44 Photonik international · 2013 Originally published in German in BioPhotonik 1/2013

In many biomedical research areas, analy-ses of dynamic processes in live speci-mens are increasingly gaining impor-tance. Methodological and technological advancements have made it possible to observe and analyse processes at the molecular and cellular level in three dimensions over time in the context of entire live specimens and cell clusters.

These methods provide images of struc-tures inside the specimens, and no longer require mechanical cutting. In addition, the use of fl uorescent markers like GFP (green fl uorescent protein) in genetic engineering and other advancements in genetics have enabled the labeling and the observation of proteins in cell cultures and in whole organs. In this context, fl uorescence microscopy techniques that

create optical cross sections of three-dimensional biological specimens are key technologies [1]. The new trend is to move away from the combination of static microscopy of fi xed and stained specimens and toward live imaging of the dynamics of specifi cally marked structures in vivo. And last but not least, recent pro-gress in computer speeds and networking

technology allows capture, storage, and further analyses of the large quantities of data that are generated by time series of 3D images.Despite these many advancements, fl uo-rescence microscopy, using live specimens remains challenging today. Even the most modern microscopes exhibit weaknesses in several key factors, which are neces-sary for the complete optical imaging of

an organism’s development over time. For example, the signifi cant scattering of visible light in biological tissue limits reso-lution, having negative impacts on image quality as the depth of the structure being imaged in the specimen increas-es. Similarly negative phototoxic effects of fl uorescence excitation, including the photobleaching of fl uorescent dyes due to the excitation light, shorten the maximum time span for recording images before the specimen is damaged. Finally, the limited temporal resolution and sensitivity of the fl uorescence detector have made it impossible to observe many interesting processes. However, the illumination concept of LSFM has undergone a renaissance in recent years [2,3,4]. And in particular, there have been ground-breaking developments in making LSFM methods usable in 3D fl uorescence microscopy of live biological objects [4]. Taken together, these steps address most of the aforementioned challenges, and as such, open up new possibilities for devel-opmental biologists. LSFM examinations enable better understanding of processes related to organ creation, organ function, and reveal the relationships between cel-lular processes that lead to embryogenesis and the creation of an organism. In many LSFM examples, the zebrafi sh Danio rerio and the fruit fl y Drosophila melanogaster serve as model organisms.

The method of using a light sheet

In its technical implementation, a light sheet microscope differs fundamentally from a conventional microscope stand – fl uorescence excitation and image acquisi-

Recent technological advancements have enabled the three-dimensional (3D) analysis of live specimen via the over 100-year-old concept of Light Sheet Fluorescence Microscopy (LSFM). Compared to other microscopy methods, LSFM provides high frame rates with low phototoxicity due to a fundamentally different arrangement of specimen illumination and image detection with independent optics. As a result, LSFM-based studies are becoming the fi rst choice in many of the life sciences, particularly when observing millimetre-sized live specimens in developmental biology and 3D cell biology.

Olaf Selchow, Carl Zeiss Microscopy GmbH, Jena, GermanyJan Huisken, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

Figure 1: The light sheet is projected onto the sample from the side (A), i.e. per-pendicular to the optical axis of the detection lens, hence illuminating the micro-scope’s entire focal plane. (B) The light sheet is generated either statically using a cylindrical lens or dynamically by high-frequency scanning of a laser beam

Light sheet fl uorescence microscopy and revolutionary 3D analyses of live specimens

Microscopy

Photonik international · 2013 45Originally published in German in BioPhotonik 1/2013

Microscopy

tion are carried out through completely separate optical paths that are arranged perpendicular to one another. The light sheet is generated by using either a cylin-drical lens or a scanning laser beam. The light sheet is aligned exactly with the focal plane of the detection lens (fi gure 1). Both methods of generating the light sheet have their advantages, depending on the application. While the static light sheet allows for lower laser intensities or shorter exposure times, the scanned light sheet produces a more homogeneous illumina-tion of the image area.The specimen is usually embedded in a transparent gel to be suspended vertically in the light sheet. A motorised mount allows the specimen to be translated as desired, as well as rotated around its verti-cal axis. By moving the specimen in a lin-ear manner across the light sheet, a series of images is recorded (known as a Z-stack) that is used to reconstruct the specimen in three dimensions.

The fl uorescent signal emitted by the sam-ple is then collected by a lens aligned perpendicular to the plane of illumina-tion using conventional wide-fi eld technol-ogy. A fast and sensitive camera serves for detection.

Minimal photobleaching – maximum effi ciency

LSFM, sometimes referred to as selective plane illumination microscopy (SPIM) uses the most effi cient method for fl uorescent imaging of optical sections: the light sheet specifi cally excites only dyes within the focal plane, and in doing so, an image of a single plane of the specimen is recorded as an optical section. Through this selective excitation, no signal is generated outside the focal plane, which minimises any con-tribution from out-of-focus structures. In addition, the dyes that are not involved in creating the image cannot photobleach or contribute to other phototoxic effects.

Desirable 3D image stacks are created and captured by stepping the specimen across the light sheet, while successively record-ing optical sections at different sample depths (a process known as Z-stacking). Typical light sheet thickness ranges from approximately 2 to 10 µm. This means that stacks of images from millimetre-sized specimens are often composed of 1000 optical sections. Especially when deal-ing with such large samples, established methods, such as confocal laser scanning microscopes (CLSM), have more negative effects on the sample. For example, when section number 1000 is recorded using an LSFM, it is illuminated for the fi rst time. When using a CLSM, it has already been illuminated 999 times before being record-ed. As such, the use of an LSFM reduces pototoxicity at least by a factor of 1000 in this simple example. Because the light sheet illuminates only the area of the specimen that is actively being recorded (fi gure 2), the benefi t of the lower light exposure scales with the relationship of the light sheet thickness to the sample thickness, or the number of optical sections required to complete-ly record the 3D volume. This extreme advantage when working with live speci-mens creates the foundation for a new form of 3D fl uorescence microscopy and enables biological researchers to conduct many new types of experiments [5].

A new way of preparing specimens

The geometry of an LSFM also ena-bles a new sample preparation method (fi gure 3), which decisively improves the maintenance of physiological environmen-tal conditions for the specimen. A speci-men is placed inside a chamber, which is then fi lled with an appropriately tempered,

Figure 2: Illuminated volume of an extended specimen while recording an optical section (A). In an LSFM, only the focal plane is illuminated and imaged all at once by the camera (B); light-induced bleaching can only occur in the focal plane (C). In a confocal laser scanning microscope, each image point is recorded individually in a sequential manner, and a cone along the optical axis is illuminated for each point (D), meaning that the entire specimen is exposed to light when creating just one optical section (E)

Figure 3: Typical chamber design for live specimens embedded in 3D polymer hydrogel (A and B): in order to embed the specimen, a low-concentration transparent polymer matrix in liquid state is drawn into a hollow cylinder, together with the specimen, and once inside, passes into a gel state in cylindrical shape. For imaging the specimen with the microscope, the polymer with the embedded specimen is pushed out of the cylinder (C) or left in an FEP tube (D) and immersed into the medium in the chamber. The result: Transmission contrast image of a mounted two-day-old zebra fi sh embryo (E)

46 Photonik international · 2013 Originally published in German in BioPhotonik 1/2013

Microscopy

aqueous medium and then can be posi-tioned as desired. In addition to the obvi-ous three axes of movement, the specimen can be freely rotated around the vertical axis, which enables not only selection of the ideal perspective for the experiment being conducted, but also recording of multiple image stacks from different view-ing angles, if so desired. The LSFM is set up around the experi-ment, and the observation lens looks sideways into the specimen chamber that has been converted into an incubator. This makes it possible to gently embed the prepared sample within a cylinder of a transparent 3D matrix (such as aga-rose, Matrigel, or a similar material). As a result, researchers are no longer confi ned to the two-dimensional world of cover slips, which poorly relate to physiological

conditions. This LSFM set up best sup-ports the undisturbed development of live specimens over long periods of time in a manner that most closely matches their natural environment [11].

Multiview image acquisition

Suitable software can combine the com-plementary information in different image stacks, which have been collected from various directions when rotating the specimen cylinder. Resultant 3D images (fi gure 4) [12] bring together all the details from each perspective. In certain cases, doing so achieves an isotropic spa-tial resolution in the processed data set, overcoming the reduced resolution along the optical detection axis, which is a well-known drawback in microscopy.

In addition, illumi-nating the specimen from both sides also compensates for det-rimental absorption and scattering effects within the tissue [13]: Instead of reaching only as far as to the middle of an extend-ed preparation with a conventional micro-scope, the second half is also accessible with such multiview images recorded by an LSFM. In this way, even biologi-cal preparations of several millimetres in size can be imaged as a whole.

Now LSFM is ready for commercialisation: Lightsheet Z.1 (fi gure 5) has been devel-oped by Zeiss especially for microimaging live specimens over extended periods of time. The closed housing protects the specimen and the incubation chamber from ambient infl uences and offers opto-mechanical stability required for long-term recordings of several days. Six laser wavelengths (405, 440, 488, 514, 561, and 638 nm) are available for fl uores-cence stimulation, and the detection optics are based on water immersion lenses. Depending on the application, either two CCD or two sCMOS cameras can be used simultaneously. Dedicated software and integrated data management support evaluation of the Multiview images.

Applications

The low level of phototoxicity and the extended depth of penetration make the specimen-friendly LSFM technology ideal for live images of larger biological sam-ples, such as embryos or 3D cell cultures. Figure 6 shows six points in time during the development of a zebrafi sh embryo during the fi rst day after fertilisation [11]. The ability to record its highly dynamic mor-phogenesis from several directions (in the image: 0° and 180°) allows researchers to continuously study a specimen’s processes. In conventional 3D fl uorescence microscopy, illumination levels are too high and the specimens are not embedded adequately for normal development over extended periods of time. Instead, the high frame rates possible by LSFM record the entire volume of the embryo in two or more views, and at one-minute intervals, making it possible to conduct completely new experi-ments, and thereby gain deeper insight into the processes of embryogenesis. Similarly, LSFM is revolutionising the meth-ods used by developmental biologists and genetic engineers, who regularly work with the model organism Drosophila mela-nogaster. Figure 7 shows fi ve embryonic development points of the fruit fl y, and does so in three views of the rendered volume data set of an 18-hour time series. Without the LSFM’s multiview recording ability and low level of light exposures, drosophila embryos cannot be record-ed at one-minute time intervals. Other specimens that can now be examined in ground-breaking new ways using light sheet microscopy include the roundworm C. elegans, many marine organisms (Ciona, squid, many types of plankton), and cell clusters cultivated in a 3D matrix.Established fl uorescence methods, such as FLIM, FCS, two-photon excitation, and

Figure 4: Multiview imaging: The light sheet penetrates into the area of a scat-tering specimen’s tissue that faces the illumination source (A). High-contrast fl uorescence images are recorded from the side facing the detection lens; one quadrant of the specimen is captured (B). The specimen is rotated to record all four quadrants (C). The individual data sets (D) are combined, resulting in a full 3D image of the entire specimen (E), I–IV: Views of a Drosophila melanogaster embryo, each recorded and then merged after turning the specimen by 90°

Figure 5: Layout of the Lightsheet Z.1 microscope

Photonik international · 2013 47Originally published in German in BioPhotonik 1/2013

Microscopy

Bessel beams can be combined with LSFM [6,7,9,10]. And both extremely small speci-mens, such as yeast cells, and extremely large (cm scale) specimens, which need to be fi xed and optically cleared, benefi t from the concept of LSFM. For the latter, researchers prefer a special variation of the LSFM, also known as an ultramicro-

scope. It is based on macroscopes and is not optimised for the microscopy of live objects [14]. The wide range of possible applications and the increasing number of publications on the topic demonstrate the enormous potential of this fundamentally new way to examine specimens under a microscope [6–11].

Outlook

LSFM has become popular in recent years; the further development of new techni-cal variations of LSFM illumination prin-ciples is an active fi eld of investigation [6,7]. Now researchers can observe highly dynamic intracellular processes (at mil-lisecond intervals) in the overall context of the tissue and during morphogenesis in developing specimens over a period of hours and days. Light sheet illumination is becoming one of the most important tools in 3D fl uorescence microscopy, not only because different methods of sample preparation and mounting are possible compared to conventional microscopes, but also because of the effi cient ways of creating optical sections of spatially extended specimens.

New areas of application, driven both by technical advancements and by funda-mental questions in biological research, may trigger a paradigm shift in the micro-scopic examination of live specimens. We expect to see improved resolution by using thinner light sheets, deeper pen-etration into scattering tissue, faster and

more sensitive detectors, experimental variations, such as tools for image manip-ulation or ablation, as well as methods such as FCS or FLIM [8,9,10]. Computer speeds and networking technol-ogy are also a driving force behind excit-ing advancements in this fi eld. Long time series of large volume data sets result in enormous amounts of data – up to several terabytes per experiment – which require a high-performance infrastructure for man-agement and storage. The computer indus-try has already been addressing this issue for some time [15]. New software, and hardware developments during the past few years, including LSFM, continue to support methodological breakthroughs in biology and computational sciences.

Literature:[1] T. Kues, S. Wiesner, M. Marx, Optical sectioning

methods in fl uorescence microscopy, Photonik international 2010

[2] H. Siedentopf, R. Zsigmondy, Über Sichtbarma-chung und Größenbestimmung ultramikoskopis-cher Teilchen, mit besonderer Anwendung auf Goldrubingläser, Ann. Physik 1903, 10:1-39

[3] A.H. Voie, D.H. Burns, F.A. Spelman, Orthogo-nal-plane fl uorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens, J Microsc, Oxford 1993, 170:229-236

[4] J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, E.H.K. Stelzer, Optical sectioning deep inside live embryos by selective plane illumination micros-copy, Science 305: 1007-1009 (2004)

[5] E.G. Reynaud, U. Krzic, K. Greger, E.H.K. Stelzer, Light sheet-based fl uorescence microscopy: more dimensions, more photons, and less photodam-age, HFSP Journal 2: 266-275 (2008)

[6] P.A. Santi, Light sheet fl uorescence microscopy: A review, Journal of Histochemistry and Cytochemis-try 59: 129-138 (2011)

[7] J. Huisken, D.Y.R. Stainier, Selective plane illumi-nation microscopy techniques in developmental biology, Development 136: 1963-1975 (2009)

[8] A.B. Arrenberg, D.Y.R. Stainier, H. Baier, J. Huisken, Optogenetic control of cardiac function, Science 2010, 330:971-974

[9] C.J. Engelbrecht, K. Greger, E.G. Reynaud, U. Krzic, J. Colombelli, E.H.K. Stelzer, Three-dimensional laser microsurgery in light-sheet based microscopy (SPIM), Opt Express 2007, 15:6420-6430

[10] T. Wohland, X. Shi, J. Sankaran, E.H.K. Stelzer, Single plane illumination fl uorescence correla-tion spectroscopy (SPIMFCS) probes inhomogene-ous three-dimensional environments, Opt Express 2010, 18:10627-10641

[11] A. Kaufmann, M. Mickoleit, M. Weber, J. Huisken, Multilayer mounting enables long-term imaging of zebrafi sh development in a light sheet microscope, Development. 2012 Sep;139(17):3242-7

[12] S. Preibisch, S. Saalfeld, J. Schindelin, P. Tomancak, Software for bead-based registration of selective plane illumination microscopy data, Nat Methods. 2010 Jun;7(6):418-9

[13] J. Huisken, D.Y.R. Stainier, Even fl uorescence excitation by multidirectional selective plane illu-mination microscopy (mSPIM), Opt Lett. 2007 Sep 1;32(17):2608-10

[14] H.U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C.P. Mauch, K. Deininger, J.M. Deussing, M. Eder, W. Zieglgänsberger, K. Becker, Ultramicroscopy: three-dimensional visualization of neuronal net-works in the whole mouse brain, Nat Methods. 2007 April; 4(6):331-6

[15] T. Hey, S. Tansley, K. Tolle (Eds.), The Fourth Para-digm Data-Intensive Scientifi c Discovery, Microsoft Research, Redmond, Washington, USA 2009

Author contact:Dr. Olaf SelchowProduct ManagerBioSciences DivisionCarl Zeiss Microscopy GmbHCarl-Zeiss-Promenade 1007745 JenaGermanyTel. +49/3641/64-2089eMail: olaf.selchow@zeiss.comInternet: www.zeiss.com/microscopy

Dr. Jan HuiskenResearch Group LeaderMax Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)Pfotenhauerstr. 10801307 DresdenGermanyTel. +49/351/210-2487eMail: huisken@mpi-cbg.deInternet: www.mpi-cbg.de/research/

research-groups/jan-huisken.html

Figure 6: LSFM microscopy of an entire zebrafi sh embryo (featuring nucleus stain-ing) from two different perspectives, between 8 and 17 hours after fertilisation

Figure 7: Embryonic development and morphogenesis of the model organism Drosophila melanogaster