Noam Ziv

Dept. of Anatomy and Cell Biology

Technion Faculty of Medicine

Haifa, Israel

Microscopy in Cell Biology:Live imaging

Topics:

• Imaging live specimens

• 6D microscopy

• Green fluorescent protein and derivatives

• FRAP

• Photoactivation

• Mouse models

• Tetracysteine-biarsenical tagging

Cellular Biology

Traditional approach

Morphometry

Cell counting

Immunohistochemistry Biochemistry Electron microscopy

But what if one wants to study the process itself

Imaging in live cells

“Here and elsewhere we shall not obtain the best insight into things until we actually see them growing from the beginning”

Aristotle, Physics, 1, 184a. 12.

“Here and elsewhere we shall not obtain the best insight into things until we actually see them growing from the beginning”

Aristotle, Physics, 1, 184a. 12.

• Cell motility• Apoptosis• Cell division • Structural rearrangements • Intracellular transport• Membrane trafficking• Organelle physiology• Ion flux (Ca2+)• Membrane potential recordings• Gene expression

Imaging in live cells

Imaging in live cells: Time lapse microscopy

Intracellular transport Axonal and dendritic motility

Cell motility and Mitosis Gene expression

• Optimal environmental conditions• Phototoxicity • Focus drift• Vital labels

Imaging in live cells: considerations

Optimal environmental conditions• Temperature (37°C)

• Heated chambers• Heated objective• Heated solutions

• CO2• Premixed “incubator gas” / Gas flow meters• Enclosure with controlled atmospheric mixture

• Sterility• Filtered solutions• On stage “hood” (positive pressure of filtered air)

• Perfusion• Gravity feed • Pumps

Our solution (others exist)

Heating resistor

ChamberCoverslip with cells

Heating resistor

Chamber temperature probe

Solution temperature probe

Harness + connector

Perspex insert

Outlet reservoir

Inlet reservoir

Microscope enclosure

•Constant Temperature•Controlled Atmosphere•Minimized focal drift

Life imaging Services, Switzerland

Phototoxicity• Intense light -> damage to tissue

• Issue greatly aggravated by labeling with fluorescent dyes

• Goal: minimize exposure duration

• Implication -> high efficiency collection of emitted light

• Implementation: • Expose only when collecting data (electronic shutters, AOTF)

• High NA objectives (oil immersion)

• High quantum efficiency detectors (PMTs, cooled CCDs)

• Minimize light loss (minimize # of mirrors, lens, filters)

Focus drift• Focus drifts gradually with time

• Issue greatly aggravated by heating

• Some compensation mechanism necessary

• Implementation: • Manual adjustments

• Collection of stacks of images collected at different focal planes

• Auto focus algorithms based on images collected

• Glass/water interface detection

0 µm-3 µm

Average intensity along line

Objective Z position

Ave

rage

inte

nsity

glass

aqueous media

Glass/water interface detection

Microscope objective

6D Microscopyor

Automated, multisite time lapse microscopy

• X• Y• Channel• Z• Time• Site

XY

6D Microscopyor

Automated, multisite time lapse microscopy

• X• Y• Channel• Z• Time• Site

Channel 1:PSD95:GFP Channel 2:FM 4-64

6D Microscopyor

Automated, multisite time lapse microscopy

• X• Y• Channel• Z• Time• Site

Z

6D Microscopyor

Automated, multisite time lapse microscopy

• X• Y• Channel• Z• Time• Site t1 t2 t3

time

6D Microscopyor

Automated, multisite time lapse microscopy

• X• Y• Channel• Z• Time• Site

Specimen

12

3

45

6

Aut

omat

ed M

ultis

ite T

ime

laps

e M

icro

scop

y

Example-digital movies created online

Site 1

Site 2

Organic compound vital labels

• Intracellular ion concentrations (fura2, fluo3…)• pH (BCECF, SNARF…)• Cell surface (DiI, DiO… )• Membrane potential (Di-4-ANEPPS, RH dyes…)• Organelles (MitoTracker, Rhodamine 123…) • Membrane trafficking (FM 1-43, TMA-DPH…)• Lineage tracers• Enzyme activity• …

(see http://www.probes.com)

Green Fluorescent protein

• Fluorescent protein derived from the Jellyfish Aequorea victoria

• In animal, converts light emitted from luminescent molecule aequorin from blue to green

• Isolated in 1962 by Shimomura et al• Cloned in 1992.• First expressed in other organisms in

1994.

• Codon optimized and mutated to give many forms. Most popular is Enhanced GFP (EGFP)

• Fluorescence of GFP requires an internal reaction in which three consecutive amino acids (Ser-Tyr-Gly) react to form the chromophore

• This reaction is spontaneous, but requires several hours and depends on O2

• Fluorescence is similar to that ofFluorescein (FITC)

• The extinction coefficient and quantum yields are ~56x103 and ~0.60 at 488 and 508 nm respectively

Transient expression of EGFP

Cultured neuron from postnatal rat hippocampus

Molecule of interest

DNAPlasmid

GFP

GFP in cell biology: Chimeric proteins

• Generate a DNA construct coding for a chimera of the protein of interest and GFP

• Insert into mammalian expression vector• Transfect cells (stably or transiently)• Assess functionality of recombinant molecule• Quantify expression levels• Perform experiments

GFP-tagged molecules in cell biology

• Cytoskeleton remodeling• Intracellular transport• Membrane trafficking• Cell adhesion• Spatiotemporal patterns of gene expression• Cell lineage• Apoptosis• Synapse formation and synaptic remodeling

Formation of new axodendritic synapse

ProSAP2:GFP5 µm

Spectral variants of Enhanced GFP

Blue FPCyan FPGreen FPYellow FP

PSD-95:GFP DsRed

Expression of multiple fluorescent molecules

Red fluorescent proteins

Red Mushroom Anemone, Discosoma sp. (“red”)

• dsRed

Sebae AnemoneHeteractis crispa

• hcRed

Other fluorescent proteins

Ptilosarcus (sea pen) GFP

Renilla mullerei (sea pansy) GFPRenilla reniformis (sea pansy) GFP

Claimed to be brighter and less harmful than EGFP

http://www.nanolight.com/

http://cgr.harvard.edu/thornlab/gfps.htm

GFP: Emerald, CoralHue™ Azami Green, CopGFP, AcGFP, ZsGreen1

BFP: GFPuv, Sapphire, T-Sapphire

CFP: Cerulean, AmCyan1, CoralHue™ Midoriishi-Cyan

YFP: Citrine, Venus, PhiYFP, ZsYellow1

Orange Proteins: CoralHue™ Kusabira-Orange, mOrange

RFP: tdimer2(12), mRFP1, DsRed-Express, DsRed2,HcRed, HcRed1, AsRed2, eqFP611, mPlum,mRaspberry, mCherry, mStrawberry, mTangerine,tdTomato, JRed

And the list goes on…

Shaner, Steinbach, Tsien. Nat Methods (2005) 2, 905-909

What XFP should I use?

Fluorescence recovery after photobleaching (FRAP)

Measurement and Quantification of:

• Diffusion and transport (in cytosol and membrane)

• Protein Turnover

Lippincott-Schwartz et al., Nat Cell Biol. 2003 Suppl:S7-14.

• Practically invisible form of GFP (488 nm excitation)• Irradiation at 405 nm (Violet light) increases absorbance x100 =>

Protein becomes brightly fluorescent.• Excellent for protein trafficking studies.

Photoactivable (PA) fluorescent proteins

Patterson GH, Lippincott-Schwartz J. A (2002) photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873-1877.

PA-GFP – Native state Activation PA-GFP -Activated

• Fluorescent protein from stony coral (Trachyphyllia geoffroyi)

• Includes a tripeptide, His-Tyr-Gly that acts as a green chromophore

• Can be converted to red by irradiation with UV / violet light (350–400 nm)

• Excitation light used to elicit red / green fluorescence does not induce photoconversion.

Kaede

http://www.mblintl.com

Dronpa

Science 306, 1370 – 1373, 2004

Before Photoactivation (-5 min)

PA + 3 min

PA + 1:15 hours

PA + 7:45 hours

0

1

2

3

4

5

0 2 4 6 8

F/Fo

0

1

2

3

4

5

6

0 2 4 6 8

F/Fo

25±10µm (n=17)50±10µm (n=25)75±10µm (n=14)105±10µm (n=10)

Time (hours)

SomaSoma

PSDs along dendritesPSDs along dendrites

Replenishment of synapses with proteins from somatic sources

0 10 20 30 µm-10-20

3

2 4 5

1

CFP

PA-GFP:Synapsin I

-1.5’

0’

10’

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 10 20 30 40 50 60time (min)

Nor

mal

ized

Flu

ores

cenc

e

1 (0 µm)

2 (8 µm)3 (14 µm)

4 (21 µm)5 (28 µm)

Reincorporation of Synapsinlost from one synapse into nearby synapses

Reporter molecules based on GFP

• Calcium indicators (Cameleon, CAMgeroo, Pericam)GFP(s) + CalmodulinMiyawaki et al. (1997) Nature 388: 882–887.Miyawaki, Griesbeck, Heim, Tsien (1999) PNAS 96, 2135-2140.Baird, Zacharias, Tsien (1999) PNAS 96, 11241-11246.Nagai, Sawano, Park, Miyawaki, (2001) PNAS 98 3197-3202.

• Voltage Sensors (FlaSh)GFP + ShakerSiegel, Isacoff (1997) Neuron 19 735-741.

• Exocytosis and Endocytosis (synapto-pHluorin)GFP + Synaptobrevin/VAMPMiesenbock, De Angelis, Rothman (1998) Nature 394, 192-195.

Ryan TA & Reuter H (2001) News Physiol. Sci 16 10-14

Synapto-pHluorin

5 µm

Nature Neurosci 8, 1263-1268

Channelrhododopsin-2 (ChR2; fused to EYFP)A light-gated cation channel from the unicellular green alga Chlamydomonas reinhardtii

Stimulating neurons with light

Feng G et al., (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41-51

XFP Trangenicmice

Trachtenberg et al., (2002) Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature, 420, 788-794.

Long-term in-vivo imaging in trangenic mice

2:e163 (2004)

Knock-in miceHomozygous for Munc-13-1:YFP

Presynaptic protein Munc-13-1 tagged with YFP at original locus in genome.

Mice prepared by Stefan Kalla& Nils Brose, MPI Goettingen

FlAsH / ReAsH (tetracysteine-biarsenical tagging)An alternative to fluorescent proteinsProblem:• GFP is a large tag (25-27kD)• Not suitable for EM (does not photoconvert well)

Solution• Tag protein with 6-20 amino acids that contain 4 Cysteines• Add a membrane permeable fluorescent substance that

binds to this motif

Advantages:• Multiple labelings• Photoconvertible• Optimal tag sequences determined

(HRWCCPGCCKTF or FLNCCPGCCMEP)

Gaietta et al., (2002) Science 296, 503 – 507. Martin et al (2005) Nat Biotechnol. 2005 23, 1308-1314.

FlAsH-EDT2 = 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2ReAsH-EDT2= 4',5'-bis(1,3,2-dithioarsolan-2-yl)resourfin-(1,2-ethanedithiol)2

Fluorescent proteins: Considerations and potential pitfalls

• Excitation and emission filters – optimize for best S/N• Phototoxicity – less severe but significant• pH sensitivity

• Expression levels• Interference with physiological function of original molecule• Multimerization • Toxicity

Summary

• Live imaging microscopy – a very powerful approach for understanding processes

• Much attention must be paid to maintaining cell viability• Many vital probes currently available• GFP and derivatives – extraordinary tool for studying

molecular and cellular physiology• 6D microscopy provides a means to collect sufficient and

satisfactory data• FRAP – quantitative tool for evaluating molecular dynamics• PA - quantitative and qualitative tool for evaluating

molecular dynamics trafficking and transport• Additional genetically encoded probes appearing daily• Bright future for live imaging!

Ziv lab, Technion, IsraelZiv lab, Technion, IsraelAmirAmir MinerbiMinerbiShlomo TsurielShlomo TsurielRan Ran GevaGevaMichal Michal SternSternRoni KahanaRoni KahanaLarissa Larissa GoldfeldGoldfeldVladimir Vladimir LyakhovLyakhov

CollaborationsCollaborationsCraig Garner, StanfordCraig Garner, StanfordEckart GundelfingerEckart Gundelfinger, , IfNIfN, , MagdeburgMagdeburgThomas Thomas DresbachDresbach, U. Heidelberg, U. HeidelbergNilsNils Brose, MPIBrose, MPI GoettingenGoettingenTobias Tobias BoeckersBoeckers, U. , U. UlmUlm

Tal Tal BreslerBreslerMika ShapiraMika ShapiraHagit Vardinon-FriedmanYaron Ramati

http://www.microscopy.fsu.edu