Leica Microsystems TCS SP8 Application Note

From Eye to Insight

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 Microscope,

Leica Microsystems AG, Switzerland

2

RNA

/

Caenorhabditis elegans

40

[1] [2]

[3]

302

1/3

15 20

°

C 1 mm 75 µm

1



DNA



/

RNA

RNAi



dsRNA siRNA

microRNA



RNAi



/ /

3

1

1.

[4]

2. a)

[5 6]

2. b)

RNA

RNAi [7] 2

ds RNA

3.

RNAi

4.

5.

2

•

→

•

→

•

→

[4]

•

→

•

→

S/N

•

60

p. 2

2

4

3

S6 [8]

LED 2500 [9] 4 S6

10

16

3

LED2500 S6 [8]

[9] 1.6

S6

5 S6 4 S6 LED2500

5

LED2500 S6

LED 2500

•

LED

•

6 M80 TL3000 ST

M80 [10] TL3000 ST [11]

6 M80 CMO

500 MC 170/190 HD [12] HD

S6 LED2500

6

RNAi

/

2 [13] CRISPR/Cas9 [14]

CRISPR 1

[6]

5

DNA

DIC [15] IMC [16]

DMi8 7 DM IL LED [17]

Narishige Instruments 3

RNA RNAi

2 ds RNA

dsRNA

si RNA

siRNA

7

DMi8

7

20

°

C

2

5 [18]

10

[19]

GFP

[20]

DsRed [20]

GFP

Rottermann contrast

[FOV]

M165 FC [21] MZ10 F [22]

TL4000 RC [11]

8

•

M165 FC TL4000

•

M165 FC MZ10 F TL4000

•

TripleBeam

S/N

[23]

→

8 M165 FC TL4000 RC Rottermann contrast

8

YouTube

[24] 1

mCherry [20] M205 FA

DMC4500

•

TL4000 RCI [11]

•

TL4000 RC

•

S/N

•

TL3000 ST TL4000 BFDF [11] TL4000 RC

9

TCS SP8

TCS SPE [25] DM6 DM2500 [26]

GFP RFP

mCherry DsRed YFP CFP [20] 9 10

Levamisol

[27]

9 GFP TCS SP8

10

10 SP2 CFP GFP YFP

DsRed DiD BC

H. Hutter [28]

[29] / /

•

S6 TL3000 ST

•

DMi8

DNA

•

MZ10 F M165 FC

TL4000 RC

•

TL4000 RC CMO

M80

•

DM6 TCS SP8

•

M205 FA TL5000 Ergo [11]

•

11

Harald Hutter BC

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.

S6

9.

10.

M50 M60 M80

11.

12.

MC170 HD

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) DIC

16.

B. Kleine, T. Veitinger

Integrated Modulation Contrast (IMC): Oblique Illumination Enhances Visibility of Living Cells

IMC

17.

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

21.

M165 FC M205 FA

22.

MZ10 F

23.

B. Fuchs

Stereo microscopes with TripleBeam Technology: Third illumination path for better signal-to-noise ratio in

fluorescence microscopy

TripleBeam SN 3

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.

26.

27.

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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Leica Microsystems TCS SP8 Application Note

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