryland, USA

Deletion, Talking Glossary of Genetic Terms, National Human Genome Research Institute (NHGRI), National Institute of Health

(NIH), Bethesda, Maryland, USA

Genome, Talking Glossary of Genetic Terms, National Human Genome Research Institute (NHGRI), National Institute of Health

(NIH), Bethesda, Maryland, USA

L.T. Reiter, L. Potocki, S. Chien, M. Gribskov, E. Bier, A Systematic Analysis of Human Disease-Associated Gene Sequences In

Drosophila melanogaster

, Genome Res., vol. 11, iss. 6 (June, 2001) pp. 1114–1125; doi: 10.1101/gr.169101

J. Herrero, How to get all the orthologous genes between two species, Ensembl Project, Cambridge, UK

F. Hoff, How to Prepare Your Specimen for Immunoﬂuorescence Microscopy, Science Lab

10.

Phenotype, Talking Glossary of Genetic Terms, National Human Genome Research Institute (NHGRI), National Institute of Health

(NIH), Bethesda, Maryland, USA

11.

D. Goeggel, The History of Stereo Microscopy – Part III: The 19th Century – Breakthrough of Modern Microscope Manufacturing,

Science Lab

12.

D. Goeggel, The History of Stereo Microscopy – Part II: The 18th Century – Greater Demands are Placed on Optics, Science Lab

13.

Objective Classes, Leica Microsystems

14.

R. Rottermann, P. Bauer, How Sharp Images Are Formed: Depth of Field in Microscopy, Science Lab

15.

D. Goeggel, Factors to Consider When Selecting a Stereo Microscope, Science Lab

16.

Genotype, Talking Glossary of Genetic Terms, National Human Genome Research Institute (NHGRI), National Institute of Health

(NIH), Bethesda, Maryland, USA

17.

C. Greb, Fluorescent Proteins – Introduction and Photo Spectral Characteristics, Science Lab

18.

Sharyn A. Endow, Detecting Gene Expression in

Drosophila

, Application Notes, Leica Biosystems

19.

D. Goeggel, A. Schué, D. Kiper, FusionOptics – Combines high resolution and depth of ﬁeld for ideal 3D optical images, Science Lab

20.

I. Maimon, L. Gilboa, Dissection and Staining of

Drosophila

Larval Ovaries, Jove, doi: 10.3791/2537

21.

J. De Bock, Mosaic Images: Bringing Together What Belongs Together, Science Lab

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