Imaging molecular dynamics in vivo – from cell biology to ... · FRET High-specificity assessment of protein–protein interactions Complicated bleed through corrections required

IntroductionAdvances in optical microscopy have produced a variety of methodssuitable for the study of protein dynamics in cultured cells. Thishas been accelerated by the development of fluorescent proteins(FPs), which enable not only selective labeling and localization ofvirtually any protein, but also allow engineering of functionalprobes that report on the activity of signaling molecules such askinases (Wang, Y. et al., 2005) and GTPases (Yoshizaki et al.,2003). FPs are increasingly incorporated into sophisticated animalmodels of disease, in which tissue-specific driver mutations can belinked to the expression of FP probes. As a result, new animalmodels that are appropriate for the investigation of moleculardynamics in vivo, such as genetically engineered mice (GEM),have been developed.

The need for in vivo imaging has emerged in conjunction withthe understanding that many cellular responses are determined bysignals within a tissue niche (Coussens and Werb, 2002; Sneddonand Werb, 2007). For example, local mediators of cellularcommunication, such as tumor necrosis factor (TNF),inflammatory cytokines, and chemokines can crucially influencethe progression and metastasis of cancer (reviewed in Wilson andBalkwill, 2002). The ability of stem cells to retain their specialproperties also appears to be regulated by features of the stem cellniche (Zhang et al., 2003). Furthermore, imaging of proteindynamics in response to therapeutic intervention has the potentialto dramatically increase the use of mouse cancer models and ourunderstanding of the molecular mechanisms of drug action (Kamb,2005). By combining advanced imaging techniques withsophisticated new animal models, we are now able to investigateboth disease and treatment at the molecular level.

This Commentary will review approaches for imaging moleculardynamics in vivo, including both imaging techniques and

experimental systems, with examples drawn primarily from theuse of mouse cancer models. We begin the discussion with generalapproaches to FP imaging and their use to study cellular dynamicsin vivo, and progress to more specialized methods for molecularlevel investigations. Then we discuss methods that complementthe use of FPs by providing contextual information that is importantfor the interpretation of results. Finally, we highlight the factorsthat constrain in vivo experimental design and the use ofintermediate experimental systems, to bridge the gap between glasscoverslips and native tissue.

Imaging cellular dynamics with fluorescentproteinsUse of FPs has expanded into developmental models, such aszebrafish and Drosophila melanogaster, following the FP revolutionof cell biology in the late 1990s. The application of FPs in vivo hasbeen limited both by the development of appropriate imagingapproaches and the understanding of what kinds of questions canbe answered using imaging approaches. Therefore, in this section,we begin with an introduction to general approaches for imagingthe dynamics of cells in tissues (summarized in Table 1).

Whole-body fluorescence imagingWhole-body imaging is useful for determining the location andsize of tumors in living mice (Fig. 1). This approach is generallynon-invasive and can be repeated many times on the same animal,which facilitates longitudinal studies of tumor growth orsenescence, metastatic progression and response to therapy (Ahmadet al., 2011; Morton et al., 2010). Basic systems provide qualitativeinformation about the size and location of fluorescent tumors onthe basis of epi-fluorescent illumination, a macroscopic lens andsensitive detection using cooled CCD cameras. Some of these

SummaryAdvances in fluorescence microscopy have enabled the study of membrane diffusion, cell adhesion and signal transduction at themolecular level in living cells grown in culture. By contrast, imaging in living organisms has primarily been restricted to thelocalization and dynamics of cells in tissues. Now, imaging of molecular dynamics is on the cusp of progressing from cell culture toliving tissue. This transition has been driven by the understanding that the microenvironment critically determines many developmentaland pathological processes. Here, we review recent progress in fluorescent protein imaging in vivo by drawing primarily on cancer-related studies in mice. We emphasize the need for techniques that can be easily combined with genetic models and complementfluorescent protein imaging by providing contextual information about the cellular environment. In this Commentary we will considerdifferences between in vitro and in vivo experimental design and argue for an approach to in vivo imaging that is built upon the useof intermediate systems, such as 3-D and explant culture models, which offer flexibility and control that is not always available invivo. Collectively, these methods present a paradigm shift towards the molecular-level investigation of disease and therapy in animalmodels of disease.

Key words: In vivo imaging, Intravital imaging, FRAP, FRET, FLIM, GFP, SHG

Journal of Cell Science 124, 2877-2890 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.085191

Imaging molecular dynamics in vivo – from cellbiology to animal modelsPaul Timpson, Ewan J. McGhee and Kurt I. Anderson*The Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK*Author for correspondence ([email protected])

ARTICLE SERIES: Imaging Commentary 2877

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systems have sufficient resolution to image single cells, and havebeen used to image events such as cancer cell extravasation(Yamauchi et al., 2006). Alternatively, advanced tomographicsystems generate quantitative fluorescence images by usingtransmitted laser light to correct for sample absorption andscattering (reviewed in Ntziachristos et al., 2005).

Confocal laser scanning microscopyConfocal laser scanning microscopy (CLSM) is the technique ofchoice for many in vitro cell-imaging applications [for a thoroughdiscussion refer to the Handbook of Confocal Microscopy (Pawley,2006)]. Optical sectioning (Box 1) in CLSM is based on theselective detection of fluorescence from a single point within thespecimen by using a spatial filter (pinhole) placed in front of adetector. The main advantages of CLSM compared withmultiphoton microscopy are its relative ease of use and itswidespread availability, and its low cost and high safety (Table 1).One disadvantage of CLSM is that the illumination methodgenerally leads to photobleaching and phototoxicity throughoutthe focal path of the laser, including regions above and below theimage plane. Such photobleaching can be minimized by reducingthe intensity of the laser beam when particularly bright or dimregions of the sample are encountered (Hoebe et al., 2007). Asecond disadvantage is that confocal detection suffers greatly iflight emitted from the focal point in the sample is scattered on itsway to the detector. Light scattering is one of the main limitingfactors in tissue imaging (Helmchen and Denk, 2005). Scatteringprimarily occurs as a result of differences in refractive indexassociated with compartmental boundaries within cells and tissues,such as nuclei, blood vessels and extracellular matrix (ECM) fibers(Fig. 1B). As a photon transits such a boundary, its direction isaltered so that it cannot be properly refocused through the detection

pinhole and its signal will be lost. Measurements have shown that,on average, a photon will be scattered once for every 47 mtraveled through adult rat brain tissue (Oheim et al., 2001), whichsuggests an effective imaging depth of <50 m for CLSM.

CLSM has been used to study the shedding of cells from thesurface of the intestinal epithelium (Watson et al., 2005) and alsoto quantify the frequency and type of movement in tumors that areformed by cells deficient for pyruvate dehydrogenase kinaseisoenzyme 1 (Pinner and Sahai, 2008). However, CLSM is morecommonly used for thinner translucent specimens, includingintermediate systems, such as cell derived matrices (Caswell et al.,2007), tissue explants (Mort et al., 2010) and organoids (Pearsonand Hunter, 2007) (refer to Fig. 1 and Box 2 for more detail). Forexample, Gaggioli et al. recently used CSLM to demonstrate thecollective invasion of carcinoma cells into an organotypic matrix(Gaggioli et al., 2007). Contextual information was provided byimaging collagen fibers using confocal reflection microscopy (adetection mode in which reflected excitation light rather thanfluorescence is imaged by the detector). Collagen is a mainconstituent of most tissues, and a common, biologically relevant(compared with glass coverslips) substrate for cell migration (Fig.1B). Confocal reflection microscopy has also been used to visualizereorganization of collagen matrices, such as local contraction orhydrolysis, during invasion (Wolf et al., 2003).

Intermediate systems such as cell-derived matrices andorganotypic gels are amenable to CLSM because of their superioroptical properties (less absorption and scattering of light) comparedwith whole tissues. Kubow and Horwitz used translucent collagengels and low-light confocal microscopy to visualize formation offaint green fluorescent protein (GFP)-labelled paxillin adhesions(imaged in fluorescence) juxtaposed with collagen fibers (imagedin reflectance) in U2OS cells and, thereby, confirmed the existence

2878 Journal of Cell Science 124 (17)

Table 1. Fluorescence microscopy approaches for in vivo imagingAdvantages Disadvantages

Imaging system

Wide-field microscopy Cheap, easy to use, widely available Poor contrast, no optical sectioningSDCM Fast imaging rates possible, sectioned images, compatible with a

range of other methods and extra-sensitive CCD camerasPoor light efficiency

CLSM Well established technology, large range of options to suitdifferent budgets, diffraction limited performance

Limited imaging depth, out-of-focus phototoxicityeffects

MPLSM Higher spatial resolution imaging in tissue when compared withconfocal microscopy, potential to combine with other non-linear optical techniques, direct imaging of collagen and otherendogenous proteins, ultra-sensitive detection possible

Requires expensive laser systems, harder to optimizeexperiments than on confocal systems

Whole-body imager Large range of magnifications, imaging range from whole-bodyto cell level, non-invasive, longitudinal studies

No optical sectioning, difficult to separateautofluorescence from skin and hair

Fluorescence technique

Optical windows Longitudinal studies at subcellular level Complicated animal guidelines to followPhotoactivation, photobleaching

and photoconversionHighly specific region of interest Limited range of fluorophores, more complex

imaging strategiesPhoto-activation of protein function Localized activation of protein function Cell behavior might be affected if protein is

overexpressedFRET High-specificity assessment of protein–protein interactions Complicated bleed through corrections required for

accurate quantification of activityFLIM Concentration independent read out of protein environment,

good read out of FRET because only one emission channel iscollected

Accuracy depends on signal strength – the more lightthe better, requires pulsed lasers and/or modulatedLEDs

Exogenous dyes Useable in short-term experiments, fast acting, range of targets Becomes dilute with cell divisionAutofluorescence Endogenous, biologically relevant signal present in all cellular

structuresDifficult to identify species because of large spectral

overlap, less signal strength

SDCM, spinning disk confocal microscopy; CLSM, confocal laser scanning microscopy; MPLSM, multiphoton laser scanning microscopy; FRET, Försterresonance energy transfer; FLIM, fluorescence lifetime imaging.

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of focal-adhesion-like structures in 3D culture (Kubow and Horwitz,2011).

Spinning disk confocal microscopy (SDCM) has emerged inrecent years as a powerful technique for live imaging of ‘semi-thick’ specimens such as Caenorhabditis elegans (Mayer et al.,2010), zebrafish (Wang et al., 2011) and Drosophila (Aldaz et al.,2010). In SDCM an array of pinholes rather than a single pinholeis scanned over the specimen and light from the pinhole array isimaged onto the surface of a CCD camera [for a detaileddescription see Pawley (Pawley, 2006)]. SDCM has been less

widely adopted for in vivo imaging in mice because of itssusceptibility to crosstalk between pinholes, which limits imagecontrast when imaging deep within highly scattering tissue (Egneret al., 2002; Wang, E. et al., 2005). Nevertheless, SDCM hasbeen used to examine the differential effects of hypoxia on T-lymphocyte and myeloid cell migration in MMTV-PyMT mice –a mammary carcinoma model (Egeblad et al., 2008) – and inconjunction with laser ablation to study the signals that guideneutrophil migration into superficial sites of inflammation in theliver (McDonald et al., 2010).

2879Imaging dynamics in vivo

Cellculture

CDMmatrigel

Ex-vivomodelsOrganotypic GEMTransplant

B

A

Cancer cell

Stromal cell

Immune cell

Extracellular matrix

Vasculature Key

Fig. 1. Experimental imaging systems. (A)Intermediate experimental systems can form a pipeline that facilitates the progression from cell culture to mousegenetic models. (A)Examples of the types of image obtained using each system. Cell culture. 2D culture of pancreatic ductal adenocarcinoma (PDAC) cells grownon glass in phase contrast (top) or expressing E-cadherin-GFP (bottom). Cell-derived matrix (CDM). PDAC cells grown on CDM imaged in phase contrast (top) orusing FLIM–FRET to show RhoA activity (bottom). The matrix can be seen as background striations in the phase-contrast image (top). Organotypic. H&E section(top) showing cell invasion from the top surface down into the matrix. In addition to collagen, the matrix includes stromal cells (bottom, red), which have beenshown to promote the migration of tumor cells (green). Ex vivo. Mouse stem cells cultured in collagen gel grow into self-organized ‘organoids’ that recapitulate themorphology of the intestinal crypt (top, phase contrast). Embryonic epidermal tissue cultured ex-vivo from a mouse genetic model in which melanoblasts expresscytoplasmic GFP. PDAC cells expressing E-cadherin-GFP (green) were grown subcutaneously in a mouse and injected with a probe to visualise matrixmetalloprotease activity in red (top, brightfield). Fluorescent cancer cells (green) with surrounding ECM were imaged using SHG (blue, bottom). Geneticallyengineered mice (GEM). Primary pancreatic tumor imaged through the abdominal wall in a GEM that exhibits tissue-specific expression of GFP (top). GFP-expressing cells can also be imaged at high resolution within the host tissue and microenvironment (bottom). Scale bars: 10 mm for whole-body mouse images and20m for all others; see Box 2 and Table 2 for additional information. (B)Intermediate experimental systems can be arranged in a pipeline offering increasinglevels of fidelity to the tissue environment under study, e.g. through the inclusion of ECM, stromal cells, immune cells and vasculature.

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Multiphoton laser scanning microscopyMultiphoton laser scanning microscopy (MPLSM) has becomethe technique of choice for imaging cellular dynamics in tissues.Unlike confocal microscopy, which generates optical sections onthe basis of selective detection of light, MPLSM generates opticalsections through the selective excitation of fluorescence withinthe sample. In MPLSM the energy required to excite an electronwithin a fluorophore (Box 1) is provided by simultaneousabsorption of two photons (Denk et al., 1990). The probability ofsuch an event is extremely small and only achieved in the focalpoint of the objective, where light from a pulsed laser is focussedto the smallest possible volume. This provides one of the mainbenefits of multiphoton over confocal microscopy, because onlythe molecules within the focal point are excited and photo-damage above and below the image plane is eliminated. The useof a pulsed laser has additional benefits, including its suitability

for second harmonic generation (SHG) and fluorescence lifetimeimaging (FLIM). A less obvious but equally important benefit isthe flexibility in detection that is inherent to MPLSM. There isno requirement to focus the emitted fluorescence back throughthe laser-scanning system onto a pinhole aperture (Box 1 and Fig.2). Instead, non-descanned detection can be used, whereby thedetector is placed as close to the objective as possible (Williamset al., 1994). This approach is more effective at detecting thescattered photons that result from fluorescence excitation deepwithin a tissue sample and, therefore, leads to a higher sensitivityand a better signal–to–noise ratio (Centonze and White, 1998;Wang et al., 2002). However, disadvantages of MPLSM includethe need for expensive, potentially hazardous, lasers andsubstantially increased photobleaching when the illuminationintensity of the sample is too high (Table 1) (Patterson and Piston,2000).


Box 1. Glossary

In vitro and in vivoOriginally, these terms referred to experiments performed outside of and within the body, respectively. However use of in vivo has beenstretched to cover experiments performed in cells, which leads to confusion and complicates online searches. Therefore, we suggest in muro(from the latin murinus) to distinguish work performed in living mice.

Laser scanning microscopy (LSM)It is possible to raster-scan the point of a focused laser beam across a specimen by using pivoting mirrors and a telecentric lens system toachieve single or multiphoton excitation of fluorescence. Images can be generated by confocal or wide-field detection of fluorescenceemission.

FluorescenceFluorescence involves absorption of a photon by a fluorophore, which raises the energy state of an electron within the molecule to an excitedstate, followed by relaxation of the electron down to ground state and loss of energy in the form of an emitted photon after a brief period oftime. The average time between excitation and emission is known as the fluorescence lifetime, which lasts between 2 and 5 nseconds formany common fluorophores.

Non-linearityProcesses such as multiphoton fluorescence and second harmonic generation (SHG) are non-linear because they require the interaction oftwo or more photons and, therefore, only occur at very high photon densities. Doubling the illumination intensity results in the four (22) or eight(23) times increase of SHG or third harmonic generation (THG), respectively.

Optical sectioningOptical sectioning refers to the ability to image a single plane with high contrast in the middle of a thick specimen.

OPOAn optical parametric oscillator (OPO) laser converts pulsed light from a Ti:sapphire laser into tuneable output (~1100–1300 nm) that has thesame repetition rate and pulse-length as the pump laser (Zhang et al., 2010).

PALM and STORMPhotoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are imaging methods that surpassthe classical resolution limit of light microscopy through sequential localization of single fluorophores.

Raman scatteringA non-fluorescent process by which a photon can lose or gain energy by interacting with a molecule. The magnitude of the energy change is afunction of the illumination wavelength and properties of the molecule.

STEDStimulated emission depletion (STED) is an imaging method that surpasses the classic resolution limit of light microscopy by using two laserbeams that are focussed in tandem to suppress fluorescence in a doughnut-shaped region around a central emission point.

TCSCPTime correlated single photon counting (TCSPC) is a laser-scanning fluorescence lifetime imaging method that requires pulsed excitation.The laser intensity is attenuated until each pulse excites no more than a single fluorophore within the sample. For each pixel, the time ofphoton detection is correlated against the time at which the pulse occurred, and many tens of thousands of events are averaged to computethe fluorescence lifetime.

Ti:sapphireThe titanium:sapphire (Ti:sapphire) laser is a sapphire crystal laser that emits pulsed light (~80 MHz repetition rate) over a broad infraredrange (~780–1080 nm). Owing to the extremely short pulse duration (<200 fseconds), moderate average power levels are delivered in pulsesof mW/cm2 to gW/cm2 intensity.

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MPLSM has revolutionized the study of immune cell dynamics(reviewed in Garside and Brewer, 2008), Ca2+ dynamics (reviewedin Gobel and Helmchen, 2007) and the mechanisms underlyingtumor cell invasion of host tissue (reviewed in Provenzano et al.,2009). In a seminal study Ahmed et al. used multi-photonmicroscopy to characterise tissue-specific expression of GFP in themammary glands of transgenic mice (Ahmed et al., 2002). Usingtime-lapse imaging, these authors could also demonstrate themigration of individual tumor cells towards blood vessels in amanner suggestive of metastatic intravasation. Much subsequentwork focused on characterizing cell migration in tumors withinliving organisms on the basis of, for example, EGFR expression(Xue et al., 2006) and the interaction of cancer cells with tumormacrophages (Wyckoff et al., 2007). In the latter study, the authorsobserved that, in MMTV-PyMT mice, intravasation only occurredwithin approximately one cell diameter (~20 m) of perivascularmacrophages; and these observations confirmed the importance ofmacrophages in promoting metastasis.

Generally, cytoplasmic expression of FPs has been used fortracking cell dynamics. However, some studies have made use ofGFP to investigate subcellular protein localization. For example,Wyckoff et al. used GFP to assess the localization of the myosinlight chain in metastatic MTLn3E cells within xenograft mousetumors (Wyckoff et al., 2006). Similarly, Giampieri et al. used GFPto visualize the location of the transcription factor and transforminggrowth factor (TGFB) effector SMAD2 to assess the influenceof TGFB signaling on the motility of MTLn3E cells in vivo(Giampieri et al., 2009). Nuclear localization of GFP–Smad2indicated TGFB signaling being active in individually migratingcells.

The redder the better in vivoLight scattering, autofluorescence and absorption by tissue (whichcan cause phototoxicity) are attenuated at longer wavelengths,which has driven the development of red FPs for in vivo use(Shaner et al., 2008; Shcherbo et al., 2007). A typical red FP, suchas mKate (Shcherbo et al., 2007), has a single-photon excitationpeak of 588 nm, suggesting a two-photon excitation peak of ~1180nm, which lies beyond the tuning range of a Ti:sapphire laser (Box1). Such far-infrared wavelengths can be generated by an OPOlaser (Box 1) (Andresen et al., 2009); however, the need for anOPO to excite red FPs adds complication and expense to an alreadysufficiently complex and expensive microscope system. Piatkevichand colleagues took a mutagenesis approach to resolve this problemby systematically changing the sequence of mKate to produceLSS-mKate1, which can be excited in the tuning range of aTi:sapphire laser but retains emission at ~605 nm (Piatkevich et al.,2010). This enabled the authors to simultaneously excite cyanfluorescent protein (CFP; blue), fluorescein isothiocyanate (FITC;green), and LSS-mKate1 (red) at 870 nm in MTLn3 cells growninto mouse xenograft tumors, and to monitor cell polarization nearand far away from the blood vasculature in response to achemotactic gradient. Finally, the infrared fluorescence protein(IFP), which is based on conjugation of a bacterial phytochromewith biliverdin (Shu et al., 2009), emits photons at even longerwavelengths (>700 nm) and should, therefore, enable even deepertissue imaging.

Imaging molecular dynamics using FPsA variety of fluorescence techniques have been developed to reporton molecular dynamics in cultured cells (Table 1). Although such

techniques have been developed for ideal experimental conditionsthat use cells grown on glass coverslips, the techniques coveredhere have also been successfully applied to study moleculardynamics in tissue environments.

PhotobleachingFluorescence recovery after photobleaching (FRAP) (Axelrod etal., 1976; Lippincott-Schwartz et al., 2003) involves rapidlyphotobleaching the fluorescence within a small region of the samplethrough intense excitation and then analysing the recovery of


Box 2. Intermediate experimental systemsCell-derived matrix (CDM) is a flexible, fibronectin- andcollagen-based matrix that is derived from fibroblasts. It is ~5–10m thick and provides a source of growth factors and receptorsfor integrin engagement (Discher et al., 2005; Ginsberg et al.,2005). CDMs have been routinely used to assess integrintrafficking and the bi-directional, physical and mechanicalinteraction of cells with an elastic fibrillar network (Discher et al.,2005; Caswell et al., 2007). Organotypic 3D collagen Imatrices consist of acid extracted native, crosslinked collagenthat is polymerized in the presence of fibroblasts (Sabeh et al.,2004; Edward et al., 2005; Raub et al., 2007). Organotypiccultures are a malleable platform for the investigation of theimportant roles of stromal cells – either directly or throughsecretion of cytokines, growth factors or ECM – in the initiationand progression of tumorigenesis. Organotypic cultures can alsoinvolve the co-culture of multiple cell types on top of the matrixprior to interaction with stromal fibroblasts during invasion. Thisway, a great deal of information on tumor growth, survival anddifferentiation has been established (Edward et al., 2005; Edwardet al., 2010; Meier et al., 2000). Ex vivo culture of organs ortissues offers the opportunity to image molecular or cellulardynamics within the complex architecture of authentic tissue withincreased sample access and stability. Ex vivo live imaging hasbeen used to examine melanoblast kinetics in embryo mouseskin, intestinal crypt activity within crypt cultures and immuneinfiltration with regards to leucocytes and dendritic migration intissue explants (Lammermann et al., 2008; Lammermann et al.,2009). Transplantation models. Tumor growth can be modeledin vivo using transplanted cells, including stably transfected celllines. Transplantation models mimic native tissue featuresincluding a 3D setting, multiple ECM interactions, formation ofvasculature networks and interactions with stromal cells. Tumorscan have both hypoxic and necrotic regions, and some modelsresult in local invasion of the surrounding tissue or bone (Canel etal., 2010a; Condeelis and Segall, 2003; Serrels et al., 2009).However, such models frequently involve transplantation of cellsinto a tissue that is different from their origin and use ofimmunocompromised animals. Genetically engineered mouse(GEM) models of disease have been engineered to recapitulatethe progression and histopathology of human disease.Increasingly, fluorescent protein expression has beenincorporated to enable the study of cellular dynamics within thesetumor models. Murine models of human cancers that use theCre–Lox technology to target expression of known oncogenesand/or tumor suppressors in parallel with fluorescent proteins tospecific tissues have recently facilitated the study of pancreatic,bladder, renal and colon cancer (Ahmad et al., 2011; Cole et al.,2010; Doyle et al., 2010; Morton et al., 2010). The use ofintermediate systems described above combined with in vivowork can substantially improve our insight into cell behavior in thegiven disease state.

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fluorescence intensity in the bleached region over time. A FRAPrecovery curve can be used to derive two primary parameters: theimmobile fraction and the half time of recovery. The immobilefraction is the fraction of bleached molecules that remains trappedin the analysis region and, therefore, limits full recovery of thefluorescence intensity. The half time of recovery is a measurementof the rate at which freely moving molecules diffuse in and out ofthe bleached region. Molecular diffusion can also be interrupted bytransient binding or ’corralling’ events that reduce the apparentdiffusion rate. FRAP acquisition tools are standard features ofmost commercial CLSM or MPLSM systems and many approachesfor data analysis have been described (Sprague and McNally,2005).

FRAP has been extensively used to assess the dynamics of GFP-tagged proteins in vitro; however, quantitative investigations ofmolecular dynamics at the subcellular level in vivo remain limited.In Drosophila, FRAP has been used to address the crosstalkbetween actin dynamics and E-cadherin. These studies identifiedtwo sub-populations of actin that regulate either clustering or lateralmovement of E-cadherin (Cavey et al., 2008). Similarly, we have

used FRAP to compare E-cadherin dynamics within A431 cellsgrown in vitro and in subcutaneous tumors in mice (Serrels et al.,2009; Timpson et al., 2009). Interestingly, the efficacy of theclinically approved Src inhibitor dasatinib, which impairs E-cadherin turnover, was substantially greater in vivo than in vitrodespite the same cells being used (Serrels et al., 2009), whichhighlights an important caveat in the use of cultured disease modelsas a replacement for living tissue.

Photoactivation and photoconversion of fluorescencePhotoactivation of fluorescence is a process during which a non-fluorescent molecule is converted into its fluorescent form, usuallythrough a chemical reaction that is driven by ultraviolet light. Forinstance, activation of photoactivatable GFP (PA-GFP) by intenseillumination at 405 nm drives decarboxylation of Glu222, whichconverts the tripeptide fluorophore at the heart of this GFP mutantfrom a neutral (dark) to an anionic (fluorescent) state (Pattersonand Lippincott-Schwartz, 2002). Photoactivation is, essentially, theinverse of photobleaching; however, it can offer improvedsensitivity of detection by creating a positive signal against a dark


840 nm Ti:sapphire + 1210 nm OPO

405/10THG

525/50GFP

630/60 SHG

Primarycrypt

cultures

840 nm Ti:sapphire

435/40SHG

525/50GFP

Live mouse onheated stage

A B

C D

Activated ROCKKinase-dead ROCKE

Section ofintestine

840 nm Ti:sapphire

435/40SHG

525/50Autofluorescence

OR

Live mouse onheated stage

Section ofskin

OR

F

Fig. 2. Use of multiphotonmicroscopy for ex vivo imaging ofcrypt cultures, intestinal crypts andskin. (A,B) Crypt cultures.(A)Schematic for dual laser excitationand three-channel detection. The 840nm laser excites GFP, whereas the1210 nm laser produces blue THG andred SHG. (B)3D reconstruction of amature crypt culture. Scale bar: 50 µm.(C,D) Intestinal crypts. (C)Schematicfor imaging intestinal crypts in live orex vivo tissue. Living or freshlyexcised tissue is incubated on a heatedstage and imaged using a long-working distance water-immersionlens. (D)Maximum intensityprojection of intestinal cryptsexpressing GFP (green) under controlof the leucine-rich repeat-containingG-protein-coupled receptor 5 (LGR5)promoter. Collagen (blue) forms thedense connective tissue layer at thebase of the crypt. LGR5-GFP micekindly provided by Nick Barker andHans Clevers. Scale bar: 25 µm. (E,F)Skin. (E)Schematic for imaging skinin living or ex vivo tissue.(F)Autofluorescence (green) can beimaged in conjunction with SHG (red).Here it is used to quantify changes inthe volume of collagen in response toRho-associated, coiled-coil-containingprotein kinase 1 (ROCK) activity inmouse skin (Samuel et al., 2011).

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background and, therefore, allowing the distribution of subcellularpools of PA-GFP-tagged protein to be tracked over time. The mainweakness of the photoactivation approach is that labeled proteinsare nonfluorescent and, therefore, difficult to target with the 405nm laser prior to activation. A solution to this problem is to labelproteins of interest with a fluorophore, such as Kaede (Ando et al.,2002) or Dendra (Gurskaya et al., 2006), which can bephotoconverted from one color to another through targetedillumination at 405 nm. A drawback of photoconversion comparedwith photoactivation is that it requires the dedication of twodetection channels (e.g. green and red) to image the labeled proteinbefore and after conversion.

As with FRAP, photoactivation and photoconversion have foundwidespread use in vitro, but their application in vivo is morelimited. At the cellular level, photoconversion of Dendra2 has beenused to track the migration and intravasation of orthotopicallyinjected breast cancer cells in conjunction with a surgicallyimplanted imaging window (Gligorijevic et al., 2009; Kedrin et al.,2008). The authors were able to repeatedly image a pre-selectedpool of cells for up to 21 days in the context of the surroundingenvironment, which included the vasculature and the ECM (seealso Gligorijevic and Condelis, 2009). Recently, this technologyhas been used to quantitatively and repeatedly measure tumor cellmotility and proliferation in response to clinically relevant anti-metastatic agents that target the integrin–Fak–Src signaling axisover long time courses (Canel et al., 2010). A weakness ofphotoactivation and/or photoconversion for long-term tracking ofcells is that the cytoplasmic signal becomes diluted with successivecell divisions. Photoactivation of gene expression (see followingsection) is, therefore, a better approach in this case. Photoactivationin vivo was used for the first time to track plasma membranedynamics, by using the membrane-targeting sequence of H-Raslinked to photoactivatable GFP (Serrels et al., 2009; Timpson etal., 2009). Proteins attached to the plasma membrane were foundto be substantially less mobile in tumor cells that had been grownin living animals compared with the same cells cultured in vitro(Serrels et al., 2009), an observation that is of crucial importancefor the initiation of signal transduction at the cell surface(Radhakrishnan et al., 2010).

Photoactivation of protein activityAdvances in protein photochemistry have recently progressedbeyond the uncaging of fluorescence and now enable the uncagingof protein activity by using targeted bursts of light. Recently, aconstitutively active mutant of Rac1 was fused to the photoreactivedomain of phototropin and the resulting steric hindrance impairedthe interaction of Rac1 with its effectors (Wu et al., 2009). Uponillumination with light at 458 nm, the photoreactive domain ofphototropin unwinds and uncages the fused protein, thereby leadingto Rac1 activation. In this way, the precise location and timing ofprotein activation can be achieved at subcellular resolution. Thismethod has recently been used in vitro to control the location ofprotrusions and direction of fibroblast migration (Levskaya et al.,2009; Wu et al., 2009), and in vivo to selectively activate Rac incells that migrate through tissue in Drosophila (Wang et al., 2010)and zebrafish (Yoo et al., 2010).

An alternative approach that is based on phytochrome 2 andCRY2-interacting bHLH from Arabidopsis thaliana has been usedto activate protein expression and Cre-recombinase-mediated DNArecombination in cultured cells (Kennedy et al., 2010). In the nearfuture, these techniques will hopefully enable new experimental

approaches, such as photoactivation of gene expression, whichwould allow the activation of oncogenes and fluorescent reportersin specific locations within tissues – such as the intestine or liver– followed by repeated imaging to study the progression of diseaseand response to drug treatment at the molecular level.

FRET and FLIMFörster resonance energy transfer (FRET) involves the directtransfer of energy from a donor fluorophore to an acceptorfluorophore. Because this can only occur when the two fluorophoreslie within close proximity (~5 nm), FRET indicates a directmolecular interaction between two fluorescently labeled proteins.This has enabled analysis of when and where proteins interactwithin cells. There are two primary means of detecting FRET,through fluorescence intensity or fluorescence lifetime.

FRET results in the loss of donor fluorescence and an increasein acceptor fluorescence, which can be quantified by imaging bothchannels and calculating their ratio. By using this approach,Kardash et al. demonstrated the subcellular activation of Rac1 andRhoA at the front of migrating germ cells during zebrafishembryonic development (Kardash et al., 2010). Ratiometricdetection of FRET is easy to implement on virtually any live cellimaging system; however, corrections are required to compensatefor issues such as excitation and emission bleed-through, anddifferences in protein expression levels (Berney and Danuser,2003). A wide variety of FRET probes now exist for theinvestigation of many aspects of cell migration including adhesion,signaling and membrane dynamics (Sabouri-Ghomi et al., 2008).

FRET can also be detected as a change in the fluorescencelifetime of the donor fluorophore by fluorescence lifetime imaging(FLIM). Fluorescence lifetime is most commonly measured bytime-correlated single-photon counting (TCSPC, Box 1), althoughmany alternative approaches, which include frequency domaindetection (Lakowicz et al., 1992a) and time-gated wide-fielddetection (Elson et al., 2004), now exist. TCSPC requires the useof a pulsed laser source and is, therefore, amenable to combinationwith two-photon excitation and non-descanned detection. A keybenefit of FLIM detection of FRET (FLIM–FRET) is that thistechnique is insensitive to the expression levels of donor andacceptor fluorophores.

Using the FLIM–FRET technique a wide variety of biochemicalinteractions have been analyzed in vitro. More recently, progresshas also been made in the application of this technique to studyliving mammalian tumor tissue. In an initial study FLIM–FRETwas used to image the interaction between GFP-tagged chemokine(C-X-C motif) receptor 4 (CXCR4-GFP) and red fluorescent protein(RFP)-tagged protein kinase C (PKCA-RFP) in mammarycarcinoma xenografts (Kelleher et al., 2009). In another study,FLIM was used to image a caspase-3-based FRET probe to examinethe development of drug-resistance in a syngenic mouse tumormodel (Keese et al., 2010). In the first case, the authors usedfluorescence lifetime to demonstrate differences in receptor bindingwhen comparing deep and shallow tumor regions. In the secondstudy changes in fluorescence lifetime were analyzed to investigatethe tumor response following drug treatment. However, in bothcases FLIM–FRET was analyzed on the basis of the response ofentire fields of cells, rather than single cells or subcellular regions.More recently, our laboratory has used FLIM–FRET to examinethe subcellular activation of RhoA in a metastatic model ofpancreatic cancer driven by mutations in KRas and p53R172H. Invivo FLIM-FRET demonstrated that Rho is activated specifically


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at the poles of invasive cells, and that treatment with dasatinib ata concentration that impairs metastasis of primary tumors to theliver, specifically abolishes polar Rho activation without affectingthe basal activation level within the cell body (Timpson et al.,2011).

Fluorescence correlation spectroscopyFluorescence correlation spectroscopy (FCS) is not a conventionalimaging method. However, this powerful technique uses the samebasic hardware as CLSM or MPLSM, and several commerciallaser scanning microscopes (LSMs) can be used for both correlationspectroscopy and imaging. FCS delivers information about theconcentration, mobility and binding affinities of fluorescentlylabeled molecules in solution, in cells and, more recently, indeveloping tissues (reviewed in Haustein and Schwille, 2007).Like in CLSM, a laser beam is focused into the sample but, unlikeCLSM, the focal point remains fixed in one position and the signalis derived from the diffusion of fluorescently labeled molecules inand out of the focal volume. Comparison of fluctuations in thefluorescence intensity on a time-scale of milliseconds to secondsgenerates an autocorrelation function that can be analyzed in manydifferent ways, including cross-correlation of fluorescent signalsfrom two different molecules [fluorescence cross-correlationspectroscopy (FCCS), reviewed in Bacia et al. (Bacia et al., 2006)].

In a ‘tour de force’ study that involved many technicalrefinements, Ries et al. used FCCS to determine the concentrationsand binding affinities of fibroblast growth factor (FGF) receptors1 and 4 for FGF8 in living zebrafish embryos undergoinggastrulation (Ries et al., 2009). This work showed that the affinitiesderived from the measurement of purified proteins weresubstantially different from the apparent affinities measured in acomplex reaction in vivo. Similar to FRET, FCCS providesinformation about the association of fluorescently labeled proteins.However, detection of molecular complexes by FCCS is not limitedby the distance and orientation between the two fluorophores. Thislimitation can lead to false-negative FRET results when twoproteins interact but their associated fluorophores are too far apartor have the wrong orientation for efficient FRET.

Complementary imaging approachesAlthough FPs are the corner-stone of in vivo imaging, their use inanimal models is limited by time-consuming and expensiveproduction and breeding strategies, particularly when complexgenetic backgrounds are required. However, a variety of techniquescan be used to complement FPs. The best complementarytechniques are easy to use in conjunction with any genetic modeland provide contextual information for the interpretation of resultsderived from experiments with FP probes.

Use of exogenous fluorescent dyesPerhaps the easiest method of generating fluorescence signals inliving organisms is through the injection of fluorescent dyes. Onesimple approach is the non-specific use of inert fluorescencemarkers, such as rhodamine–dextran (Wang et al., 2002), or theuse of quantum dots (reviewed in Zrazhevskiy et al., 2010) tovisualize, for example, the vascular (Larson et al., 2003; Stroh etal., 2005) and lymphatic networks (Kim et al., 2004). Aninteresting functional aspect of using quantum dots is that, as aresult of their larger size, they tend to only extravasate underpathological conditions (Kim et al., 2009; Stroh et al., 2005).Therefore, the detection of a fluorescence signal within the

surrounding tissue provides physiologically relevant informationabout vascular leakage. One of the oldest specific labels to beused for fluorescence imaging in tissues are Hoechst dyes. Theylabel DNA through intercalation, have a high LD50 in mice, andwere originally used to label the nuclei of vascular endothelialcells (Chaplin et al., 1985; Olive et al., 1985). They can also beused effectively to label cell nuclei in the skin and intestinalcrypt.

Some drugs contain delocalized electron structures that conveyfluorescence. These include anthracycline-intercalating agents, suchas doxorubicin (Olive et al., 2009) and epirubicin (Featherstone etal., 2009); photosensitizing agents, such as porphyrins (Pathak etal., 1995); acriflavine (Kiesslich et al., 2007), and psychoactivecompounds including LSD (Fisher et al., 2003). This drugautofluorescence offers the ability to directly visualize thepenetration of therapeutic agents into their target tissue, which isimportant for many tumor types in which poor vascularization maylimit drug efficacy (Olive et al., 2009).

Injection of fluorescently labeled cells has also proven to be aneffective labeling approach. Seminal multiphoton imaging of T-and B-cell interactions was performed by purifying and labelingeach population from donor mice, injecting cells back into recipientmice and allowing them to migrate into lymph nodes prior toharvesting the nodes for ex vivo observation (Bousso et al., 2002;Miller et al., 2002; Stoll et al., 2002). Similarly, the uptake ofquantum dots into the cytoplasm has been used to label cells inculture that were subsequently injected into mice and imaged onthe basis of nano-particle fluorescence (Stroh et al., 2005; Voura etal., 2004).

Use of functional dyesOver the past 10 years there has been an active development offunctional probes, in which fluorescence is activated in responseto environmental or enzymatic properties of the sample. Weisslederand colleagues have developed a class of activatable probes thatfluoresce in the infrared range only after specific enzyme cleavage(Bremer et al., 2001). One such probe is MMPSenseTM680, whichfluoresces following cleavage by metalloproteinases – includingmatrix metallopeptidases 2, 3, 9 and 13 (Bremer et al., 2001) (Fig.1). A related probe, ProSenseTM680, is activated by proteases suchas cathepsin B, L or S and plasmin (Nahrendorf et al., 2007).Instead of measuring enzymatic activity, Blum et al. have designeda fluorescent probe that labels and inhibits cysteine cathepsins ofthe papain family in mouse tumors (Blum et al., 2007). Kuchimaruand colleagues have taken an alternative approach by using Halotagchemistry to conjugate an infrared fluorophore to the oxygen-dependent degradation and protein-transduction domains (ODD–PTD) of hypoxia-inducible factor (HIF1A) to generate a probethat is actively taken up by cells in hypoxic environments(Kuchimaru et al., 2010; Palmer et al., 2010). These functionaldyes can provide valuable information about the local tumorenvironment and are easy to combine with any genetic model.

Imaging of auto-fluorescenceCells and tissues contain a number of endogenous chromophoresthat generate autofluorescence. These include melanin, keratin,lipofuscin, flavin adenine dinucleotide (FAD) and reducednicotinamide adenine dinucleotide (NADH). Autofluorescence istypically encountered as a problem that hinders detection offluorescent probes through the generation of high backgroundfluorescence, although such background can be effectively removed


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using spectral unmixing (reviewed in Levenson and Mansfield,2006).

Alternatively, autofluorescence can be used as a primary signalto visualise biologically relevant processes (Zipfel et al., 2003). Ofparticular interest is the use of NADH fluorescence to read out theoxidative state in cells and as a marker of hypoxic regions intumors (reviewed in Provenzano et al., 2009). It has beenestablished that free NADH has a short fluorescence lifetime ofaround 400 pseconds, whereas the lifetime increases to between1000 and 3700 pseconds when it is enzyme bound (Lakowicz etal., 1992b; Niesner et al., 2008). This difference enables theproportions of free and enzyme-bound NADH to be determined byFLIM. This approach has been used at the tissue level todemonstrate that the gradient in the redox potential, which isassociated with healthy epithelial tissue, is lost in pre-canceroustissue in the DMBA-treated hamster cheek pouch model of oralcancer (Skala et al., 2007). Lifetime imaging of autofluorescencehas also been used to image a variety of tumor types (McGinty etal., 2010) and has been applied to image the differences betweenbenign and malignant breast (Tadrous et al., 2003) and colorectal(Zavadil et al., 2005) human tissue biopsies, as well as betweenbasal cell carcinomas and surrounding healthy tissue (Galletly etal., 2008).

Second harmonic generationSome crystalline materials are able to promote the combination oftwo photons of one wavelength into a single photon that possessestwice the energy and half the wavelength. This non-linear process,known as second harmonic generation (SHG; Box 1) depends onthe electric dipole properties of the crystal and only occurs underextremely intense coherent illumination. SHG is essentially non-toxic because no energy is absorbed by the sample in the process,and it is easy to combine with multiphoton excitation offluorescence because both require the high illumination intensitiesderived from a pulsed infrared laser (Fig. 2) (Brown et al., 2003).

Because of their chiral, semi-crystalline structure, collagen fibrilsare efficient second harmonic generators (Verbiest et al., 1998).Collagen SHG is routinely used to complement MPLSM of FP-labelled tumor cells by providing contextual information aboutlocal tissue structure (Wolf et al., 2009; Wyckoff et al., 2006;Zipfel et al., 2003). Collagen SHG can also be used to identifyspecific features of a tissue, such as the dense irregular connectivetissue that marks the base of the intestinal crypt (Fig. 2D). Imagesof collagen SHG have been analysed in order to derive a range ofparameters including collagen age (Williams, 2005), tissue stiffness(Raub et al., 2007; Raub et al., 2008) and remodelling of the ECM(Perentes et al., 2009). We recently used this approach to assesscollagen density and stiffness in mouse skin and, thereby,demonstrated an unexpected feedback between cancer cells andsurrounding ECM components (Samuel et al., 2011) (Fig. 2E).Through a similar non-linear optical process, third harmonicgeneration (THG; the conversion of three photons into one photonthat has three times the energy and, therefore, one third the originalwavelength) can be generated at interfaces such as the plasmamembrane (Müller, 1998). SHG and THG have been successfullycombined to study cell division in early zebrafish development(Olivier et al., 2010). In this study, the authors used SHG frommitotic spindles to identify the centre of embryonic cells and THGfrom the plasma membrane to identify cell-cell boundaries, inorder to generate a lineage tree of cell divisions over the course ofseveral hours.

Experimental designImaging experiments in vivo differ from in vitro experiments inmany ways. Not only do cells look and behave differently but alsothe questions asked are often subtly different. Furthermore, imagingcells within tissues presents many technical challenges thatconstrain in vivo experimental design and influence the choice ofimaging approach. In this section, we consider some of the issuesthat relate to in vivo experimental design and offer suggestionshow to create an experimental pipeline that promotes successful invivo imaging.

Use of intermediate systemsIn vivo experimental design presents many challenges that are notencountered in vitro, including surgical preparation of animals,anaesthesia, suppression of motion artefacts arising from breathing,heartbeat and muscle twitching, a limited timescale of observation,a reduced number of experimental observations, difficultysynchronizing experiments, and reduced optical sensitivity andresolution. Three dimensional tissue culture systems (Fig. 1; Box2) offer useful intermediates for the development of in vivo imagingapproaches and, compared with 2D cell culture, allow differentquestions to be addressed. For instance, organotypic cultures (Box2) are better approximations of the in vivo cellular environmentthan glass coverslips, and offer superior optical properties andgreater ease of use than live animals. Crucially, they are moreamenable to experimental manipulation and, therefore, provide auseful system in which to characterize probes, perform controlsand undertake initial experiments prior to in vivo work. Thedevelopment of sophisticated intermediate systems, includingrecently published models for the intestinal crypt (Sato et al.,2009) (Fig. 2B) and optical cup (Eiraku et al., 2011), suggest thatmany tissue environments will soon be amenable to in vitro study.Such models will aid the development of in vivo imagingexperiments by promoting an experimental pipeline (Fig. 1A; Table2) in which similar questions are asked, using a progressive seriesof experimental systems that are characterized by increasingbiological fidelity and decreasing experimental tractability.

Choice of experimental set-upIn this Commentary we have presented a variety of imagingtechniques and intermediate model systems, which are characterizedby many different strengths and weaknesses (Tables 1 and 2). Theart of successful experimental design lies in matching an appropriateimaging approach with the required experimental system to obtainclear and meaningful results. Fig. 3 presents a flowchart to assistin the selection of experimental systems and imaging techniques.One guideline to use in conjunction with this is to ‘start as youintend to finish’. Although wide-field CCD imaging is generallysuperior for imaging cells in culture, we have found it importantto use the same probes and imaging techniques throughout anexperimental pipeline. This helps to ensure that results arecomparable across experimental platforms. Furthermore, in thisway in vitro imaging provides a ‘best-case example’ for thesensitivity and resolution that can be achieved in vivo.

Design of cell migration experimentsThe study of cell migration serves to illustrate differences in theconstraints of in vitro and in vivo experimental design. Migrationhas long been studied using cells grown on glass coverslips, whichhas led to the analysis of features such as cell area, polarization ofshape, and the rate and persistence of migration on the basis of


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statistics derived from large numbers of cells. Recent progress inin vivo imaging, however, has highlighted the difficulty ofobserving specific events in vivo, which results in small numbersof observations and difficulties in planning and synchronizingexperiments. Imaging cell migration through tissue has also led togreater interest in the mechansims by which cells move through3D environments and the environmental signals that promotemigration. Such questions have been effectively addressed usingreconstituted collagen meshworks to study the requirement forMMP activity to hydrolyze – rather than simply squeeze through– the local environment (Wolf et al., 2007). Likewise, organotypiccultures have been useful for investigating the role of stromal cellsin promoting tumor cell invasion (Gaggioli et al., 2007). In both ofthese examples, results that were first obtained using intermediatemodel systems were subsequently confirmed in vivo.

A further difference between in vitro and in vivo design is theneed to collect information about the cellular environment invivo. An example of this is the extensive use of confocalreflectance and SHG to image collagen in cell migration studies.More such approaches are needed to image other importantenvironmental parameters, such as hypoxia and localconcentrations of growth factors and cytokines. Because the timeand place of many important disease-related events isunpredictable, in vivo studies also require the development ofsteady-state molecular read-outs as surrogates for directobservation of rare events; for example, monitoring GTPase

activation or cell adhesion dynamics as surrogates for cellmigration. Intermediate culture systems can help to overcomesome of these problems through the characterization of surrogatesunder controlled conditions.

Future perspectivesThere are many developments on the horizon that have the potentialto impact in vivo imaging. New imaging approaches, such asoptical frequency domain imaging (Vakoc et al., 2009) andstimulated Raman scattering [SRS (Saar et al., 2010)], have thepotential to complement FP imaging by visualizing tissue structurein the absence of exogenous labeling. As a Raman technique (Box1), SRS is particularly attractive because it offers the possibility ofidentifying bio-molecules label-free on the basis of their chemicalstructure. Great progress has recently been made in the developmentof super-resolution techniques including PALM, STORM andSTED (Box 1) (reviewed in Schermelleh et al., 2010); however,challenges remain in applying these methods to image dynamicstructures in living tissue. As a laser scanning technique, STEDseems to have the greatest potential for dynamic, in vivomeasurements. Single-plane illumination microscopy [SPIM(Huisken et al., 2004; Keller et al., 2008)] has produced impressiveresults in developmental systems, such as zebrafish and Drosophila;however, this approach requires embedding the entire sample in amedium such as agarose and, therefore, may be of limited use formouse cancer models.


Table 2. How to choose the right experimental systemAdvantages Disadvantages

Intermediate systems

CDM Low cost; easy and quick to make; provides complex source of growthfactors, fibrillar ECM components and receptors for integrinengagement; provides a pliable surface for the assessment of cellbehaviour; can be used in conjunction with most imaging platforms,techniques and staining protocols typically used for tissue culture.

Use for invasion studies is limited because of shallowdepth, lack of crosslinked ECM components and largepore size; lacks stromal cell interaction.

Matrigel Intermediate cost; easy and quick to make; provides complex source ofgrowth factors and ECM components; can provide a thick 2D or 3Denvironment in which cells can be grown on top or embedded withinmatrix; can be titrated to assess effects on stiffness and signalling;can be used with many imaging platforms, techniques and stainingprotocols.

Lack of crosslinked ECM components, short time coursefor invasion studies, lacks stromal cell interaction.

Organotypic culture Low cost; provides complex source of growth factors and ECMcomponents including native fibrillar crosslinked collagen I; containsstromal fibroblast providing vital tumor-stroma interactions in a 3Dcontext; can facilitate co-culture of many cell types, which allowsdirect or indirect interactions to be studied; ideally suited forlongitudinal studies; can be used with many imaging platforms,techniques and staining protocols including SHG imaging of intactECM components.

Time consuming and difficult to make; lacks vasculatureand some ECM components found in vivo.

Ex-vivo explant tissue Intermediate cost, excellent way of recapitulating important features oftissue architecture, removal and ex-vivo culture enhances imageresolution of organs and tissue that are normally inaccessible toimaging.

Limited sample thickness affects oxygenation andnutrient penetration as well as sample transparency andimage stability; tissue-type-specific limitation forlongitudinal ex-vivo culture and study.

In vivo

Transplantation Intermediate cost; mimics many key features of in vivo tissue,including 3D-host setting, multiple ECM components andinteractions; contains vasculature, areas of hypoxia, normoxia andcan harbour zones of necrosis and or invasive borders.

Inherent density and complexity of in vivo tissue posesproblems with light scattering, autofluorescence,sample stability and tissue penetration during imageacquisition; lacks complete immune system; growth ofcells outside their endogenous environment may affectinterpretation of results.

GEM Excellent method of recapitulating cell behaviour in host settingincluding intact immune system and local microenvironment withadvanced in vivo fluorescence imaging. Allows longitudinal study ofdisease progression to be monitored.

High cost, time consuming to make, organ- or tissue-specific problems with imaging as described above.

CDM, cell-derived matrix; GEM, genetically engineered mice.

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The development of automated Ti:sapphire lasers has broughtMPLSM to a wider range of users, and automated OPO lasers –for excitation of red Fps – are likely to continue this trend (Box1). However, there is an urgent need for better red FPs thatovercome photostability and toxicity problems, and can be usedfor applications requiring multiple labels – especially red FRETreporters. The success of Raichu probes for monitoring the activityof small family GTPases suggests that there are opportunities todevelop sensors for other forms of protein activity, especiallykinases involved in signal transduction. Other MPLSMdevelopments with the potential to increase the resolution andsensitivity of MPLSM at greater tissue depth are adaptive optics(Girkin et al., 2009), which increases the efficiency of excitation,and ‘total emission detection’ (Combs et al., 2010), which increasesthe sensitivity of light detection. Finally, new approaches toaccessing living tissue in vivo are required to extend the list of(murine) tissues accessible to live imaging. This includesincreased refinement of observation windows (Gligorijevic et al.,2009), exteriorizing (Coppieters et al., 2010) and stabilizing(Toiyama et al., 2010) internal organs, and stick lens (Alencar etal., 2005) and endoscopic (Hsiung et al., 2008) approaches, whichare compatible with a variety of wide-field and LSM imagingmodalities.

The combination of advanced fluorescence imaging techniqueswith genetically engineered mouse models presents a new paradigmfor research on the molecular level in vivo. Previously, cell culturehas served as a proxy because most experiments were nottechnically feasible in vivo. However, it is increasingly clear thatsome approximations made in vitro fundamentally limit the validityof results, and that basic molecular responses might be differentwithin the same cells cultured in vitro compared with those in vivo

(Serrels et al., 2009; Timpson et al., 2011). To fully realize thepotential at hand a few things have to happen. First, we mustcontinue to adapt existing methods of imaging molecularinteractions for use in living animals. Second, we must developintermediate model systems that more closely recapitulateconditions in vivo. Last, we have to develop new experimentalconcepts that are based on the synthesis of imaging technology,intermediate systems, and new mouse models.

AcknowledgementsWe thank Haley Bennett for critical reading of the manuscript, andBojona Gligorievic for critical reading of the manuscript and helpassembling the reference library. We also thank Pat Caswell, JenniferMorton, David Huels and Yafeng Ma for images provided.

FundingP.T. was supported by a fellowship from AstraZeneca, E.J.McG. andK.I.A. were supported by a Cancer Research UK core grant. Theauthors have no conflict of interest.

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Imaging molecular dynamics in vivo – from cell biology to ... · FRET High-specificity assessment of protein–protein interactions Complicated bleed through corrections required