Download Light-Sheet-Based Fluorescence Microscopy for Three

Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
Topic Introduction
Light-Sheet-Based Fluorescence Microscopy for ThreeDimensional Imaging of Biological Samples
Jim Swoger, Francesco Pampaloni, and Ernst H.K. Stelzer
In modern biology, most optical imaging technologies are applied to two-dimensional cell culture
systems; that is, they are used in a cellular context that is defined by hard and flat surfaces. However, a
physiological context is not found in single cells cultivated on coverslips. It requires the complex
three-dimensional (3D) relationship of cells cultivated in extracellular matrix (ECM) gels, tissue
sections, or in naturally developing organisms. In fact, the number of applications of 3D cell cultures
in basic research as well as in drug discovery and toxicity testing has been increasing over the past few
years. Unfortunately, the imaging of highly scattering multicellular specimens is still challenging. The
main issues are the limited optical penetration depth, the phototoxicity, and the fluorophore bleaching. Light-sheet-based fluorescence microscopy (LSFM) overcomes many drawbacks of conventional
fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The basic idea is to illuminate the specimen from
the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence
microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small
volume close to the focal plane are intrinsic properties of LSFM. We discuss the basic principles of
LSFM and methods for the preparation, embedding, and imaging of 3D specimens used in the life
sciences in an implementation of LSFM known as the single (or selective) plane illumination microscope (SPIM).
TRADITIONAL OPTICAL IMAGING TECHNIQUES
An understanding of modern developmental biology requires us to monitor the complex 3D relationship of cells, whether cultivated (e.g., in an ECM-based gel), in developing embryos, or in tissue
sections (Pampaloni et al. 2007). However, the observation and the optical manipulation of multicellular biological specimens remains a challenge, principally for two reasons:
•
Such specimens are optically dense. They scatter and absorb light; thus, the delivery of the probing
light and the collection of the signal light tend to become inefficient.
•
In addition to the fluorophores, many endogenous biochemical compounds absorb light and suffer
phototoxicity, which can induce damage or can kill the specimen (Pampaloni et al. 2007). The
situation is particularly dramatic in confocal fluorescence microscopy. Even when only a single
plane is observed, the entire specimen is illuminated. Recording stacks of images along the optical
z-axis thus illuminates the entire specimen once for each plane. Hence, cultured cells are illuminated 10–20 times and fish embryos are illuminated 100–300 times more often than they are
observed. As we will see in the next section, this can be avoided by a simple change to the
optical arrangement.
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011.
© 2014 Cold Spring Harbor Laboratory Press
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
1
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
J. Swoger et al.
Traditionally, biological imaging is dominated by wide-field imaging techniques, in which the full
field of view is captured in a single exposure. Although invaluable for two-dimensional applications,
conventional wide-field fluorescence microscopy provides no optical sectioning. This means that any
image recorded in a particular plane in a thick specimen will contain both the in-focus and the out-offocus signals from the fluorophores above and below the plane of focus. This is the fundamental
limitation of conventional wide-field fluorescence light microscopy and makes the construction of
quantitative 3D models even of moderately thick specimens difficult, if not impossible, without some
form of further innovation.
One obvious way to avoid the problem of out-of-focus light in wide-field fluorescence microscopy
is to physically section the sample so that material that would be a source of out-of-focus light is
physically removed. Traditionally, the sample is mechanically cut into thin slices, which are treated as
two-dimensional specimens, whose images can be computationally assembled into a 3D reconstruction. However, because it is destructive, the technique is not useful for the live time-lapse imaging that
is essential in the modern life sciences. In addition, physical sectioning is a low-throughput technique
that is not suitable for automated scanning of many specimens.
Over the last few decades, confocal fluorescence microscopy (Pawley 2006) has become a standard
tool for biological imaging in which 3D data sets are required. This is a technique whereby, through
the use of a confocal pinhole, images containing mainly in-focus information are acquired. Therefore,
an optical rather than a physical sectioning of the specimen is achieved. By using confocal fluorescence
microscopes to localize fluorophore distributions in three dimensions, studies of complex biological
structures can be performed with fixed and live samples. Although confocal fluorescence microscopy
does provide 3D voxel data sets, the resolution along the axial direction of a confocal fluorescence
microscope is always lower than along the lateral directions (Pawley 2006). This is because of the
physics of diffraction. This resolution anisotropy can be reduced by working with high-numericalaperture (NA) objective lenses. However, in practical lens design, a very large NA is accompanied by a
small working distance (generally substantially less than a millimeter). In addition to the mechanical
limitations imposed by the working distance of the lens, the image quality in confocal data stacks
degenerates rapidly with depth because of the high optical scattering caused by the refractive index
heterogeneity of thick biological specimens. Taken together, these properties limit the suitability of
confocal fluorescence microscopy for imaging thick 3D samples.
An alternative to confocal fluorescence microscopy that provides optically sectioned data sets
through a different physical mechanism is multiphoton microscopy (Denk et al. 1990; Konig
2000). Instead of using a pinhole to discriminate against out-of-focus light, nonlinear interactions
between the excitation light and the fluorophores are used to ensure that only molecules exposed to
very high illumination intensities (i.e., those located directly in the focal plane) are excited. The
resulting optically sectioned images produced by multiphoton microscopy are, in many respects,
similar to those recorded with confocal fluorescence microscopy. However, because of the longer
wavelengths used, the resolution is considerably worse along all three dimensions than in a confocal or
a conventional fluorescence microscope (Stelzer and Lindek 1994).
Several other imaging methods have been used successfully in developmental biology. Optical
coherence tomography, a technique based on interferometric detection of low-coherence illumination, has received considerable attention in the past decade as a technique for noninvasive biological
imaging. Magnetic resonance imaging (MRI; Bamforth et al. 2011; Metscher 2011; Ruffins and Jacobs
2011), ultrasound imaging (Foster and Brown 2011), and microcomputed tomography (μCT; Quintana and Sharpe 2011) all allow imaging of relatively large samples and are, therefore, suitable for
imaging the later stages of development of specimens such as mouse embryos. Although, in many
ways, these are ideal methods for imaging a range of samples, a common drawback is that they cannot
produce images using the wide range of highly specific chemical and genetically encoded fluorophores
that have become invaluable in light microscopy (Lichtman and Conchello 2005). Optical projection
tomography (OPT; Gu et al. 2011; Quintana and Sharpe 2011) is one of the few optical imaging
techniques suitable for developmental biology that is capable of operating with both fluorescence and
absorption contrasts.
2
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
LSFM for 3D Imaging
PRINCIPLES OF LSFM
The basic idea of LSFM is to use a light sheet that illuminates the specimen from the side and that
overlaps with the focal plane of a wide-field fluorescence microscope (Huisken et al. 2004). In contrast
to an epifluorescence arrangement, which uses the same lens, a light-sheet-based orthogonal/
azimuthal fluorescence arrangement uses two independent lenses for illumination and detection.
An LSFM system is able to provide optical sectioning without phototoxic damage or photobleaching
outside of a small volume located close to the focal plane. LSFM takes advantage of modern chargecoupled device (CCD) camera technologies to generate images with a signal-to-noise ratio (SNR) that,
for a given fluorophore, objective lens, and specimen, is at least one order of magnitude better than
that of a confocal fluorescence microscope (Keller et al. 2008). (Note that good-quality confocal
fluorescence images have an SNR between 10 and 40 [given by the square root of the number of
detected photons; see the Scientific Volume Imaging-Wiki, http://support.svi.nl/wiki/WikiWikiWeb].
CCD cameras used in fluorescence microscopy have an SNR between 100 and 1000 [defined as the
square root of the full-well capacity]. For example, the ORCA-AG [Hamamatsu] has a full-well
capacity of 18,000 electrons, which gives an SNR of 130.)
The idea of using light-sheet illumination for microscopy has occurred to various groups at
different times and has been implemented in a number of configurations. These are summarized
in Table 1. All of these techniques intrinsically provide optical sectioning and, except for the original
implementation of ultramicroscopy (Siedentopf and Zsigmondy 1903), which works with incoherent
illumination and scattered light contrast, all rely on laser illumination focused in one dimension to
excite fluorescence. Here, we refer to an instrument known as a single (or selective) plane illumination
microscope (SPIM). The optical properties of LSFM can be calculated by following the approach in
Engelbrecht et al. (2007) and Stelzer and Lindek (1994). In SPIM, the sample is illuminated by a sheet
of laser light. A laser array, in combination with dichroic mirrors and an acousto-optical tunable filter
(AOTF), is used to provide a variety of wavelengths for the excitation of different fluorescent dyes
(Fig. 1A). Cylindrical optics (Huisken et al. 2004; Greger et al. 2007) or a scanning mechanism in the
digital scanned laser light-sheet fluorescence microscopy (DSLM) (Keller et al. 2008) is used to focus a
collimated laser beam along one dimension, thereby creating a single plane of light. The specimen is
embedded in a cylinder of agarose or other mounting gel, which is generally immersed in an aqueous
medium. The fluorescence emitted from the narrow volume of the specimen that is excited by the light
TABLE 1. Some methods that use LSFM
Name
Distinctions/features
Reference(s)
Scattering ultramicroscopya
Developed to study subwavelength
colloidal particles (these are illuminated
from the side by an incoherent
illumination)
Point-scanning device using an orthogonal
or, in general, azimuthal optical
arrangement
Applied to fixed, chemically cleared
samples
Developed for imaging very large and
chemically cleared organs, such as the
guinea pig cochlea
Used in microbial oceanography
Developed for live, fast, multiple-view and
multiple-dye imaging of embryos, cell
clusters, and single cells
Illumination optics mechanically coupled
to detection objective lens
Siedentopf and Zsigmondy 1903
Confocal θ fluorescence microscopya
Fluorescence ultramicroscopy
Orthogonal-plane fluorescence optical
sectioning device
Thin laser light-sheet microscopy
SPIM, scanned light-sheet microscopy
(DSLM)
Objective-coupled planar illumination
microscopy
Stelzer and Lindek 1994
Dodt et al. 2007
Voie et al. 1993; Buytaert and Dirckx
2009
Fuchs et al. 2002
Huisken et al. 2004; Keller et al.
2008
Holekamp et al. 2008; Turaga and
Holy 2008
The first two examples (original ultramicroscopy and confocal θ fluorescence microscopy) are forerunner techniques exploiting the idea of
side illumination of the specimen.
a
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
3
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
J. Swoger et al.
A
Camera
Detection
TL
FW
OL
LA
AOTF
BEO
S
CO
Light-sheet illumination
B
C
Single-plane acquisition
Conventional and confocal
Illumination
Light-sheet microscopy
Detection
3D data acquisition
Light-sheet microscopy
Detection
Single view
i)
Illumination
Multiview
Photodamage
0°
ii)
Specimen
60°
Advantages of the light-sheet-based approach:
i) Intrinsic optical sectioning, i.e., capability of 3D imaging
ii) Reduction of photodamage in 3D image stacks by a factor
of n, where n is equal to the number of planes in the stack
120°
Improved axial resolution
reconstruction of large specimen
FIGURE 1. Principles of light-sheet-based microscopy. (A) The overall layout of an SPIM. The emission from a laser
array (LA) of various wavelengths for exciting fluorescence is combined into a single beam. An acousto-optic tunable
filter (AOTF) is used to select the wavelengths and the intensity of the excitation beam. The beam expanding and
cylindrical optics (BEO and CO) create the light sheet that illuminates the sample (S). Detection is through a wide-field
fluorescence microscope consisting of an objective lens (OL), a filter wheel (FW) that contains filters for rejection of
scattered excitation light and background fluorescence, a tube lens (TL), and a camera. (B) Comparison of sample
illumination and fluorescence detection in conventional/confocal microscopy and in LSFM. (i) The entire region of
interest in the specimen is illuminated in conventional/confocal microscopy, although only a single plane in the
specimen is being observed. (ii) In contrast, no photodamage is inflicted outside the in-focus plane of the detection
system in the LSFM. (C ) Three-dimensional imaging in LSFM is performed by moving the specimen step by step
through the light sheet while recording two-dimensional images. In multiple-view imaging, the same volume inside
the specimen or even inside the entire specimen is recorded along several angles. The resulting multiple-view
information is combined into a single image stack by a fusion algorithm. (B,C, Adapted, with permission, from
Keller and Stelzer 2008.)
sheet is imaged onto a camera using standard fluorescence microscopy optics: an objective lens, a filter
wheel, and a tube lens (Fig. 1A). The sample can be moved along the detection axis (i.e., perpendicular
to the plane of the light sheet) and imaged in a stepwise fashion to generate a 3D data set. The SPIM
setup usually includes a mechanism for rotating the specimen, so that its orientation can be optimized
for imaging in a particular experiment or for acquiring image stacks from multiple directions
(Fig. 1C).
With an SPIM, optical sectioning is obtained in a direct and efficient way. The sectioning capability
is defined by the thickness of the light sheet (typically 2–6 µm, depending on the extent of the field of
4
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
LSFM for 3D Imaging
view). By illuminating only the plane of interest, no out-of-focus light is created, and there is no need
for a confocal pinhole for the discrimination of this background (Fig. 1Bi). Therefore, photobleaching
and photodamage are less than with confocal or wide-field fluorescence microscopy, in which the
complete specimen is illuminated even when observing only a single plane (Fig. 1Bii). Objective lenses
with a long working distance and a relatively low NA can be used for detection when imaging large
specimens (a few millimeters). The lateral resolution is defined by the NA of the detection objective
lens, whereas the axial resolution of the system is determined by the light-sheet thickness, when it is
thinner than the axial detection resolution. The combination of optical sectioning, long working
distances, and minimal photodamage make LSFM ideal for imaging of samples typically of interest
in developmental and 3D multicellular biology. A schematic of an LSFM setup can be found in Keller
et al. (2011).
One of the advantages of an SPIM is that the sample can be rotated and can be observed from
multiple orientations (Fig. 1C). Three-dimensional stacks can be acquired from different directions,
and the images can be computationally fused into a single representation of the sample (Swoger et al.
2007; Verveer et al. 2007). This has the advantage of improving the resolution in small transparent
specimens and of improving the uniformity of image quality in larger specimens for which absorption
and/or scattering are significant limitations to imaging. Examples are provided in the next section.
SPIM APPLICATIONS
Figure 2 illustrates the effect of multiple-view image fusion on a grain of paper mulberry pollen. The
same grain of pollen has been imaged along 18 directions, two of which are shown in Figure 2, A and
B. Note that although the light-sheet illumination improves the axial resolution over wide-field or
confocal fluorescence microscopy with the same detection objective lens, the resolution is anisotropic.
This is indicated schematically by the red ellipses: These point-spread functions (PSFs) are narrowest
along the direction with the highest resolution. In the image shown in Figure 2A, the resolution is
good vertically but poor horizontally; in Figure 2B, which was acquired with the sample oriented at
90˚ to the orientation in Figure 2A, the situation is the reverse.
By recording 18 three-dimensional data sets (i.e., a rotation of the sample in 20˚ steps), aligning
them, and taking the average, the image shown in Figure 2C is obtained. It can be seen that the
resolution (the PSF is represented by the red circle) is quite high along all directions and approximately equal to the resolution along the best axis in either Figure 2, A or B. A further improvement in
resolution and contrast can be obtained by using a multiple-view image deconvolution (Verveer et al.
2007) rather than the simple average used in Figure 2C. The result is illustrated in Figure 2D, in which
the interior details and the quasispherical surface layer or sporoderm of the pollen grain can be
clearly seen.
If the sample is nearly optically transparent (e.g., the pollen grain in Fig. 2) and the different
multiple-view data sets overlap, then the multiple-view reconstruction compensates for the anisotropy
FIGURE 2. SPIM of paper mulberry pollen, multiview reconstruction. Slices from volumetric data stacks of an autofluorescent grain of paper mulberry pollen are imaged with an SPIM. (A,B) Single-view images along orthogonal
directions. (C ) Combination of 18 views by averaging. (D) Combination of the same 18 views as in (C ) by multipleview deconvolution. Scale bar, 5 µm. (Image acquisition by Klaus Greger, EMBL, Heidelberg.)
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
5
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
J. Swoger et al.
in each single view and provides an isotropic resolution. In the case of a more translucent or opaque
sample (e.g., the Drosophila melanogaster embryo in Fig. 3), additional views provide information
about regions of the sample that is not visible in a single view. Here, the multiple-view reconstruction
combines the information into a complete image of the sample. It can be seen in the single-view images
(Fig. 3A,C) that some regions are not well resolved (arrowheads) or are entirely missing (arrows)
because they were imaged through thick optically scattering tissue. When a multiple-view reconstruction is performed (Fig. 3B,D) the processing combines the best-resolved features from each individual
view into a single 3D representation with uniform high-resolution coverage of the sample.
Imaging 3D Cell Biology Specimens with a SPIM
Increasingly, cell biologists appreciate that conventional two-dimensional cell cultures do not provide
a physiological environment for the cell (Pampaloni et al. 2007; Mazzoleni et al. 2009). The surfaces of
plastic and glass substrates are flat and rigid. In contrast, a tissue is a 3D soft-matter structure. There
are many instances in which cells have drastically different phenotypes in two dimensions than in
three dimensions. For example, fibroblasts cultured in two dimensions have a flat shape dissimilar
from the bipolar/stellate shape found in tissue. Strikingly, culturing fibroblasts in 3D collagen induces
their tissue-specific phenotype (Beningo et al. 2004; Rhee et al. 2007; Rhee and Grinnell 2007). It is
currently acknowledged that establishing 3D cell–cell interactions and using ECMs or 3D gels reduces
the gap between cell culture and real tissue. There are strong indications that toxicity assays based on
3D cultures of human cells provide more accurate results than two-dimensional cultures (Pampaloni
et al. 2009). Three-dimensional cultures are promising for toxicity screening of chemicals and to sort
out toxic substances at early stages of drug discovery. Imaging of fluorophore-tagged targets provides
the specificity that is required to perform detailed studies at the molecular level in live 3D cultures.
Novel advanced fluorescence imaging technologies are required to study the behavior of living 3D
cultures. LSFM provides an excellent tool to study the live behavior of 3D cellular systems with high
spatial resolution and for long periods of time. Light-sheet illumination provides optical sectioning
deep inside large 3D tissue. The extremely low illumination level induces minimum photobleaching
and photodamage.
Embedding in agarose is a well-established approach to stably mount specimens for imaging with
an SPIM. The agarose concentration used is 0.5%–1% (w/v) in buffer or culture medium. Agarose is a
nearly ideal embedding medium because it is optically clear and introduces very limited aberrations.
Agarose is nontoxic and, in most cases, does not interfere with the physiology of a living specimen. For
detailed protocols for embedding and imaging with an SPIM, see Imaging Cellular Spheroids with a
FIGURE 3. SPIM of a D. melanogaster embryo. Maximum-value projections through volumetric data stacks of a fruit fly
embryo with the trachea labeled with green fluorescent protein. (A) Single-view data set, projected along the dorsoventral axis. (B) Multiple-view fusion of 12 views similar to that shown in (A) but taken with different orientations of the
sample. (C,D) Projections corresponding to those in (A) and (B) but along the lateral axis of the embryo. Scale bar, 100
µm. (Sample courtesy of Ferenc Jankovics and Damian Brunner, EMBL, Heidelberg; image acquisition by Klaus
Greger, EMBL, Heidelberg.)
6
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
LSFM for 3D Imaging
Single (Selective) Plane Illumination Microscope (Swoger et al. 2014a) and Imaging MDCK Cysts
with a Single (Selective) Plane Illumination Microscope (Swoger et al. 2014b).
SUMMARY
We have described the SPIM, a microscope that uses the principles of LSFM for imaging 3D specimens, such as are commonly studied in developmental biology and in 3D cell culturing. The principal
advantages of SPIM over techniques such as wide-field or confocal microscopy are as follows:
•
•
•
reduced photobleaching and phototoxicity
the ability to achieve high resolution with long working distance objectives, which allows imaging
of thick 3D specimens
the option of achieving high isotropic resolution via multiple-view reconstructions
Autofluorescent pollen grains and genetically labeled D. melanogaster embryos were used to show
the advantages of multiview reconstructions, which (to the best of our knowledge) is not a technique
available in systems other than SPIM. The ability of SPIM to generate high-resolution images of live
samples as they develop over time will lead to novel types of experiments in developmental biology
and 3D cell cultures.
REFERENCES
Bamforth SD, Schneider JE, Bhattacharya S. 2011. High-throughput analysis
of mouse embryos by magnetic resonance imaging. In Imaging in developmental biology: A laboratory manual (ed. J Sharpe, RO Wong). Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Beningo KA, Dembo M, Wang YL. 2004. Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors. Proc Natl Acad Sci 101:
18024–18029.
Buytaert JAN, Dirckx JJJ. 2009. Tomographic imaging of macroscopic
biomedical objects in high resolution and three dimensions using
orthogonal-plane fluorescence optical sectioning. Appl Opt 48: 941–
948.
Denk W, Strickler JH, Webb WW. 1990. Two-photon laser scanning fluorescence microscopy. Science 248: 73–76.
Dodt HU, Leischner U, Schierloh A, Jahrling N, Mauch CP, Deininger K,
Deussing JM, Eder M, Zieglgansberger W, Becker K. 2007. Ultramicroscopy: Three-dimensional visualization of neuronal networks in the
whole mouse brain. Nat Methods 4: 331–336.
Engelbrecht CJ, Greger K, Reynaud EG, Krzic U, Colombelli J, Stelzer EH.
2007. Three-dimensional laser microsurgery in light-sheet based microscopy (SPIM). Opt Express 15: 6420–6430.
Foster SF, Brown AS. 2011. Micro-ultrasound and its application to longitudinal studies of mouse eye development and disease. In Imaging in
developmental biology: A laboratory manual (ed. J Sharpe, RO Wong).
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Fuchs E, Jaffe J, Long R, Azam F. 2002. Thin laser light sheet microscope for
microbial oceanography. Opt Express 10: 145–154.
Greger K, Swoger J, Stelzer EH. 2007. Basic building units and properties of a
fluorescence single plane illumination microscope. Rev Sci Instrum 78:
023705.
Gu S, Jenkins MW, Watanabe M, Rollins AM. 2011. High-speed optical
coherence tomography (OCT) imaging of the beating avian embryonic
heart. In Imaging in developmental biology: A laboratory manual (ed. J
Sharpe, RO Wong). Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EH. 2004. Optical
sectioning deep inside live embryos by selective plane illumination
microscopy. Science 305: 1007–1009.
Holekamp TF, Turaga D, Holy TE. 2008. Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled
planar illumination microscopy. Neuron 57: 661–672.
Keller PJ, Stelzer EH. 2008. Quantitative in vivo imaging of entire embryos
with digital scanned laser light sheet fluorescence microscopy. Curr
Opin Neurobiol 18: 624–632.
Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EH. 2008. Reconstruction of
zebrafish early embryonic development by scanned light sheet microscopy. Science 322: 1065–1069.
Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EHK. 2011. Imaging the development of entire zebrafish and Drosophila embryos with digital scanned
laser light-sheet fluorescence microscopy (DSLM). In Imaging in developmental biology: A laboratory manual (ed. J Sharpe, RO Wong). Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Konig K. 2000, Multiphoton microscopy in life sciences. J Microsc 200:
83–104.
Lichtman JW, Conchello JA. 2005. Fluorescence microscopy. Nat Methods 2:
910–919.
Mazzoleni G, Di Lorenzo D, Steimberg N. 2009. Modelling tissues in 3D:
The next future of pharmaco-toxicology and food research? Genes Nutr
4: 13–22.
Metscher BD. 2011. X-ray microtomography (microCT) imaging of vertebrate embryos. In Imaging in developmental biology: A laboratory
manual (ed. J Sharpe, RO Wong). Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Pampaloni F, Reynaud EG, Stelzer EH. 2007. The third dimension bridges
the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:
839–845.
Pampaloni F, Stelzer EH, Masotti A. 2009. Three-dimensional tissue models
for drug discovery and toxicology. Recent Pat Biotechnol 3: 103–117.
Pawley JB. 2006. Handbook of biological confocal microscopy. Springer,
New York.
Quintana L, Sharpe J. 2011. Optical projection tomography of vertebrate
embryo development. In Imaging in developmental biology: A laboratory
manual (ed. J Sharpe, RO Wong). Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Rhee S, Grinnell F. 2007. Fibroblast mechanics in 3D collagen matrices. Adv
Drug Deliv Rev 59: 1299–1305.
Rhee S, Jiang H, Ho CH, Grinnell F. 2007. Microtubule function in fibroblast
spreading is modulated according to the tension state of cell-matrix
interactions. Proc Natl Acad Sci 104: 5425–5430.
Ruffins SW, Jacobs RE. 2011. MRI in developmental biology and the construction of developmental atlases. In Imaging in developmental biology:
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
7
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
J. Swoger et al.
A laboratory manual (ed. J Sharpe, RO Wong). Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Siedentopf H, Zsigmondy R. 1903. Über Sichtbarmachung und Größenbestimmung ultramikroskopischer Teilchen, mit besonderer
Anwendung auf Goldrubingläser. Ann Phys 10: 1–39.
Stelzer EHK, Lindek S. 1994. Fundamental reduction of the observation
volume in far-field light microscopy by detection orthogonal to the
illumination axis: Confocal θ microscopy. Opt Commun 111: 536–547.
Swoger J, Verveer P, Greger K, Huisken J, Stelzer EHK. 2007. Multi-view
image fusion improves resolution in three-dimensional microscopy.
Opt Express 15: 8029–8042.
Swoger J, Pampaloni F, Stelzer EHK. 2014a. Imaging cellular spheroids with
a single (selective) plane illumination microscope. Cold Spring Harb
Protoc doi: 10.1101/pdb.prot080176.
8
Swoger J, Pampaloni F, Stelzer EHK. 2014b. Imaging MDCK cysts with a
single (selective) plane illumination microscope. Cold Spring Harb
Protoc doi: 10.1101/pdb.prot080184.
Turaga D, Holy TE. 2008. Miniaturization and defocus correction for
objective-coupled planar illumination microscopy. Opt Lett 33: 2302–
2304.
Verveer PJ, Swoger J, Pampaloni F, Greger K, Marcello M, Stelzer EHK.
2007. High-resolution three-dimensional imaging of large specimens
with light sheet-based microscopy. Nat Methods 4: 311–313.
Voie AH, Burns DH, Spelman FA. 1993. Orthogonal-plane fluorescence
optical sectioning: 3-dimensional imaging of macroscopic biological
specimens. J Microsc 170: 229–236.
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168
Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by
Cold Spring Harbor Laboratory Press
Light-Sheet-Based Fluorescence Microscopy for Three-Dimensional Imaging of
Biological Samples
Jim Swoger, Francesco Pampaloni and Ernst H.K. Stelzer
Cold Spring Harb Protoc; doi: 10.1101/pdb.top080168
Email Alerting
Service
Subject
Categories
Receive free email alerts when new articles cite this article - click here.
Browse articles on similar topics from Cold Spring Harbor Protocols.
Cell Imaging (495 articles)
Imaging Development (218 articles)
Imaging/Microscopy, general (562 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press