Wideﬁeld vs. confocal microscope · 2016. 9. 4. · Antonio Miranda 18 Live imaging E5.5 mouse embryos with Z1 Matthew Stower • Two image volumes (at 180°) every 5 min • 200

Confocal and Lightsheet imaging

2016 Live Imaging WorkshopUniversity of São Paulo

Shankar SrinivasProfessor of Developmental Biology

Dept. Physiology Anatomy & GeneticsUniversity of Oxford

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Widefield vs. confocal microscope

http://www.olympusmicro.com

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A pinhole blocks out of focus light

http://www.leica-microsystems.com

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Optical sectioning with a confocal microscope

Watanabe, Biggins et al. Development 2014

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Confocal time-lapse microscopy

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Confocal vs. lightsheet microscopy

Supatto et al. 2011

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Light sheet microscopy – the basics

Huisken and Stainier 2009

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Light sheet microscopy

Keller et al. 2008

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Light sheet microscopy

Keller et al. 2008

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Light sheet microscopy – the basics

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Faster and less damaging


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Sample mounting

1965REVIEWDevelopment 136 (12)

Glossary in Box 1) or the higher the laser power needs to be, whichcan lead to noise and fluorophore saturation (see Glossary in Box 1),respectively. The excitation light is not axially confined, i.e. the wholedepth of the sample is exposed to the fluorescence excitation light, andhence the sample is potentially bleached or damaged even when onlya single image is acquired. These negative effects scale with thenumber of planes that are acquired: In a stack of 100 planes, the lastplane imaged has already been exposed 99 times. In addition, theefficiency of the photomultipliers (see Glossary in Box 1) of aconfocal microscope is approximately two to three times worse thanthe efficiency of modern cameras. Nevertheless, the confocalmicroscope is a routine instrument found in many laboratories. Itsoptical sectioning properties make it ideally suited for the 3D imagingof tissue sections of thickness in the order of 100 µm and for time-lapse experiments of tissue dynamics near the surface of relativelytransparent embryos, such as those of the zebrafish (e.g. Haas andGilmour, 2006; Rembold et al., 2006).

In the third approach to optical sectioning, fluorescence is excitedonly where it is needed, which leaves the remainder of the sampleunexposed and therefore free of photo-bleaching and -damage. Thisapproach is taken in multi-photon microscopy and in fluorescencelight-sheet microscopy in two very different ways. In multi-photonmicroscopy, a tightly focused high-intensity infrared laser pulsepenetrates the sample and excites fluorescence only in a smallvolume in the focus of the beam. This nonlinear multi-photonexcitation prevents excitation outside of the focal plane. The use oflonger wavelengths employed for multi-photon microscopyincreases the penetration depth (up to 700 µm) (Helmchen andDenk, 2005) but reduces the resolution compared with single-photonconfocal microscopy. In addition, high power laser pulses arerequired, and over time the whole sample can suffer from thedetrimental effects of light, such as heating, photo-bleaching andphoto-damage. Absorption of infrared light can severely damagespecimens, one of the reasons why zebrafish embryos are routinelytreated to be pigment-free when imaged with multi-photonmicroscopy (e.g. Kamei and Weinstein, 2005). In light-sheetmicroscopy, however, a different approach is taken to selectivelyilluminate the area of interest while at the same time preventing thesample from damage, as discussed in the following section.

Light-sheet fluorescence microscopy: concept andcomponentsThe idea behind SPIM and other light-sheet-based microscopytechniques is to illuminate the sample from the side in a well-definedvolume around the focal plane of the detection optics (see Box 2).Even though there are many different implementations of this idea(see below and Table 1), the common general principles remain thesame and are illustrated in Fig. 1. The following sections describethe illumination, detection and photo-manipulation units in SPIMand illustrate the major benefits of this approach, such as opticalsectioning, reduced photo-bleaching and high-speed acquisition (seeBox 3 for a historical perspective of the concept).

IlluminationThe ideal scenario, a perfectly thin optical section, would beobtained if a sheet of light illuminated only the focal plane of thedetection objective. Preferably, this sheet should be as thin aspossible and uniform across the field of view (see Glossary in Box1). However, diffraction sets a limit on how thin the illuminatedvolume can be. In SPIM, a currently widely adopted light-sheetmicroscope system, cylindrical optics are used to create a sheet oflight of varying thickness. The light converges towards the sample

and diverges away from it. The waist of the light-sheet is positionedin the center of the field of view. The dimensions of the light-sheetcan be adapted to different sample sizes: for smaller samples (20-100 µm), the light-sheet can be made very thin (~1 µm); whereas forlarger samples (1-5 mm), the sheet has to be thicker (~5-10 µm) toremain relatively uniform across the field of view (Engelbrecht andStelzer, 2006).

Alternatively, the sheet of light can be generated by focusing a laserbeam to a single line and by rapidly scanning it up and down duringthe exposure time. This concept was recently introduced as digitalscanned laser light-sheet fluorescence microscopy (DSLM) (Keller etal., 2008). The benefit of this technique is the uniformity of the light-sheet intensity profile and the ability to control its height throughcomputer-controlled scan mirrors (see Glossary in Box 1). In otherlight-sheet-based techniques, the laser light is expanded and croppedwith apertures to cover the field of view of the detection lens, resultingin a less uniform intensity profile. Owing to the sequential lineillumination in DSLM, the beam illuminates only a fraction of thefinal image at any one time. Therefore, the sample is typically exposedto a local light intensity that is ~300 times higher than in SPIM in order

Box 2. SPIM basicsThe basic principle of SPIM is to illuminate the sample from the sidein a well-defined volume around the focal plane of the detectionoptics (Fig. 1). The illumination and the detection path are distinctand perpendicular to each other. The illuminated plane is aligned tocoincide with the focal plane of the detection objective. Hence, theillumination light can be shaped and positioned independently of thedetection optics and thus does not share the same constraints, suchas numerical aperture and working distance. The sample is placed atthe intersection of the illumination and the detection axes. Theillumination sheet excites fluorescence in the sample, which iscollected by the detection optics and imaged onto a camera. For asingle 2D image, no scanning is necessary. To image the 3D extentof the sample, the sample is moved along the detection axis in astepwise fashion, and a stack of images is acquired.

Sample

Objective lens

Fluorescence

Illumination

Focal plane

Light sheet

Excitation

Detection

Sample

Focal plane

Light sheet

Detectio

Fig. 1. The concept behind fluorescence light-sheet microscopy. Inlight-sheet microscopy, fluorescence excitation (blue arrow) anddetection (green arrow) are split into two distinct optical paths. Theillumination axis is orthogonal to the detection axis. A microscopeobjective lens and common widefield optics are used to image thesample onto a camera (not shown). The illumination optics aredesigned to illuminate a very thin volume around the focal plane of thedetection objective. Many different implementations of this principleexist, however, the most common one is the generation of a sheet oflaser light that illuminates the sample in the focal plane from one side.


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Sample presentation


Air

Water

Oil

Medium

Embryo

Agarose

Plug

Support

FEP tube

Matthew StowerAntonio Miranda

Tomoko Watanabe

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Stelzer mDSLM

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Lightsheet Z1

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Matthew Stower

• 256 sections • 1µm interval • Exp 29.97ms • 1x zoom • lightsheet 4.56 µm thick • acquisition <40s • laser: 488 & 561 • dual side illumination

Zeiss Z1 compared to Zeiss LSM710

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Apical projections from VE cellsLifeAct-GFP embryo

LifeAct-GFP embryo

Antonio Miranda

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Live imaging E5.5 mouse embryos with Z1

Matthew Stower

• Two image volumes (at 180°) every 5 min • 200 sections @ 1 uM interval • 1% 30mW 488nm laser • 30 ms exposure per section • 4 hour duration

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500 µmAntonio Miranda

5x#Air#Objective#0.5x#Zoom#1100#slice#4#min#Acquisition#E8.5#488#Phaloidin#

Large field of view

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20x#objective#1.0x#Zoom#0.48#um#interval#139#µm#total#1.27#min Antonio Miranda

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Multiple illumination and views

Weber and Huisken 2011

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Multiple illumination

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Matthew Stower


View 1 View 2

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Matthew Stower


View 1 View 2

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Challenges

• Sample mounting (particularly from mouse)

• Image processing – terabytes of data

• Automated segmentation

• Data visualisation

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Acknowledgments

Satish Arcot JayaramDi HuKarolis LeonaviciusAntonio MirandaEleni PanousopoulouChristophe RoyerMatthew StowerRichard TyserTomoko Watanabe

George TrichasBradley Joyce

Tristan RodriguezRoland Wedlich-Soldner

Ian DobbieEva WegelIlan Davis

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Wideﬁeld vs. confocal microscope · 2016. 9. 4. · Antonio Miranda 18 Live imaging E5.5 mouse embryos with Z1 Matthew Stower • Two image volumes (at 180°) every 5 min • 200

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