From Eye to Insight

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS

AUTHORS

James DeRose, Ph.D.

Scientific Writer, Applied Microscopy Marketing,

Leica Microsystems AG, Switzerland

Martin Gamerdinger, Ph.D.

Scientific Project Leader, Molecular Microbiology,

University of Constance, Germany

Heinrich Bürgers, Ph.D.

Product Manager, Life Science Research Stereo Microscopy,

Leica Microsystems AG, Switzerland

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WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS2

Abstract

For scientists, technicians, and teachers working with the worm C. elegans in the research lab or classroom, this report is intended to give

useful information to help improve their daly work. The aim is to make the work steps of worm picking, transgenesis, RNA interference,

screening, and functional imaging efficient. It also details the various possibilities for equipping a research worm lab or biology classroom/

teaching lab explaining worm methods.

Introduction

The roundworm Caenorhabditis elegans has been used as a model organism for neuroscience, developmental and molecular biology, and

genetics for over 40 years [1]. It was the first multicellular organism to have its entire genome sequenced [2] and, until the present time, is

the only organism whose connectome, i.e., diagram of neuronal connections in the nervous system, is fully mapped [3]. The worm is a eutelic

organism with a genetically determined number of cells which remain constant at the end of the larval stage and its nervous system contains

302 neurons (nerve cells). The worm is used as a model organism for studies in neurological development, cellular differentiation, apoptosis

(programmed cell death), aging, etc. A large number of genes in C. elegans can function in similar ways to those of mammalians. About 1/3 of

genes in the C. elegans genome are homologous (derived from common ancestors) with some human genes and, as a result, make the worm

useful as a model organism for the study of human-related diseases. The worms are quite easy and fast to cultivate in the laboratory and are

usually kept just below room temperature (15-20°C). Adult worms are about 1 mm in length and less than 75 µm in diameter. They are kept on

agar plates, i.e., an agar gel inside a petri dish, with bacteria (E. coli) as food.

Figure 1: Work steps typically done in worm laboratories. See the text on the following pages for more details.

Methods for Worms (C. elegans)

Worm Picking and Brightfield

Screening

Choose worms for

study, transfer between agar plates

Transgenesis and Microinjection

Inject Transgene

(DNA)

Work Steps

Functional Imaiging

Maintain Stable Line

Check Transgene

Development/

Neuronal Activity/ HighResolution

Observation

RNA Interference (RNAi)

Introduce dsRNA

(siRNA, microRNA)

Fluoro-Screening

Check Transgene Expression or RNAi

gene supression

Worm Biology

Biochemistry/

Genetics/ Molecular Biology

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS 3

General work steps for C. elegans

There are several common steps for routine laboratory work with

worms (Fig.1):

1. Picking worms [4];

2. a) Transgenesis [5,6]: microinjection of a transgene into the worm

to modify its genome;

2. b) RNA interference (RNAi) [7]: introduction of double stranded

(ds) RNA into the worm to inhibit gene expression;

3. Screening: assessing worms to detect successful RNAi or

transgenesis;

4. Study the molecular biology, biochemistry, genetics of the

worm with various methods;

5. Documentation or functional imaging of worms, often done

with confocal or compound microscopy.

Figure 2: Photos of a well-established C. elegans laboratory (Molecular Microbiology, University of Constance, Germany). Flasks, petri dishes, electrophoresis equipment, and centrifuges are commonly used for worm cultivation and analysis.

Key considerations for optimizing work efficiency

Optimization of the work efficiency will depend on application and use, namely whether laboratory research or teaching in a classroom.

Worm cultivation and imaging in general

There are several points to consider when cultivating, picking, screening worms, etc.:

• avoid refocusing when changing agar plates → same amount of agar (same height ) in each one

• get sharper microscope images of worms → lower concentrations of bacteria (food) on agar

• reduce fluorescence background (autofluorescence) for worm imaging plates → with a thin agar layer and no peptone [4]

• worms must be distinguished from agar → good contrast is important with transmitted light

• detect a fainter fluorescence signal for better image results → good fluorescence signal-to-noise (S/N) ratio and dark background critical

• ensure good contrast and high resolution when imaging worms at higher total magnification (above 60x)

The work steps shown on page two are not necessarily done in a

linear sequence, i.e., a non-linear “workflow”. First, worms must

be selected and “picked”, next comes the manipulation of gene

or protein expression, followed by breeding worms for a period of

time. Then the worms are distinguished by phenotype (observable

characteristics) to find those with the sought-after traits. If there are

too few of these worms, the previous steps may be repeated. When

there is a sufficient number of worms having the traits of interest,

they are “picked” again, immobilized, and finally functional imaging

is performed.

The photos below (Fig. 2) show a typical worm lab at the Dept. of

Molecular Microbiology of the University of Constance in Germany.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS4

Worm picking

Research labs

When picking suitable worms and preparing them for genetic or

biochemical analysis, often the worms are selected using a stereo

microscope and picked with a small platinum wire or eyelash (Fig.

3). Then, for genetic modification via microinjection in the worms‘

gonads, they are placed onto a small, dry agar pad on a glass slide

(Fig. 3).

Classroom and teaching laboratory

Teaching requires different considerations than research applications. The risk of contaminating the microscope optics with the agar or worm

picking utensils needs to be reduced. The Leica S6 stereo microscope [8] for instance, offers a convenient working distance, high magnification,

and a large depth of field to help cope with those challenges and work efficiently. In combination with the LED 2500 illumination [9] (Fig. 4)

the Leica S6 provides a good solution for worm picking in classrooms and teaching laboratories. Inexperienced students tend to prefer high

magnification over high resolution. So eyepieces with the higher magnification of 16x are recommended instead of the more common ones

with only 10x.

Figure 3: Wooden stick with eyelash glued to its end used for worm „picking“ from the agar plate surface (left). Agar gel pad on a glass slide used for worm microinjection (right).

Worm screening in Brightfield

Efficient screening of worms for faster throughput can be achieved with several stereo microscope systems from Leica Microsystems. The

Leica S6 routine stereo microscope [8] with the LED2500 light stand, or small illumination subbase is very practical for worm screening [9]. It

allows an even higher magnification and resolution when operated with the 1.6x front lens accessory. Due to its compact design, the S6 also

takes up less space in the laboratory. Worm images with high contrast, even at low magnification, are easily made with these microscopes.

They are especially good solutions for teaching staff.

Figure 5: Image (left) of C. elegans on agar recorded with a Leica S6 and illumination subbase (right). Image taken with one-sided darkfield illumination using the diffuse reflection mirror of the subbase to enhance contrast of the worms.

Figure 4: Image (right) of C. elegans on agar recorded with a Leica S6 and LED2500 light stand (left). Sharp contrast in the image allows the worms to be easily distinguished from the agar.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS 5

Comparison of Advantages: Light Base and Subbase for Leica S6Leica LED 2500 light base Leica Illumination subbase

• Simplifies specimen handling, due to enough space to hold

multiple agar plates at the same time, minimizing the chance

that a plate drops off inadvertently

• Practical for doing worm picking, microinjection, etc., with

sufficient place for users’ hands to work around and under the

objective lens

• Clean setup for student courses, because there are no external

lamps, cables, nor equipment that can fall off the base

• Good contrast and even illumination for specimen imaging due to

a centered LED

• Easy storage when not in use, as the entire microscope setup

easily fits onto a closet shelf

• Strong image contrast: both brightfield and one-sided darkfield

illumination

• Simple to operate, so ideal for teaching and training

• Small footprint: the entire microscope setup takes up a small

space on the table

Figure 6: Image (left) of C. elegans on an agar plate taken with the Leica M80 using a TL3000 ST light base (right). Darkfield helps to better view the worms.

Efficient worm screening can be also achieved with the Leica M80 stereo microscope [10] using a TL3000 ST light base (Fig.6) [11]. The Leica

M80 has a common main objective (CMO), so there are no tilted focus planes and the entire field of view is in sharp focus for both eyes.

Also the system is modular and allows you to flexibly add, exchange or replace components very quickly. If specimen image observation by

multiple users simultaenously is desired, along with the recording of high resolution images for reports, then a digital camera can be installed.

For example, a Leica MC 170/190 HD camera [12] having 5 or more megapixles produces both high definition (HD) live and recorded images.

The table below lists the different advantages for worm screening in brightfield with the Leica S6 microscope when using either the Leica

LED2500 light base or subbase illumination.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS6

Transgenesis, Microinjection, and RNAi

There are several ways to alter the gene/protein expression of a worm. Stable and transient gene modification is carried out with microinjections

into the worm. The two main methods are: transposons [13], also known as jumping genes, or the CRISPR/Cas9 [14] system. The transgenic

yield with CRISPR is lower and requires one generation cycle for selection, but the transgene insertion is more stable. The transposon method

is efficient with higher yields, requiring fewer worms to be injected, but it is not always clear where the transgene integrates into the genome.

Once worms have been placed onto a dried agar pad on a glass slide, it is important to work quickly, otherwise the worms may desiccate and

die during the procedure. To slow down desiccation of a worm, normally it is covered with oil, such as halocarbon or paraffin [6]. Typically, the

microinjection should be completed in less than 5 minutes. The slide with agar pad is placed onto the stage of an inverted microscope, a worm

is located, and the transgene (DNA) is injected into the distal gonad.

Then the worm is covered with recovery buffer solution [6], picked with a platinum wire or eyelash and placed onto an individual agar plate

for further cultivation and propagation. This work step is usually carried out on a manual, inverted compound microscope equipped with

differential interference contrast (DIC) [15] or integrated modulation contrast (IMC) illumination [16], such as the Leica DMi8 (Fig. 7) or the DM

IL LED [17]. It is strongly recommended to have an anti-vibration setup under the microscope for higher yield. For precise positioning of a needle

for microinjection, it is practical to use a high precision, 3-axis, oil manipulator, such as one from Narishige Instruments.

RNA interference (RNAi) is used to alter protein expression at

the translation level. It is carried out by feeding the worms with

transgenic bacteria expressing double stranded (ds) RNA. The worms

ingest the dsRNA, it enters into all of the worm’s cells (except the

neurons), it gets processed by the cellular biochemistry, and then

short interfering (si) RNA is produced. Once present in the cells, the

siRNA alters protein expression.

Figure 7: Leica DMi8 inverted compound microscope with a customized oil-micromanipulator used for microinjection into the worm's gonads.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS 7

Transgenic worm breeding and fluorescence screening

After injection, the worms are further cultivated and the next

generation is used for experiments. The worms are kept at 20° C for

propagation. Desiccation of the worms must be avoided, so the agar

plates are usually stored in plastic boxes. To get very high numbers of

worms, liquid cultures in an Erlenmeyer flask are a second possibility.

Approximately five worms are sufficient for a western blot analysis

(identify proteins) [18]. However 100,000 worms are required for

differential gradient centrifugation to separate cellular organelles

and proteins by density [19].

Because the transgenes are usually combined with green fluorescent

protein (GFP) [20] they can be selected using fluorescence stereo

microscopes. Other fluorescent markers like DsRed [20] can be toxic

at high expression levels, so usually GFP is the marker of choice.

Worms have to be located using brightfield and Rottermann contrast

(large field of view [FOV]), checked for a fluorescence signal, and

then picked for additional experiments. For imaging, younger

worms are generally preferred as older ones usually exhibit higher

autofluorescence, especially in the gut. The chosen worms are

placed onto special, small agar plates with no peptone nor bacteria

to minimize the autofluorescence. The agar layer is often very thin to

reduce the autofluorescence even further.

Efficient fluorescent screening of worms can be done with the Leica

M165 FC [21] or MZ10 F [22] fluorescence stereo microscope using

the TL4000 RC [11] light base (Fig. 8).

Advantages:

• Single cells of a worm are easily distinguished, due to the many

illumination contrast possibilities available with the Leica M165

FC/TL4000 setup.

• Fast switching of the contrast for advanced documentation and

imaging makes the Leica M165 FC or MZ10 F/TL4000 combination

very practical and efficient for users.

• Intense fluorescence signal due to highest signal-to-noise (S/N)

ratio with TripleBeam technology [23] → separate beam paths for

excitation and observation.

Figure 8: Image (left) of C. elegans taken with the Leica M165 FC using a TL4000 RC light base (right). Rottermann contrast enables the worms on the agar background to be cleary visible.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS8

A video of C. elegans worms on an agar plate can be viewed via YouTube, just click on the video below. Only the emitted light from fluorescence

is detected, no other illumination is used (dark background). Most worms seen in the video show some autofluorescence in the gut [24]. Only

one worm is double positive and also shows a bright signal from the fluorescent protein mCherry [20] expressed in its pharynx. The video is

recorded with a Leica M205 FA fluorescence stereo microscope using a Leica DMC4500 digital camera. The video playback speed is slightly

faster than the original recording speed, so the worms may appear to move faster than in reality.

Important considerations for fluorescence screening

• To achieve the highest contrast and resolution, a transmitted light base with its sophisticated construction acts like a light condenser at

high magnification.

• Little or no background light “noise” for fluorescence imaging is produced by the Leica TL bases

• Better contrast and higher resolution images at high magnification when using the Leica TL4000 RCI [11] base with the concave side of its

double-sided mirror.

• Avoid heating the worms when undesired using the Leica TL4000 RC base with external light source.

• For the case of high autofluorescence, reducing fluorescence excitation intensity will increase the S/N ratio. In many cases, the fluore-

scence signal from the worms is maximal and cannot increase more, but the background always can.

• Depending on the budget and demands of individual users, the Leica TL3000 ST, TL4000 BFDF [11], and TL4000 RC light bases can all be

used.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS 9

Functional imaging

Confocal and compound microscopes, such as the Leica TCS SP8 or TCS SPE confocal system [25] and the Leica DM6 or DM2500 upright

compound microscope [26], are normally used for imaging and documentation of worms to obtain high resolution images of subcellular and

macromolecular structures.

For confocal imaging, the worm is expressing fluorescent proteins, i.e. GFP, red fluorescent protein (RFP, mCherry, or DsRed), yellow fluorescent

protein (YFP), or cyan fluorescent protein (CFP) [20], as mentioned above (Fig. 9 and 10).

Confocal microscopes with the upright configuration are preferable, because the worms are on the top of the agar layer in the plates. The

worms move relatively quickly, so they are usually anesthetized by the drug Levamisol and then a coverslip is placed over them for observation

by liquid immersion objectives.

Young worms in the larval stage can be very sensitive to light, so using the confocal resonant scanner [27], due to the short dwell rate, can

reduce the problem of phototoxicity to a minimum. The resonant scanner allows for long term observation of C. elegans larvae without

noticeable damage.

Figure 9: Brightfield (left) and confocal (right) image of the pharynx of C. elegans expressing GFP. Both images were taken with a Leica TCS SP8 confocal microscope system.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS10

Figure 10: Confocal image (Leica SP2) of the brain and nervous system of C. elegans (viewed from ventral side of worm). The neuron cells are expressing the fluorescent proteins CFP, GFP, YFP, and DsRed, and additionally labelled with the white lipophilic tracer dye DiD. Image courtesy of H. Hutter, Dept. of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada [28].

Summary and Conclusions

Common methods for C. elegans [29] involve multiple work steps using stereo, compound, or confocal microscopy. The demands of a worm lab

can vary. Several configurations and instruments can be used to address specific tasks done in the work steps.

Below is a list of commonly used microscope solutions:

• Worm picking is often done with a Greenough stereo microscope, such as the Leica S6, using a transmitted light base producing high

contrast, e.g. the Leica TL3000 ST;

• Transgene microinjection is achieved with an inverted compound microscope, such as the Leica DMi8, using a micromanipulator to the

position the needle and an injector for the DNA. In addition, integrated modulation contrast is the illumination method of choice;

• Fluorescence screening can be accomplished with a range of fluorescence stereo microscopes, e.g. the Leica MZ10 F or M165 FC, using a

transmitted light base achieving very good contrast, such as the Leica TL4000 RC;

• Brightfield screening is usually performed with a common main objective (CMO) stereo microscope, such as the Leica M80, having a

transmitted light base with high contrast, for example the TL4000 RC ;

• Functional imaging, i.e., the characterization of transgenic worms for studies like cellular differentiation, apoptosis (programmed cell death),

aging, neuronal activity, etc., is done with upright compound or confocal microscopy, such as the Leica DM6 or TCS SP8;

• Immobilized worms can also be imaged at reasonable resolution with a higher performance fluorescence stereo microscope, such as the

Leica M205 FA, and transmitted light base, e.g. TL5000 Ergo [11];

• To image worm embryos, a confocal resonant scanner is recommended for low phototoxicity; and

• For classrooms, instruction laboratories, and training, the microscope setup requirements can vary from those for research laboratories.

This short report can be a useful reference or guidelines when setting up a worm lab and explain the various instruments needed to carry out

the necessary work steps efficiently.

WORK EFFICIENTLY IN DEVELOPMENTAL BIOLOGY WITH STEREO AND CONFOCAL MICROSCOPY: C. ELEGANS 11

Acknowledgement

We would like to thank Prof. Harald Hutter, Dept. of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada for supplying the

confocal image of the C. elegans brain and nervous system.

Additional Reading

1. S. Brenner, Nature’s Gift to Science, Nobel Prize Lecture (December, 2002) The Nobel Foundation

2. The C. elegans Sequencing Consortium, Genome Sequence of the Nematode C. elegans: A Platform for Investigating Biology, Science,

vol. 282 (1998)

3. S.W. Emmons, The beginning of connectomics: a commentary on White et al. (1986) ‘The structure of the nervous system of the

nematode Caenorhabditis elegans’, Philosophical Transactions of the Royal Society B, vol. 370, iss. 1666 (2015), DOI: 10.1098/

rstb.2014.0309

4. T. Stiernagle, Maintenance of C. elegans, Wormbook: The Online Review of C. elegans Biology (2006)

5. P.J. Schweinsberg, B.D. Grant, C. elegans gene transformation by microparticle bombardment, Wormbook: The Online Review of C.

elegans Biology (2013)

6. T.C. Evans, Transformation and microinjection, Wormbook: The Online Review of C. elegans Biology (2006)

7. J. Ahringer, Reverse genetics, Wormbook: The Online Review of C. elegans Biology (2006)

8. Leica S6, Product Page, Leica Microsystems Website

9. Leica Stereo Microscopes Illumination, Leica Microsystems Website

10. Leica M50, M60, M80, Brochure, Leica Microsystems Website

11. Leica Transmitted Light Bases, Leica Microsystems Website

12. Leica MC170 HD Digital Camera, Product Page, Leica Microsystems Website

13. L.A. Pray, Transposons: The jumping genes, Nature Education, vol. 1 iss. 1, p. 204 (2008),

14. F.A. Ran, P.D. Hsu, J. Wright, V. Agarwala, D.A. Scott, F. Zhang, Genome engineering using the CRISPR-Cas9 system, Nature Protocols,

vol. 8, pp. 2281–2308 (2013), doi:10.1038/nprot.2013.143

15. W. Ockenga, Differential Interference Contrast (DIC), Science Lab

16. B. Kleine, T. Veitinger, Integrated Modulation Contrast (IMC): Oblique Illumination Enhances Visibility of Living Cells, Science Lab

17. Leica Inverted Compound Microscopes, Product Pages, Leica Microsystems Website

18. T. Mahmood, P.-C. Yang, Western Blot: Technique, Theory, and Trouble Shooting, N. Am. J. Med. Sci. vol. 4, iss. 9, pp. 429–434 (2012),

doi: 10.4103/1947-2714.100998

19. T. Mašek, L. Valášek, M. Pospíšek, Polysome analysis and RNA purification from sucrose gradients, Methods Mol. Biol. vol. 703, pp.

293-309 (2011), doi: 10.1007/978-1-59745-248-9_20

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

21. Leica M165 FC, M205 FA, Brochure, Leica Microsystems Website

22. Leica MZ10 F, Brochure, Leica Microsystems Website

23. B. Fuchs, Stereo microscopes with TripleBeam Technology: Third illumination path for better signal-to-noise ratio in fluorescence

microscopy, Science Lab

24. Z. Pincus, T.C. Mazer, F.J. Slack, Autofluorescence as a measure of senescence in C. elegans: look to red, not blue or green, Aging, vol.

8, no. 5, pp. 889-898 (2016)

25. Leica Confocal Systems, Product Pages, Leica Microsystems Website

26. Leica Upright Compound Microscopes, Product Pages, Leica Microsystems Website

27. Leica Confocal Scanner Technology, Product Pages, Leica Microsystems Website

28. H. Hutter, Five-colour in vivo imaging of neurons in Caenorhabditis elegans, Journal of Microscopy, vol. 215, pt. 2, pp. 213 –218 (2004),

doi: 10.1111/j.0022-2720.2004.01367.x

29. Worm Book: The Online Review of C. elegans Biology

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