brief communications
Four-dimensional imaging and quantitative
reconstruction to analyse complex
spatiotemporal processes in live cells
Daniel Gerlich*, Joël Beaudouin†, Matthias Gebhard*, Jan Ellenberg† and Roland Eils*‡
*Intelligent Bioinformatics Systems Department, German Cancer Research Centre, 69120 Heidelberg, Germany
†Gene Expression and Cell Biology/Biophysics Programmes, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
‡e-mail: [email protected]
Live-cell imaging technology using fluorescent proteins
(green fluorescent protein and its homologues) has revolutionized the study of cellular dynamics1,2. But tools that
can quantitatively analyse complex spatiotemporal
processes in live cells remain lacking. Here we describe a
new technique — fast multi-colour four-dimensional imaging combined with automated and quantitative time-space
reconstruction — to fill this gap. As a proof of principle,
we apply this method to study the re-formation of the
nuclear envelope in live cells3–6. Four-dimensional imaging of three spectrally distinct fluorescent proteins is used
to simultaneously visualize three different cellular compartments at high speed and with high spatial resolution.
The highly complex data, comprising several thousand
images from a single cell, were quantitatively reconstructed in time–space by software developed in-house.
This analysis reveals quantitative and qualitative insights
into the highly ordered topology of nuclear envelope formation, in correlation with chromatin expansion — results
that would have been impossible to achieve by manual
inspection alone. Our new technique will greatly facilitate
study of the highly ordered dynamic architecture of
eukaryotic cells.
wo-dimensional time-lapse live-cell recordings are used widely for the investigation of dynamic, fluorescently labelled
structures with high spatial resolution7–9. A crucial step in the
interpretation of dynamic imaging data has been the development
of computational methods for automated quantitative analysis and
time-space visualization10–13. However, complex dynamic processes
are ideally studied in three spatial dimensions over time (4D imaging), which requires imaging approaches based on either advanced
confocal or deconvolution microscopes. Such approaches generate
large and complex data sets, typically of the order of several gigabytes or up to 5,000 single images per experiment. These are commonly visualized by a sequence of z-stack projections to single
planes, whereby spatial information along the z axis is lost completely. Visualization can be performed by volume rendering, but
this technique does not allow a quantitative analysis14.
Here we describe a technique for fully automated quantification
and visualization of surfaces from dynamic 3D fluorescent structures in live cells, which enhances both temporal and spatial resolution. A visualization module allows the user to explore cellular
structures from different perspectives while they are animated.
Importantly, a wealth of quantitative parameters of the reconstructed structures including mean or total grey values, object position and volume or surface area is immediately accessible for
detailed 3D kinetic analysis.
Live-cell recordings are generally limited by the total light exposure used during in vivo observation to avoid disruption of cellular
T
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Figure 1 Three-dimensional reconstruction of LBR–YFP recruitment to
patches on chromatin surface. a, Optical slice of a middle section of the image
stack from two daughter nuclei at late anaphase, where LBR (green) aggregates on
chromatin (red). Yellow arrowhead points to a distinct LBR patch. b, The same optical slice after diffusion filtering. c, Segmentation of chromatin (yellow outline) and
LBR patches (blue outline) in this slice. d, Three-dimensional stack of filtered consecutive optical sections. Shown are 8 out of a total of 15 sections; the 3-µm slice
corresponds to a–c. Yellow arrowhead points to the same membrane patch as in
a–c. The two patches marked by blue and yellow arrowheads are detectable in several consecutive optical slices. e, Maximum intensity projection of the entire zstack. f, Three-dimensional reconstruction of the entire z-stack. The reconstruction
is based on five interpolated subslices per z-slice, thereby increasing spatial resolution. Contours at optical slices are highlighted. Arrowheads point to the same LBR
patches as in a–e, and show that LBR patches are reconstructed from contours of
several optical slices. See Supplementary Information for a video illustrating the
reconstruction procedure. Scale bars, 4 µm.
processes and bleaching of the fluorescent marker. Thus, the signalto-noise ratio and, more importantly, the number of time–space
sections taken in a particular experiment are reduced considerably.
Moreover, acquisition speed and light efficiency of the microscope
hardware typically limits the number of space sections to maintain
high temporal resolution.
To minimize hardware limitations, we have used a custom-built
confocal laser scanning microscope optimized for the 4D imaging
of multi-colour fluorescent proteins. To compensate for low-image
quality, we applied a highly sensitive anisotropic diffusion filtering
that only smoothes in areas with homogeneous image information
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Figure 2 High-speed 4D imaging of early LBR patch formation on chromatin
surface. a, A time series of 53 stacks each comprising 15 z-slices was captured
simultaneously for LBR–YFP (green) and H2B–CFP (red) every 10 s over a time
course of 8 min and 40 s. Both the LBR signal and the chromatin signal were segmented automatically. Three-dimensional reconstructions of LBR patches and chromatin domains are shown at 10 representative time steps from top left to bottom
right. At time step 0 s, initial tubular patches are formed on the distal side of both
daughter nuclei. Note that a central region stays free from LBR–YFP patches for
the first 60 s (marked by star at time step 0 s). See also video in Supplementary
Information. b, A triple-colour 4D image sequence was captured simultaneously for
LBR–YFP, H2B–CFP and γ-tubulin–RFP, comprising 190 stacks with 10 z-sections
and a time lapse of 8 s. Three-dimensional reconstructions show chromatin (red),
LBR patches (green) and centrosomes (blue). c, LBR–YFP protein distribution in ER
and nuclear envelope. Quantitative analysis of LBR–YFP targeting to ER and nuclear
envelope during nuclear reassembly was performed for a 35-min time series. The
total LBR–YFP intensity in patches was normalized by the total LBR–YFP intensity in
the ER at time step 0 min, when initial patch formation was observed. Every observation is based on three independent experiments. Scale bars, 2 µm.
without perturbing overall morphology10,15. The high noise levels in
rapidly acquired confocal images limit the application of many
advanced segmentation tools. Sophisticated edge-based object
detection10, which has the advantage of being independent of
absolute intensities cannot be applied properly in our data.
Therefore, object contours could be readily identified in z-slices by
thresholding.
To overcome the suboptimal z resolution, we used advanced
interpolation techniques to reconstruct 3D surface models on the
basis of object contours. Because linear interpolation does not yield
a smooth surface, we made use of cubic B-splines, which are commonly used for approximation of curves and surfaces to sample
points16. They have the advantage of second-order continuity and
are stable in a geometric sense, as they do not tend to oscillate even
when there are many sample points. The surface reconstructions
are then displayed in a powerful virtual reality viewer, which makes
the interior of the sample fully accessible by using either transparent surface materials or interactively hiding parts of the reconstructed surfaces. In this way, objects of interest for further quantitative analysis can easily be selected in the complex 3D structure of
the cell.
We have used our imaging approach to analyse the reconstitution of nuclear organization after mitosis. In higher eukaryotes,
nuclear architecture is reversibly broken down during
prometaphase: nuclear membrane proteins are dispersed in the
endoplasmic reticulum (ER), the nuclear lamina depolymerizes,
and the nuclear envelope no longer associates with the condensing
chromosomes3,17. Reassembly of these nuclear structures begins in
late anaphase, where the chromosomes of the daughter nuclei are
sealed rapidly by nuclear membranes. In vitro studies have shown
the molecular interactions of inner nuclear membrane proteins
with the lamina and chromatin; these interactions are thought to be
essential for the structural maintenance of the nuclear envelope, the
higher level organization of chromosomes during interphase, and
nuclear envelope disassembly and re-formation during mitosis5,6,18.
We used fusions of fluorescent proteins (cyan, CFP; yellow, YFP;
and dsRed19,20 (RFP)) targeted to chromosomes, centrosomes and
inner nuclear membrane to simultaneously monitor the dynamics
of these three cellular structures during nuclear assembly in triplecolour 4D confocal imaging experiments. Exploiting the power of
our versatile software tools, we could detect a precise pattern of
inner nuclear membrane assembly on chromatin during late
anaphase. Moreover, we could analyse quantitatively the recruitment of an inner nuclear membrane protein to the chromosome
surface and analyse the volume expansion rates of nuclear envelope
and chromosomes in both daughter cells.
Previous work has shown that lamin B receptor fused to GFP
(LBR–GFP) is a suitable marker to visualize the inner membrane of
the nuclear envelope in vivo3,4. LBR is distributed homogenously
throughout the cytoplasmic ER after nuclear envelope breakdown
in prometaphase (ref. 3; and data not shown). To monitor the very
early stages of nuclear envelope reassembly on chromatin from the
ER, dual-colour confocal 4D images of histone 2B fused to CFP,
and LBR fused to YFP were captured at very high temporal resolution. These images were filtered and segmented to generate a 3D
surface reconstruction (Fig. 1; see also Supplementary Information
Video 1 and Fig. 1).
We found that LBR–YFP concentrated at specific sites on the
chromatin surface in late anaphase. A comparison of the surface
reconstruction with the projected image shows that the detailed
topology of LBR patch formation can be observed only in the 3D
reconstructed visualization. The advantage of surface reconstruction
rendering is the very distinct display of small-scale features; however, this is compromised by the truncation of some low-intensity
regions from the object (for example, parts of the chromatin extensions visible in Fig. 1e). Because such a possible truncation effects all
time steps equally, it does not bias the interpretation of the dynamic
topology changes (see Supplementary Information Fig. 4 and
below). Although LBR appears to be widely distributed on the distal
part of the bottom daughter nucleus in the projection, the 3D reconstruction shows unequivocally that LBR is localized in patches organized around the central distal area of this nucleus and that the central
part is not covered by LBR patches at this time step (Fig. 1e, f).
High-speed time-lapse recordings show that these structures
initially have an extended tubular morphology, which rapidly
expands to larger sheets that almost completely enclosed and sealed
chromatin within 5 min (Fig. 2a; see also Supplementary
Information Video 2). The first detectable concentrations of LBR in
patches on the chromatin surface appeared virtually simultaneously in a time frame of less than 30 s in both daughter nuclei. The distribution of these patches occurred in a reproducible and symmetric pattern in both daughter nuclei, beginning at the most distal
and peripheral regions of the chromatin caps. In a second experiment, triple-colour GFP confocal 4D imaging was performed to
visualize simultaneously chromatin, nuclear envelope patches and
centrosomes. The distal central region on chromatin that remained
free of LBR until most nuclear membrane had formed correlated
precisely with the position of the centrosomes (Fig. 2a, b).
The 3D object detection allowed precise, quantitative measurements of LBR recruitment from the ER compartment into the
nuclear membrane patches by measuring the grey values of the raw
data voxels corresponding to the segmented objects in the reconstruction. The concentration of GFP-labelled proteins is directly
proportional to the grey value intensity in the recorded images21.
The total amount of LBR–YFP in patches on chromatin and later in
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Figure 3 Synchronized expansion of nuclear envelope and chromatin
domain. Time–space interpolated (morphed) reconstructions of chromatin surface
and nuclear envelope were computed from a series of 46 z-stacks with 15 slices in
each channel captured over 61.5 min, beginning at the complete coverage of chromatin with nuclear envelope (time step 0 min). The time lapse between two consecutive time steps was 30 s in the early frames and 120 s in the later frames. Four
intermediate states of chromatin and nuclear envelope surfaces per time frame
were interpolated, thereby increasing temporal resolution. Three representative
intermediate reconstructions at indicated time points are displayed. a, Chromatin
domain. See also Supplementary Information for a video showing the animated
reconstructions over time. b, Transparent overlay of nuclear envelope and chromatin domain. c, Expansion of nuclear envelope and chromatin surface. Volume
measurements for the area enclosed by the LBR signal were performed only after
the nuclear envelope completely sealed the chromatin (time step 0 min). d,
Expansion of chromatin domain in two daughter nuclei. The difference in absolute
volumes of nuclear envelope and chromatin is due to the segmentation procedure:
using a threshold to detect the hollow structure of the nuclear envelope returns the
outer contour and therefore overestimates the volume, but this does not impair relative volume measurements in the temporal evolution. e, Comparison of relative expansion of nuclear envelope and chromatin in six daughter nuclei. Scale bar, 5 µm.
the sealed nuclear envelope was measured over a time course of 35
min. Total LBR–YFP fluorescence was first determined in the diffuse ER pool before patch formation. Next, the relative amount of
LBR–YFP fluorescence in reforming nuclear envelope was quantified. The recruitment reproducibly followed a discrete biphasic
profile (Fig. 2c).
During the first 2–3 min of nuclear assembly, about 70% of LBR
was recruited to the chromosome surface in new nuclear membrane sheets. This time corresponds to the initial tubular patch formation and expansion into sheets (Fig. 2a, 0–200 s). About a 7-min
plateau followed this initial fast phase. In this lag time, the membrane sheets fused and sealed the chromatin. After sealing, nuclear
expansion started, leading to a second slower phase of LBR recruitment increasing its nuclear membrane pool slowly over 30 min
until it reached a final plateau. Intriguingly, this biphasic behaviour
is consistent with a prediction of the diffusion and retention model
of nuclear membrane protein targeting8. In this model, nuclear
membrane proteins such as LBR would have free access to diffuse
from the ER into the directly connected inner nuclear membrane
before the nuclear envelope seals. This would correspond to the fast
first phase of recruitment.
After sealing, however, the only connection between the ER —
which at that time still holds ~30% of the nuclear LBR pool — and
the inner nuclear membrane is at the nuclear pore complexes. Its
narrow lateral channels22 are likely to constrain diffusional access of
membrane proteins, resulting in the second slower recruitment
854
phase during nuclear expansion. Notably, this kinetic quantification would not have been possible with 2D imaging methods,
owing to the strong deformation and movement along the z axis of
the nuclei during anaphase (see Supplementary Information Figs 2
and 3 for a comparison with 2D + time quantification).
After complete sealing of the chromatin by nuclear membrane
sheets, both nuclear envelope and chromatin expand to about double size. We have processed 4D imaging data to analyse the timing
of chromatin and nuclear envelope volume expansion from
anaphase to G1. To inspect the process of chromatin unfolding and
nuclear envelope expansion visually, we carried out a morphed surface reconstruction. Interpolation (morphing) over time is especially useful when the experiment is carried out over longer time
courses, which thus increase the time lapse between consecutive
image stacks. Global translational or rotational movements of the
nuclei often impair the visualization and quantification of surface
dynamics; therefore, we automatically corrected for rotational and
translational movements by a rigid transformation approach,
which does not influence the local topology of the objects.
A 3D registration algorithm was implemented that iteratively
minimizes the mean squared distances of an object’s surface
points23. The series of morphed reconstructions was animated
within the interactive graphical viewer. Three representative frames
from the reconstructions of nuclear envelope and chromatin surface are shown in Fig. 3a and b (see also Supplementary
Information Video 3). The quantification of volume expansion
revealed that chromatin and nuclear envelope expand synchronously with very similar rates (Fig. 3c). Notably, the onset of chromatin expansion coincided precisely with the complete coverage by
LBR (at time step 0 s). In a set of six daughter nuclei from three
independent experiments, we measured a relative expansion of
2.12 ± 0.37 for chromatin and 2.03 ± 0.20 (means ± s.d.) for the
nuclear envelope (Fig. 3e). To evaluate further the reliability of our
segmentation with thresholding, we compared the quantitative
results obtained by different threshold values. We found that
whereas the absolute volumes show a dependence of the threshold
value, the kinetic profiles are largely independent of the specific
threshold (see Supplementary Information Fig. 4).
The mechanism underlying nuclear expansion at anaphase to
G1 transition remains an open issue. Visual inspection of 2D timelapse sequences has suggested that chromatin expansion only takes
place after complete coverage with nuclear envelope membranes3.
However, these studies were limited by the visual inspector, and
volume measurements in 2D image sequences are generally of poor
accuracy because of the strong deformation in z direction during
expansion. Our reconstructions of dual-colour 4D imaging data
provided automated accurate volume measurements and visualization of this process.
We found that chromosome expansion takes place only after
complete wrapping of chromatin with nuclear envelope membranes, suggesting that nuclear compartmentalization is required
before chromatin decondensation. In addition, the expansion of
the two daughter nuclei was synchronized even after cytokinesis
(Fig. 3d). This behaviour suggests that nuclear expansion operates
under the same limiting principles in the two independent cells
after mitosis. Nuclear envelope and chromatin expansion were
strongly correlated in our measurements, suggesting that both
processes are tightly linked. This is consistent with chromatin
decondensation providing the driving force for nuclear expansion.
By contrast, an expansion mechanism that would increase
nuclear volume by influx of proteins through nuclear pores without decondensing chromatin would generate nuclear space devoid
of chromosomes and a lagging of chromatin expansion behind
nuclear membrane expansion. We never observed nuclear areas
devoid of chromosomes or kinetic uncoupling in our analysis of
nuclear assembly. Such behaviour is seen in prophase, when chromatin condensation precedes nuclear disassembly. In addition, the
initial nuclear expansion does not seem to depend on a fully
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formed nuclear lamina, as lamins have been shown to be recruited
to the nuclear envelope significantly after sealing of nuclear membranes (data not shown; but see refs 24–26).
We have shown that the techniques described here are a valuable
tool for a detailed spatiotemporal analysis of dynamic cellular
processes such as the complex interplay between integral nuclear
membrane proteins and chromosomes, which display a specialized
targeting pattern. An essential feature of our imaging approach is
the capability of interpolation in space and time, because the total
amount of captured slices and therefore both the z and temporal
resolution are limited in live-cell recordings. Moreover, we have
analysed quantitatively 4D sequences regarding changes in volumes, object distances (data not shown) and grey value kinetics of
objects, which would not have been possible by an interactive/manual approach. At present, we have not been able to quantitatively
investigate plastic deformations of cellular structures. To overcome
these limitations, we are currently extending our software modules
with affine and elastic matching algorithms. The capabilities of our
imaging approaches will prove invaluable in future studies of complex spatiotemporal dynamic processes in live cells.
Methods
Cells and DNA constructs.
NRK cells transfected using FuGene 6 (Roche, Mannheim, Germany) were imaged at 37 °C in LabTekII
chambered No. 1 coverglasses (LabTek, Naperville, IL) in CO2-independent medium (GibcoBRL) containing 20% fetal calf serum and 1 mg ml−1 ascorbic acid. LBR–YFP and H2B–CFP have been
described27. γ-Tubulin–RFP was made by inserting the full-length complementary DNA (a kind gift
from B. Oakley) into a pdsRed-N1 plasmid (Clontech).
Dual- and triple-colour fast confocal 4D imaging.
Four-dimensional imaging was performed on a custom built Zeiss LSM510 equipped with a z-scanning stage (HRZ 200) for fast 4D acquisition using a PlanApochromat 63× DIC oil immersion objective. Double labelling of CFP and YFP used 413-nm Kr and 514-nm Ar lines for selective excitation,
alternating the two lasers line by line using dual-directional scanning at a high scan speed (0.88 µs per
pixel). Detection was performed on the same photo multiplier with a double bandpass
422–500/525–630 emission filter (Chroma Technology, Brattleboro, VT). Crossover with this set-up
was below 1% in both channels, with minimized loss of emission from both fluorophores. Triplecolour imaging of CFP, YFP and dsRed was achieved by alternating the 413 nm, 514 nm lines and
using an additional 543 nm HeNe line in the same manner. Here, emissions were split with a primary
NFT 560 dichroic to a LP 560 emission filter (dsRed), and again with a secondary NFT 505 dichroic to
a LP 525 (YFP) and a BP 440-505 emission filter (CFP), respectively (Chroma Technology). Again,
crossover with set-up was less than 1% in all channels. Neither configuration requires the movement
of any mechanical parts in the microscope other than the xy (the speed of which limits acquisition
time) and z scanners, which enables ultrafast 4D confocal imaging. The minimum time for a 256 ×
256 × 15 double-labelled stack is 3 s.
Image segmentation and reconstruction of surface models.
A highly sensitive filtering process is applied to selectively smooth noisy structures while preserving
essential edge information. This constrained image-smoothing depends on local image properties and
is controlled by an object scale-dependent edge-stopping function10,15. Thereby, regions where the signal is of constant mean are selectively diffused in contrast to regions where the signal changes rapidly.
Segmentation is then performed by grey value thresholding. Three-dimensional surface models are
reconstructed from image stacks at individual time sections. First, the binarized object representation
is transformed into a parameterized contour representation, by the use of cubic B-splines16. Contours
in adjacent z-sections are assigned to a defined 3D object by a fuzzy logic-based tracking algorithm10.
The parameters of the tracking algorithm are set to allow a defined maximal angle of the contour’s
gravity centres belonging to an individual 3D object. Thereafter, a continuous surface reconstruction is
obtained by B-spline interpolation between corresponding equally parameterized contour points from
contours of neighbouring z-sections. Interpolation between 3D surfaces over time gives a continuous
reconstruction of the entire 4D data set (morphing). Global rotational and translational movements
are corrected in a time series by a rigid transformation approach that iteratively minimizes the mean
squared distances of an object’s surface points23. The local topology is not influenced by the matching
algorithm used. The animated surface reconstruction is embedded in a powerful multi-dimensional
graphical viewer that allows real time user interaction (OpenInventor SceneViewer, Template Graphics
Software Inc., San Diego, CA). The binarized object representation is used directly to measure volume
over time. Moreover, the grey values inside the segmented volume of corresponding original image
stacks are measured to determine the amount and concentration of fluorescently labelled protein in
the segmented ER or nuclear envelope compartment. The entire image analysis and image reconstruction software is integrated into the TILLvisTRAC system (T.I.L.L. Photonics, Munich, Germany).
RECEIVED 26 FEBRUARY 2001, REVISED 1 MAY 2001, ACCEPTED 14 JUNE 2001,
PUBLISHED 16 AUGUST 2001.
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ACKNOWLEDGEMENTS
We thank B. Oakley for the γ-tubulin cDNA; N. Daigle for technical assistance; J. Mattes for providing
the matching algorithm; and C. Conrad and C. Athale for critical comments on the manuscript. The
bioinformatics group acknowledges the support of the German Federal Ministry of Education and
Research (BMBF) through the BioFuture grant (AZ 11880A) and of the German Research Council
(Ei 358/2-1 and Ei 358/1-1). J.B. was supported by a fellowship through EMBL’s international Ph.D.
programme. Part of this work was performed in collaboration with T.I.L.L.-Photonics, Munich,
Germany.
Correspondence and requests for materials should be addressed to R.E. Supplementary Information is
available on Nature Cell Biology’s website (http://cellbio.nature.com).
NATURE CELL BIOLOGY VOL 3 SEPTEMBER 2001 http://cellbio.nature.com
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Figure S1. Detailed description of 3-D reconstruction technique. (a-f) Automated
detection of fluorescent structures. (a) Single optical slice from a 4-D imaging
experiment. Chromatin labeled by H2B-YFP. (b) Line profile of grey values as indicated by green line in suppl. fig. 1a. (c) Same slice processed by anisotropic diffusion
filtering. (d) Line profile from suppl. fig. 1c. Note that the noise is strongly reduced
without perturbing essential edge information. (e) Segmentation of two daughter
nuclei by thresholding. (f) Line profile from suppl. fig. 1c. (g-i) Comparison of visualisation techniques. The chromatin signal from one daughter nucleus is visualised
using surface or volume rendering. (g) Surface reconstruction using linear interpolation. After segmentation, the object outlines in individual optical slices are represented by discrete pixels. The reconstruction of a continuous surface requires a
parameterised contour running through all pixels of each outline. This can be
achieved most easily by a connection with straight lines of neighbouring outline pixels. Equally parameterised contour points from adjacent slices were then connected by straight lines. Thereby, a continuous surface is obtained. The triangulated
mesh is overlaid on the surface model. (h) Surface reconstruction with B-spline
interpolation. Cubic B-splines were used to generate smooth curves connecting all
outline pixels. Then, equally parameterised contour points on the neighbouring outlines were connected, again using B-splines. From these curves, five intermediate
contours per optical slice were obtained to increase spatial resolution of the surface model and avoid sharp edges as visible in suppl. fig. 1a. (i) Conventional volume rendering of the chromatin from one daughter nucleus. While the overall morphology appears similar with all three methods, only the reconstruction with Bspline interpolation achieves a smooth and realistic visualization of the data, considering the limited z-resolution typically obtained in 4-D live cell recordings.
Figure S2. Integration of LBR into patches and the NE measured in single slices
versus 4-D analysis. While suppl. fig. 1 shows the advantages of interpolated surface reconstruction, the most important point is the direct accessibility of quantitative data after segmentation and reconstruction. We have tried to address the problem of kinetics of membrane recruitment during nuclear assembly by conventional
methods in the past3. However, we were not able to resolve two kinetic phases by
traditional 2-D time-lapse and manual quantification. To demonstrate the advance
made possible by 4-D imaging and quantification, we directly compared the 4-D
analysis of LBR recruitment with quantification of single optical slices using the
same experimental data. Suppl. fig. 2 shows the grey value kinetics in selected
focal planes that were pre-processed and segmented the same way as in the 4-D
analysis. The kinetic profiles show very discrepant kinetics in the different focal
planes. This effect can be explained by the strong deformation and movement of
the nuclei along the z-axis. While the upper slices (e.g. slice 5) show a steep
increase in grey value sum in the first 2 min, they reach zero level after about 10
min again. This is due to the flattening of the daughter nuclei and movement
towards the coverslip. In contrast, the lower slices do not show the steep increase
during the first minutes, since the daughter nucleus is only cut at the very bottom
edge. Later, most signal is detected in the lower slices, which explains the large
increase after 5 minutes. None of the kinetic profiles in single optical slices is representative for the kinetics of LBR integration into membrane patches and later into
the NE inner membrane. In contrast, the measurement of grey values in a segmented volume (4-D analysis) is independent of deformation and movements along the zaxis as long as the nuclei remain within the recorded stacks.
© 2001 Macmillan Magazines Ltd
supplementary infomation
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Figure S3. Comparison of volume expansion quantified with 4-D methods versus 2D analysis in projected image stacks. A straightforward method to reduce artefacts
due to focal shifts is the projection of recorded image stacks to single planes for
further 2-D quantification. We compared 4-D volume quantification with 2-D+time
area measurements to evaluate the specific differences between the two methods.
Suppl. fig. 3 shows the relative expansion measured by 2-D+time and 3-D+time
analysis of the chromatin from an expanding nucleus. While the plateau of nuclear
Volume [µm3]
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expansion is reached between 30-40 min in both cases, the initial slope during the
first five minutes differs by a factor greater than two: the 4-D method measures 4.1
%/min expansion rate, while 2-D+time gives 9.0 %/min. Additionally the total expansion is overestimated much higher with the 2-D+time (3.0 times) as compared to 4D (2.2 times). These differences are again most likely explained by the strong flattening of the daughter nucleus during the beginning of nuclear expansion.
MOVIES
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Movie 1 Assembly of image stack from optical slices and reconstruction of a 3-D
surface model of LBR-EYFP patches (green) on chromatin surface (red). First, the
pre-processed optical image slices of a single image stack are displayed sequentially. Then, they are removed stepwise to reveal the 3-D reconstructed surface
model. For further details see fig. 1.
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Movie 2 Animated sequence of 3-D reconstructions of early LBR patch formation
on chromatin surface. For details see fig. 2.
Reference threshold
Plus 4%
Plus 8%
Plus 12%
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Movie 3 Morphed reconstruction of NE and chromatin expansion. Four intermediate states of chromatin and NE surfaces per time frame were interpolated, thereby increasing temporal resolution. For details see fig. 3.
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Time [min]
Figure S4. Reliability of 4-D quantification with threshold segmentation. A general
problem of segmentation by thresholds is the arbitrary and user-dependent choice
of the specific threshold value. The threshold value has a strong influence on the
absolute values of volumes and grey value sums measured in a segmented single
image stack. However, we assumed that the kinetics should remain mostly unaffected by the specific choice of threshold value. To critically assess this assumption, we compared the kinetic analysis of nuclear expansion using different threshold values. In fact, we found that the absolute volume measures are shifted, but the
kinetics appeared largely unbiased by the variant thresholds.
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