Post on 16-May-2023
Automated microaxial tomography of cell nuclei after specific labelling
by fluorescence in situ hybridisation
M. Kozubeka,*, M. Skalnıkovaa, Pe. Matulaa, E. Bartovab, J. Rauchc,F. Neuhausc, H. Eipelc, M. Hausmannc
aLaboratory of Optical Microscopy, Faculty of Informatics, Masaryk University, Botanicka 68a, CZ-60200 Brno, Czech RepublicbInstitute of Biophysics, Czech Academy of Sciences, Kralovopolska 135, CZ-61265, Brno, Czech Republic
cKirchhoff Institute of Physics, University of Heidelberg, Albert-Uberle-Str. 3-5, D-69120 Heidelberg, Germany
Received 20 January 2002; revised 16 April 2002; accepted 19 April 2002
Abstract
Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with
an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial
information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial
tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much
operator time. In order to solve this problem the Heidelberg 2p-tilting device has been mounted on the Brno high-resolution cytometer as an
attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that
drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been
developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed
software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological
example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the
first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.
q 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Microaxial tomography; Automated microscopy; High-resolution cytometry; Fluorescence in situ hybridisation imaging; Interphase cell nuclei
1. Introduction
The idea of observing the same object from different
angles of view and subsequent reconstruction of its three-
dimensional (3D) structure from angular series of images is
in principle not new in optical microscopy. The first
attempts appeared more than 25 years ago and were based
on specimen tilting, i.e. the whole microscope slide with
objects fixed to it was rotated (Skaer and Whytock, 1975).
The tilting angle was however, very limited in this case (up
to 308) and did not allow obtaining object information with
isotropic (equal axial and lateral) resolution for which at
least a 908 tilting or even more is needed (Bradl et al.,
1996a). Consequently, the next idea was to place cells into a
glass capillary that could be rotated by 908 or even more (up
to 3608) and to observe them using a water immersion
(Shaw et al., 1989) or an oil immersion (Bradl et al., 1992,
1994) objective.
Tilting 3608 by a capillary had the advantage that after
appropriate refraction index matching by immersion fluids
imaging of the specimen under the optimum perspective, i.e.
the perspective of highest resolution, was always possible. The
specimen preparation and fixation in a glass capillary,
however, was difficult, especially for fluorescence in situ
hybridisation (FISH) labelling of cell nuclei which had to be
done in suspension. In addition, optical aberrations caused by
the curved glass layer of the capillary in the light path could not
always be avoided, therefore the glass capillary was replaced
by a glass fibre (Bradl et al., 1996b,c; Rinke et al., 1996). In this
case the cell nuclei were attached to the surface of the fibre so
that the large tilting angle was preserved and a standard
coverglass was placed between the cell nuclei and the
objective lens to obtain better imaging properties. In all
these previous studies, either ‘flat’ cell nuclei fixed by
methanol–acetic acid or fluorescent beads were investigated.
0968-4328/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0 96 8 -4 32 8 (0 2) 00 0 23 -9
Micron 33 (2002) 655–665
www.elsevier.com/locate/micron
* Corresponding author. Tel./fax: þ420-5-41512467.
E-mail address: kozubek@fi.muni.cz (M. Kozubek).
To handle the fibres and capillaries under defined
microscopic conditions, a so-called 2p-tilting device was
developed in Heidelberg some years ago (Bradl et al., 1994,
1996a,b,c). Perpendicular to the observation direction one
could rotate the capillary or fibre around its own axis by
means of a stepper motor and a flexible shaft. Therefore this
technique is called microaxial tomography.
In parallel with the hardware development, methods for
the reconstruction of 3D structures from an angular series of
images have been developed (Shaw et al., 1989; Satzler and
Eils, 1997; Heintzmann et al., 2000). It has been shown that
the reconstructed images can be superior to single-angle
confocal ones due to improved 3D-resolution (obtained
after appropriate image processing) which is in the case of
microaxial tomography close to the best resolution among
all spatial axes (usually it is close to the lateral resolution
because the axial one is substantially worse). Therefore, also
non-confocal angular series of images can yield high-
resolution results. Moreover, microaxial tomography allows
a considerable increase in 3D localisation precision of
fluorescently labelled point-like objects (Bradl et al., 1996b,
c) which is a fundamental prerequisite to overcome
diffraction limited resolution in fluorescence microscopy
by Spectral Precision Distance Microscopy (Cremer et al.,
1999).
So far, a drawback of the microaxial tomography
approach has been a complicated image acquisition and
registration procedure and the necessity to track the objects
of interest as they rotate when the rotation axis fluctuates
and causes an object drift during rotation. Moreover, if
the diameter of the fibre is larger than the field of view of the
camera, it is necessary also to move the stage during the
rotation process. Therefore image acquisition and analysis
have been done interactively by a work-loaded procedure
and, consequently, the method has not been adapted for
processing a large number of objects, e.g. FISH labelled cell
nuclei.
On the other hand, conventional and confocal slide-based
microscopy has been well automated for this task using
appropriate hardware and software (Kozubek, 2001b). In
some applications not only image acquisition but also image
analysis of specifically labelled cell nuclei has been
automated (Netten et al., 1997; Ortiz de Solorzano, 1998;
Kozubek et al., 1999, 2001; Kozubek, 2001b). In Brno
recently a high-resolution cytometry (HRCM) technique has
been developed that enables automated acquisition and
analysis of thousands of cells per one microscope slide in
conventional as well as confocal mode (Kozubek et al.,
2001).
For studies of the nuclear chromatin architecture (Cremer
et al., 2000) it appears to be useful to combine the
microaxial tomography and the HRCM techniques so that
a large number of FISH-stained cell nuclei could be
processed with superior resolution (close to the lateral
resolution limit) in all spatial axes. In order to accomplish
this task it has been necessary to automate both acquisition
and analysis of microaxial tomographic data. This study
describes the automation of the acquisition process; the
automation of the analysis process is still under develop-
ment and will be the subject of a separate article. In
addition, experimental findings about the precision of
rotation are described and also a new protocol for
preparation and FISH of 3D conserved cell nuclei fixed
onto glass fibres is presented.
2. Specimen preparation
For the experiments either fluorescent beads of 900 nm
diameter (Polyscience Inc.) or FISH labelled cell nuclei
from culture cells were used. The preparation is described in
the following sections.
2.1. Glass fibres
The former experiments (Bradl et al., 1996b,c) showed
that the use of glass fibres is superior to the use of capillaries
due to preparation and handling advantages. The glass fibres
used in our experiments were made of borosilicate glass.
Typically they were 70–80 mm long and had different
diameters in the range of 120–200 mm. The fibres were
carefully pulled in order to obtain a homogenous diameter
and a good linearity along the whole fibre length. Before use
each glass fibre was cleaned in ethanol/ether (1:1 v/v)
overnight and coated with 0.1% poly-L-lysine (Sigma) by
incubating the clean fibre in this solution for 15 min.
Then the glass fibre was washed with deionised water and
dried in air.
2.2. Cell preparation
The human leukemic promyelocytic cell line HL-60 was
obtained from the American Type Culture Collection
(Manassas). HL-60 cells were maintained in Iscove’s
modified Dulbecco’s medium (IMDM) (Sigma) sup-
plemented with 10% fetal calf serum (PAN, Germany)
and 2 mM glutamine (Sigma) at 37 8C in a humidified
atmosphere containing 5% CO2. The cells were fixed in
3.7% formaldehyde with 0.5% Triton X-100 and HEPEM
(65 mM/l PIPES, 30 mM/l HEPES, 10 mM/l EGTA,
2 mM/l MgCl2, pH 6.9) (Neves et al., 1999) for 10 min at
room temperature and thoroughly washed in PBS (twice for
4 min). The dense cell suspension of 300 ml in PBS buffer
was sucked into a preparation glass capillary (inner diameter
0.5 mm, length 80 mm). A glass fibre coated with poly-L-
lysine was inserted into this glass capillary and incubated in
the cell suspension. The cells attached to the surface of the
glass fibre in about 5–8 min without drying. Then the fixed
cells were immediately permeabilised for in situ hybridis-
ation. The cell permeabilisation was performed in 0.1%
Triton X-100/0.1N HCl/PBS at room temperature for
15 min, followed by 0.1 M Tris (pH 7.8) for 10 min, 0.2%
M. Kozubek et al. / Micron 33 (2002) 655–665656
Saponin/PBS for 10 min, PBS (twice for 3 min), 20%
glycerol for 20 min and PBS (twice for 2 min).
2.3. Fast fluorescence in situ hybridisation
A digoxigenin-labelled DNA probe for alpha-satellite
sequences of the centromeric region of chromosome 4
(Oncor, UK) was used. Fast FISH was performed according
to a procedure described elsewhere (Durm et al., 1996; Haar
et al., 1996; Durm et al., 1998; Bartova et al., 1999). Briefly,
20 ng of the labelled DNA probe in 1.5 ml buffer was added
to a hybridisation mix containing 1.5 ml of a PCR buffer
(100 mM Tris–HCl, 500 mM KCl, and 25 mM MgCl2,
Roche) and 1.5 ml of 20 £ SSC diluted in deionised water
to a final volume of 15 ml. This hybridisation mixture with
the DNA probe was sucked into a preparation glass
capillary. The glass fibre with the permeabilised cell nuclei
was inserted into this glass capillary and sealed with rubber
cement and placed in a specially designed closed stainless
steel chamber. Thermal denaturation of the specimen and
the probe was performed simultaneously at 95 8C for 5 min.
For hybridisation, the steel chamber with the glass fibre in
the glass capillary was placed into a water bath at 76 8C for
120 min. Then the glass capillary was removed from the
steel chamber and the glass fibre was taken out and washed
at 37 8C in 2 £ SSC/0.1% Igepal-CA-630 (Sigma) (three
times for 3 min).
For immunodetection, approximately 15 ml of rhoda-
mine-labelled anti-digoxigenin anti-bodies (Appligene,
UK) were sucked into another preparation glass capillary
and the glass fibre was inserted into this capillary. After
20 min incubation at 37 8C in a humidified plastic chamber
the glass fibre was washed again at 37 8C in 4 £ SSC/0.1%
Igepal (three times for 3 min). DAPI (0.2 mg/ml) dissolved
in Vectashield (Vector Laboratories, CA) anti-fade solution
was used for counterstaining. Approximately 15 ml of DAPI
in Vectashield was sucked into a preparation glass capillary.
The glass fibre was inserted into this glass capillary and
stored at 4 8C or inserted into the mounting microaxial
tomography adapter for the microscope stage.
3. Microaxial tomography
3.1. Mounting adapter for fibres
To mount the fibre on the microscopy stage, the
2p-tilting device described earlier was used (Bradl et al.,
1996b,c; Rinke et al., 1996). One end of the glass fibre was
glued into a brass bearing that was mounted into an
aluminium frame of the size of a standard object slide
(76 mm £ 22 mm). The weight of the whole mounting
adapter was 29 g, so that it could also be used on
galvanometer stages of confocal laser scanning micro-
scopes. The region of investigation was located in a groove
of the plastic inlay. DAPI of 20 ml (0.2 mg/ml) in
Vectashield was added into the groove and the groove
with the fibre was covered with a coverglass. A schematic
drawing of the specimen geometry (fibre within the
2p-tilting device) is shown in Fig. 1.
3.2. Microscope and its accessories
The whole automatic system is based on the Brno HRCM
instrument (Kozubek et al., 2001; http://www.fi.muni.cz/
lom) consisting of a Zeiss Axiovert 100S inverted
microscope (Carl Zeiss, Jena, Germany) equipped with a
MicroMax cooled digital CCD camera (Princeton Instru-
ments, Trenton, NJ, USA), a piezo-electrical objective
nano-positioner PIFOC P721 (Physik Instrumente, Wald-
bronn, Germany), a Ludl motorised x–y stage and Ludl 6-
position excitation and emission filter wheels (Ludl
Electronic Products, Hawthorne, NY, USA). Optical
sectioning and 3D imaging is performed by means of a
CARV confocal unit (Atto Instruments, Rockville, MD,
USA) based on a Nipkow disc. The whole microscope
system is driven by a 2-processor computer.
In order to perform microaxial tomography, the follow-
ing components were added to the HRCM system: an
external stepper motor with a gear for 40,000 steps/3608 for
fibre rotation (ZSS 32.200.1, Phytron-Elektronik, Groben-
zell, Germany), an SMS-7000 stepper motor controller
(ELV, Leer, Germany) and a Wasco Witio-48-ext IO card
(Messcomp Datentechnik, Wasserburg am Inn, Germany)
for communication between the computer and SMS-7000. A
special drive with extendable shaft (Nadella, Stuttgart,
Germany) and two cardan joints (RS Components, Morfel-
den–Walldorf, Germany) has been designed and modified
by our workshop. This drive precisely and torsion free
translates the stepper motor rotation to the fibre axis over
space. A picture of the stepper motor, the 2p-tilting device
placed on the microscope stage and the drive shaft
connecting them is shown in Fig. 2. Whereas the motor is
fixed on an external holder, the stage with the 2p-tilting
Fig. 1. Geometry of the fibre within the 2p-tilting device. The light passes
through a high NA oil-immersion objective lens (1), immersion oil layer (2)
and coverglass (3) to the cells or cell nuclei fixed onto the fibre (4). The
fibre is surrounded with a suitable embedding medium (5). The fibre and the
embedding medium are placed into a groove made in the centre of the
microscope slide. The slide (6) can be made from glass or plastic. The
rectangle represents the field of view accessible by the objective lens. For
more details see (Bradl et al., 1994, 1996a,b,c; Rinke et al., 1996).
M. Kozubek et al. / Micron 33 (2002) 655–665 657
device can be freely moved in all three directions thanks to
the cardan joints and the extendable shaft.
For some experiments concerning the rotation precision
the microaxial tomography mounting and driving devices
described earlier were implemented into an upright Zeiss
Standard 25 fluorescence microscope with a computer
controlled stage (Marzhauser, Wetzlar, Germany). This
instrument was equipped with a PlanAPO 63 £ /NA 1.4
objective, an image magnification element (2 £ ), a stepper
motor driven z-drive and a cooled b/w CCD-camera
(CF8RCC, Kappa Messtechnik, Gleichen, Germany) (for
details of the microscope set-up see Bradl et al., 1996b,c).
3.3. Image acquisition strategy
The acquisition procedure has been designed in such a
way that the images of all cell nuclei on the fibre can be
acquired as quickly as possible. Therefore, the acquisition
procedure does not track an individual nucleus, it rather
tracks all nuclei simultaneously within a certain angular
range. The angular range is at least 908. This value fulfils
optical requirements described later (d ) and is assumed to
be sufficient to get an isotropic (equal lateral and axial)
image resolution. The cell nuclei are imaged only when they
are close to the objective and they are not imaged when they
are far from it or even behind the fibre (if viewed from the
objective side). This reduces optical aberrations involved in
the imaging process.
The acquisition procedure is based on an approach that
could be called static volume of view. This means that a
static camera field of view (no stage movements) and a
static z-coordinate range, i.e. static observation intervals in
all axes (x, y, and z ), are used for all observation angles of a
particular part of the fibre. Stage movement is used only
when moving to the neighbouring part of the fibre. The x-
and y-range (field of view) are given by the total
magnification, camera pixel size, and camera resolution.
For the Brno HRCM the field of view is approximately
160 £ 120 mm for a 63 £ objective. The 63 £ magnifi-
cation is necessary for high-resolution FISH imaging in
order to have sufficiently high sampling frequency. A
typical z-range used is 60 mm. The volume of view is then
aligned with the fibre so that the largest range (x-range of
160 mm) is aligned with the direction of the fibre axis and
the fibre is centred within the second largest range (y-range
of 120 mm) (Fig. 3).
Only those nuclei that lie within the volume of view are
acquired each time. For each nucleus, a rectangular 3D
region of interest is computed from the counterstain image
and recorded (see the small cube in Fig. 3). Hence, only
selected sub-volumes of the whole volume of view are
stored and further processed by means of high-resolution 3D
image analysis after multi-spectral acquisition of all colour
planes containing the signals of labelling sites. This
substantially reduces the disk space consumption. More-
over, small 3D images are much easier to handle than huge
ones.
After the acquisition, the fibre is rotated by a certain
angle (e.g. 10 or 208) and a new set of cell nuclei is acquired.
The new set contains some of the nuclei acquired before and
rotated by the given angle as well as some additional nuclei
that have just entered the volume of view. Also some nuclei
leave the volume of view completely (Fig. 4a versus b). This
Fig. 2. Picture of the 2p-tilting device mounted to the Brno high-resolution
cytometer. The 2p-tilting device is placed onto the microscope stage
instead of the microscope slide (bottom central part of the photograph). An
inverted Zeiss Axiovert 100S microscope is used; therefore, the objectives
are located under the microscope stage and are not visible. A stepper motor
for fibre rotation (top left corner) is fixed on an external holder. The glass
fibre and the motor axes are connected with a drive shaft with two cardan
joints and an extendable shaft. This enables to move the microscope stage
with the 2p-tilting device freely in all three directions of space.
Fig. 3. The static volume of view approach. For the observation of cell
nuclei (small spheres) attached to the glass fibre (the large cylinder) static
observation intervals in all axes (x, y, and z ) are used (shown as a large
parallelepiped). This means that the camera field of view and z-coordinate
range remain constant during the fibre rotation. In other words, no lateral
stage movements are performed and the z-movement of the stage or
objective is kept within the same limits for each observation angle. The
volume of view is aligned with the fibre so that the largest range (x-range) is
aligned with the direction of fibre axis and the fibre is centred within the
second largest range (y-range). Only those cell nuclei within the volume of
view (the highlighted ones) are acquired each time. For these nuclei,
rectangular 3D regions of interest (shown as a small cube for one of the
highlighted cell nuclei) are computed and recorded. Hence, only selected
sub-volumes of the whole volume of view are stored.
M. Kozubek et al. / Micron 33 (2002) 655–665658
procedure is repeated until full (3608) rotation is accom-
plished. As a result each cell nucleus is acquired several
times at different angles as it travels through the volume of
view.
3.4. Geometrical and optical constraints
The angular range in which a cell nucleus is observed can
be given by simple geometrical rules. Let us denote the
nucleus diameter as D, the fibre radius as R, the depth of
penetration beyond the top of the fibre as P, the length of the
chord corresponding to the given penetration depth as L and
the angle corresponding to the given penetration depth as a
(Fig. 4a). The penetration depth P is a sub-interval of the
z-range of the volume of view. The full z-range goes below
the penetration range as well as above the nucleus lying on
the top of the fibre (i.e. the z-range is larger than P þ D ).
The y-range is also larger than L. The extra space in y- and
z-directions is provided in order to capture also tilted nuclei
that have their weight centre lying at the ends of the
a-interval (see nucleus number 1 in Fig. 4a) and also to
allow for small fibre axis fluctuations.
The value of D is typically about 10 mm for most cell
types. The value of R is adjustable (glass fibres can be made
of any radius with the precision of about 5 mm). However,
the smaller the radius R, the more difficult the fibre
manipulation during the preparation procedure is. The
smallest reasonable value of R is about 60 mm. In order to
assess the largest reasonable value of R it is necessary to find
out what its relationship to a is. The values of the remaining
three variables are restricted by the following constraints:
90 # a # 1808; L # 100 mmðLmaxÞ;
P # 30 mmðPmaxÞ
The first constraint says the angular range should be at least
908 in order to ensure equal image resolution in lateral and
axial directions and it should not exceed 1808 because
imaging through the fibre should be avoided. The second
constraint is given by the camera field of view (y-
resolution). In the set-up of the Brno HRCM used here the
y-resolution of the camera is 120 mm, therefore
Lmax ¼ 100 mm leaving 10 mm extra space on both sides.
The third constraint is given by the working distance of the
objective (typically 90–100 mm for high NA objectives)
and the range of the piezo-electrical objective nano-
positioner (100 mm). But most importantly P should be as
small as possible in order to suppress optical aberrations in
deeper focal planes that cause for instance light attenuation
and worsening of resolution with increasing depth below the
coverglass (Pawley, 1995; Kozubek, 2001a; Sheppard and
Wilson, 1979; Torok et al., 1997). For practical reasons
Pmax ¼ 30 mm was chosen. This yields a z-range of 60 mm
(P þ D þ 2 £ 10 mm extra space on both sides).
The defined variables apparently satisfy the following
equations:
L ¼ 2 R sinða=2Þ; P ¼ Rð1 2 cosða=2ÞÞ
The second and third constraints applied to these equations
yield:
a # 2 arcsinðLmax=2=RÞ for R . Lmax=2;
a # 2arccosð1 2 Pmax=RÞ for R . Pmax
If we now apply the first constraint ða $ 908Þ to the above
expression we obtain:
R # Lmax=ffiffi
2p
; R # Pmax=ð1 2 1=ffiffi
2p
Þ
This means R # 71 and #102 mm, i.e. R # 71 mm is
feasible for the acquisition process. For the fibre manipu-
lation the smallest acceptable value of R is about 60 mm.
This leads to 60 # R # 71 mm: In practice, fibres with R of
60–65 mm were used for biological experiments. The actual
values of R as measured under the microscope were
62–63 mm. For this radius P < 18 mm is sufficient for a ¼
908: Alternatively, with P ¼ 30 mm a < 1178 is obtained.
Fig. 4. The geometry of the fibre, the cell nuclei and the volume of view in
the y–z plane. In these drawings a y–z cut of Fig. 3 is shown. The large
circle depicts the fibre, the small circular shapes depict the nuclei attached
to the fibre. The solid rectangle depicts the y- and z-ranges of the volume of
view. Those nuclei that are visible within the volume of view are
highlighted. Only those cell nuclei that completely fall into the volume of
view are acquired each time, incomplete cell nuclei are ignored (in the left
picture, for example, nucleus number 1 is acquired whereas nucleus number
2 is not). Two successive angles of fibre rotation are shown. In this case, the
angular step is 208 yielding 18 steps per one full rotation. After the rotation
by one step, the volume of view contains some nuclei already acquired and
rotated by the given angle (208) as well as some other nuclei that just
entered the volume of view or were incomplete in the previous step (such as
nucleus number 2); on the other hand, some nuclei leave the volume of view
(such as nucleus number 1). The rotation procedure is repeated until full
(3608) rotation is accomplished. As a result, each cell nucleus is acquired
several times at different angles as it travels through the volume of view. In
the left picture, some notations used in the text are introduced: cell nucleus
diameter (D ), fibre radius (R ), depth of penetration beyond the top of the
fibre (P ), length of the chord corresponding to the given penetration depth
(L ) and angle corresponding to the given penetration depth (a ). Note that
the y-range is slightly larger than L and the z-range is slightly larger than
P þ D (denoted by dashed lines). This extra space is provided in order to
capture also tilted cell nuclei that have their weight centre lying at the ends
of the a-interval (such as nucleus number 1 in the left drawing) and also to
allow for small fibre axis fluctuations.
M. Kozubek et al. / Micron 33 (2002) 655–665 659
4. Software and image processing
4.1. Software environment
The software for microaxial tomography was written in
the C/Cþþ programming languages as a module to the
existing software system FISH 2.0 (Kozubek et al., 1999,
2001) used for HRCM operation. The software supports
both microscope set-ups: the HRCM based one in Brno and
the Zeiss Standard 25 based one in Heidelberg. It runs in
Microsoft Windows 95/98/2000/NT environment and con-
trols all microscope parts (filters, stages, objectives, etc.) as
well as the different CCD cameras (exposure time, gain,
readout speed, binning, etc.). The extension of the software
includes the control of the stepper motor that rotates the
glass fibre. The main goal of the software system is to
automate the acquisition and analysis of images of multi-
colour FISH labelled cell nuclei. Various modes of
operation are available for different purposes. All modes
of operation used in slide-based imaging are described
elsewhere (Kozubek et al., 1999, 2001). In this article, a new
module that supports fibre-based imaging will be described.
4.2. Image acquisition algorithm
A flow diagram of the algorithm for image acquisition is
shown in Fig. 5. First, parameters necessary for the
acquisition are set: angular step for fibre rotation (f ),
penetration depth (P ), approximate object (nucleus) diam-
eter (D ), z-range of the static volume of view, z-step for
coarse sampling (Dz_coarse), z-step for fine sampling
(Dz_fine), minimal object size, maximal object roundness,
colour channels corresponding to individual fluorochromes
to be acquired, exposure times for each colour channel
and other acquisition parameters specific to the
camera (gain, offset, readout speed). Some of these
parameters have the same value for all experiments
performed using the same hardware set-up and are pre-set
therefore as constants in the software (P ¼ 30 mm,
D ¼ 10 mm, readout speed ¼ 5 MHz). All other parameters
are specified by the user for each fibre separately. The user
also has to find the fibre interactively in the oculars, centre it
within the y-range of the camera field of view and manually
focus on the cell nuclei located on the top of the fibre (i.e. on
those closest to the objective). After this, the focus is
automatically moved by P/2 ( ¼ (P þ D )/2 2 D/2) deeper
into the fibre in order to reach the central plane of the whole
z-range.
After all parameters are specified and the volume of view
is correctly positioned, the automatic part of the algorithm is
launched. The first automatic step is coarse sampling of the
counterstain image. In this step, a low-resolution 3D image
of the whole volume of view in the counterstain channel is
acquired in order to obtain a global view of the whole scene.
Large z-step for coarse sampling (Dz_coarse, typically
1 mm) is used and the whole z-range is scanned. In order to
increase the sampling step also in x- and y-directions,
camera binning is used (usually 4 £ 4 is appropriate).
Binning significantly reduces the amount of data to be
processed, increases the readout rate and reduces the
exposure time (16 times for 4 £ 4 binning). Therefore, the
acquisition of the low-resolution image is very quick
(typically several seconds if a piezo-electrical objective
nano-positioner is used; for stepper motors this would take
more time).
The second automatic step is coarse image analysis. The
cells or cell nuclei are segmented using local thresholding
approach described elsewhere (Kozubek et al., 2001). For
each object, coordinates of the centre of mass, size,
roundness and other parameters are computed. Those
objects that do not satisfy user-specified criteria (minimal
size, maximal roundness) or are incomplete are excluded
from the result. For all remaining objects, rectangular 3D
regions of interest (i.e. parallelepipeds) are computed. Each
region contains the object itself and its immediate
Fig. 5. Flow diagram of the image acquisition algorithm.
M. Kozubek et al. / Micron 33 (2002) 655–665660
surroundings. The size of each parallelepiped is chosen so
that the number of background voxels is slightly larger than
the number of object voxels. This is important for
subsequent image analysis. The computed regions deter-
mine those sub-volumes of the whole volume of view that
should be recorded.
The third automatic step is high-resolution multi-spectral
acquisition. In this step, a high-resolution acquisition is
performed within the sub-volumes computed in the previous
step. Fine z-step (Dz_fine, typically 0.2–0.3 mm) and full
camera resolution without binning are used. For each sub-
volume a set of grey-scale 3D images corresponding to
individual fluorochromes (including counterstain) are
acquired. If z-ranges of two or more sub-volumes overlap in
axial direction, then 2D image slices at all z-positions that
belong to this overlap interval are acquired simultaneously for
all such sub-volumes. In other words, if we focus onto a certain
z-plane, then the camera simultaneously acquires all sub-
volumes intersecting the given z-plane at this z-position. After
all z-planes of all sub-volumes have been acquired the
microscope filters are changed for the next colour channel
(fluorochrome excitation and emission spectra) and new z-
stacks are acquired for all sub-volumes. The procedure is
repeated for all colours specified by the user. This method has
been chosen because the time required to change the filters is
substantially longer than the time necessary to move the
objective in axial direction (<100 versus<10 ms in our case).
The next automatic step is the rotation of the fibre by the
user-defined angle (f ). After this step a check is performed
whether the total rotation angle has reached 3608 or not. If
not then the algorithm goes back to the first automatic step.
After full 3608 rotation has been accomplished, the
microscope stage is moved along the x-axis by 95% of the x-
range of the volume of view in order to reach the neighbouring
part of the fibre. The 5% overlap (8 mm in our case) between
two neighbouring parts of the fibre is provided in order not to
miss the nuclei that were incomplete in the previous volume of
view. On the other hand the overlap is small enough as
compared to the average nucleus diameter value (D, typically
10 mm) so that no nuclei are acquired twice in different
rotations. After the lateral stage movement a check is
performed whether enough image data have been collected
or not. If not then the algorithm continues with the first
automatic step, otherwise the acquisition is finished. The
amount of data or the number of neighbouring parts of the fibre
(total x-range) to be acquired is specified by the user before the
automatic part of the algorithm is launched. Alternatively, the
user can stop the acquisition algorithm manually. In this case,
the acquisition of the current part of the fibre (all angles of
view) is finished before stopping the acquisition process.
4.3. Image analysis
After the collection of image data, the image
analysis phase follows. The primary goal of this phase is
image registration. This means that the corresponding
sub-volumes (i.e. sub-volumes containing the same object)
among all angles of view have to be found. After image
registration, an image fusion takes place. In this step, one
high-resolution 3D image out of several mutually tilted 3D
images (that have high-resolution in lateral direction but
low-resolution in axial direction) is computed. This step
yields a final image with high-resolution in all axes (Satzler
and Eils, 1997) and is usually accomplished using point-
wise maximum in Fourier space (Shaw et al., 1989).
Alternatively one could speak also of a model fusion. This
means that one precise model out of several mutually tilted
less precise models is computed. Model is a vector
representation of the real world and can consist of a cell
nucleus boundary description, positions (spatial coordi-
nates) of FISH-stained dots as well as boundaries of FISH-
stained regions in our case. Presently the software for image
analysis according to this strategy is under development,
therefore a detailed description will be the subject of another
article.
5. Experimental results and discussion
5.1. Preparation and FISH labelling of cell nuclei on fibres
In principle the glass fibres could be fixed on standard
glass slides and standard preparation protocols could be
applied. However, the loss of cell nuclei not fixed on slides
was not acceptable. Moreover, the nuclei mostly adhered on
the top of the fibre and not continuously around the fibre.
Therefore, new protocols for preparation, fixation, and FISH
were developed. These protocols as described in Section 2
are based on protocols available for glass slides, however,
with the modification of fibre handling.
Several different preparation capillaries are necessary for
one fibre and it is important to move the fibre from one
capillary into another as quickly as possible in order not to
let the cells dry in the air. If they dry at a certain step, the
spatial structure of the cell nuclei is distorted and the nuclei
collapse. Since this change of preparation capillaries
requires a skilled person, it might be helpful to develop a
special device in future where the fibres could be treated
more easily.
The capillary method introduced in this study works well
for all fibre diameters (120–200 mm range tested in this
study) and allows the preparation of FISH-stained cell
nuclei fixed onto the fibre surface from all sides. The
feasibility of the described approach is demonstrated on raw
image data for methanol–acetic acid fixation and formal-
dehyde fixation (Fig. 6). The confocal image of a
formaldehyde-fixed cell nucleus clearly shows the well-
conserved 3D shape in all spatial directions. In contrast, the
wide-field image of a cell nucleus fixed by methanol–acetic
acid clearly reveals the flatness of its shape in YZ projections
if this cell nucleus is sufficiently tilted. If the nucleus is
viewed on the top of the fibre close to the objective, its shape
M. Kozubek et al. / Micron 33 (2002) 655–665 661
Fig. 6. Examples of raw images acquired by automated axial tomography. HL-60 cell nuclei were fixed onto the fibre surface and stained using the modified
Fast-FISH method: centromere of chromosome 4 was stained with Rhodamine (red colour) and the interior of the cell nucleus was counterstained with DAPI
(blue colour). Methanol–acetic acid fixation (flat cell nucleus, left three columns) is compared to formaldehyde fixation (spatially conserved cell nucleus, right
three columns). In both cases the cell nucleus was rotated by 908 with a 108 angular step. Three orthogonal maximum-projection views (YZ, XY, and XZ ) are
shown for each cell nucleus and each angle of view. The rotation was performed around the x-axis. Therefore, the two red signals rotate around the centre of
nucleus in YZ projection, whereas in XY and XZ projections they move up and down as the cell nucleus rotates. The flat cell nucleus (left three columns) was
acquired in wide-field mode to show that also in this mode it is possible to observe the flatness if the nucleus is rotated. The spatially preserved cell nucleus
(right three columns) was acquired in confocal mode to prove the spatial structure also along the axial direction. Both nuclei were acquired with the sampling
step of 0.1 mm laterally and 0.3 mm axially. However, the dimensions of the cell nuclei differ although their volumes are similar: the flat nucleus fills a volume
of about 18 £ 18 £ 2 mm, the spatially preserved one of about 9 £ 9 £ 9 mm. Therefore, the side of the square in the left three columns was set to 24 mm
whereas in the right three columns it was set to 12 mm to include also some surroundings but keep the nuclei as large as possible at the same time.
M. Kozubek et al. / Micron 33 (2002) 655–665662
appears to be round also in XZ and YZ projections due to the
blurring effect typical of wide-field imaging. This indicates
that one should be very careful when analysing single-angle
wide-field 3D images. The real shape of 3D objects can only
be obtained by optical sectioning in a confocal mode or the
multi-angle approach of microaxial tomography. In addition
microaxial tomography considerably reduces resolution
unisotropy typical for confocal optical sectioning alone.
5.2. Rotation precision
Microaxial tomography of cell nuclei requires not only
special handling of the biological specimen but also
appropriately manufactured glass fibres that can be mounted
in the microscope adapter and precisely be rotated by the
stepper motor driven device. The requirements are (a) a
homogenous thickness along the fibre axis, (b) no twist or
bending of the fibre during manufacturing and specimen
preparation, and (c) correct axial fixation in the mounting
adapter so that the fibre has no translational movement (drift
in y- or z-direction according to Fig. 3) during image
acquisition.
In order to get an estimate about the rotation precision,
the y-movement of the bary centre of microspheres (900 nm
diameter) fixed on a fibre of ð200 ^ 20Þ mm diameter was
measured using the set-up based on the Zeiss Standard 25
microscope. If the fibre rotates exactly around its axis
without any translational movement, the images of the
microsphere show a movement with a y-component
according to
yðwÞ ¼ R½sinðwþ w0Þ2 sinðw0Þ�
with R the fibre radius, w the rotation angle and w0 the
starting angle of rotation. Fig. 7 shows a typical result of
measured values and the theoretical fit curve. From this
curve, the exact local radius at the position of the
microsphere was determined. In all experiments using
different fibres of the same specification, the measured local
radii at different positions were below the tolerances
(10 mm) given by the manufacturing process. This indicates
that the fibres were produced and handled without bending
or twisting and that the fibres can be mounted into the
adapter in such a way that drifts in the fibre axis are
negligible.
In these bead experiments also several distances between
two beads (typically about 30 mm) were measured for
different rotation angles from 0 to 908 in steps of 38. Using a
focus series of 10 images the error in the distance
measurements was about 1–3 nm at 08 (when the two
beads were located at the top of the fibre close to the
objective) and about 60 nm at 908 (when the two beads were
about 100 mm deeper). This corresponds to a theoretical
precision of the localisation of the intensity bary centre of a
900 nm bead of 1–2 nm at 08 and about 40 nm at 908.
Although the localisation precision depends on the photon
statistics and beads are bright objects, these data verify the
data of Bradl et al. (1996b) showing the usefulness of this
technique for Spectral Precision Distance Microscopy
(Cremer et al., 1999). Moreover, the results obtained here
verify experimentally that axial tomography allows distance
measurements between fluorescence objects always with the
best resolution if the rotation angle of 908 or even more is
feasible.
5.3. Automated image acquisition
Using the Brno HRCM set-up, the novel algorithm for
automated image acquisition of axial-tomographic speci-
mens has been tested on several fibre preparations. Both
wide-field and confocal modes were tried out. The algorithm
worked well in both cases and proved to be a very useful tool
for generating images for isotropic high-resolution studies.
The wide-field mode is naturally less time consuming (about
five times) as compared to the confocal mode due to the
shorter exposure times. With current hardware, the typical
acquisition time for full rotation of 3608 with 208 angular
steps is about 1–5 h for wide-field and 5–20 h for confocal
mode. The exact value depends on the density of nuclei on the
fibre, on the number of probes and on the desired axial
sampling step during high-resolution acquisition (Dz_fine).
The rate is limited entirely by the exposure times for
individual fluorochromes, therefore brighter dyes could
improve the speed. The acquisition time is quite long but
the instrument is capable of working automatically for a long
time, e.g. overnight or even several days.
For obvious reasons, the value of the angular step was
always chosen so that 3608 was divisible by this value. If we
omit values smaller than 108 (too fine steps) and bigger than
458 (too big steps) we get the following set of possible
angular steps: {10, 12, 15, 18, 20, 24, 30, 36, 40, 45}.
The appropriate value must be determined from the
results of the image analysis phase that is being developed.
The preliminary results (based on FISH experiments of
Fig. 7. Example of the measurement of the y-movement Dy of the bary
centre of a micro-sphere of 900 nm diameter rotated by a fibre with a
specified diameter of 200 mm. The rotation started at w0 ¼ 258 in steps of
Dw ¼ 28: The experimental values follow the fit curve (dashed line) yðwÞ ¼
R½sinðwþ w0Þ2 sinðw0Þ� with an apparent radius R of 102.2 mm.
M. Kozubek et al. / Micron 33 (2002) 655–665 663
centromere loci) suggest that steps smaller than 208 do not
bring much additional information, which is in accordance
with Satzler and Eils (1997).
Optical aberrations were significantly reduced in the
described design as compared to the previous publications.
The cell nuclei were acquired only at a certain angle range
so that they were close to the objective. The penetration
depth P (Fig. 4) was only 30 mm. No cell nuclei were
acquired through the fibre. A standard oil–coverglass-
embedding medium approach was used, i.e. the same
conditions as in the normal fluorescence microscopy.
Moreover, thanks to the rotation process, the worsening of
axial resolution in deeper layers was not important.
Fortunately, the lateral resolution (unlike the axial one)
does not change significantly with increasing depth below
the coverglass (Kozubek, 2001a). This phenomenon is very
important in axial tomography.
For the automated acquisition clusters of cell nuclei
caused some problems, if the density of cell nuclei on the
fibre was too high. In the present version of the algorithm,
such clusters are excluded from the coarse image segmenta-
tion results and are not recorded. Unfortunately, if clusters
of cell nuclei are not rare on the fibre surface, the yield of the
acquisition process is not as high as it could be if the cell
nuclei were spread uniformly on the fibre surface.
Especially in the case of clinical specimens that are limited
in the number of nuclei available, the preparation has to be
done in such a way that the cell nuclei are distributed more
uniformly and not too densely on the fibre surface.
Another point that has to be considered for image
acquisition in practice is the apparent drift in the fibre axis
position during the fibre rotation caused by inhomogeneous
local radii or tolerances in the fibre fixation in the mounting
adapter. As shown earlier this drift was, however, less than
10 mm in both lateral and axial directions for fibres of 120–
200 mm diameter. In order to take this apparent drift into
account, the extra space in y- and z-directions (Fig. 4) was
set to 10 mm on each side and the image analysis algorithm
was developed in such a way that it can compensate for the
drifting ½y; z� position of the fibre axis.
5.4. Conclusion and possible hardware modifications
In principle the fusion of the 2p-tilting device developed
in Heidelberg and the Brno HRCM with its automated
image acquisition software has been realised successfully.
The preparation and FISH procedure of cell nuclei has been
optimised for glass fibres and applied with fixation
conserving the 3D shape of the nuclei. Precise rotation
of the fibre in the mounting adapter is possible and
the image acquisition strategy developed appears to be
well feasible for large-scale imaging. Nevertheless, the
application shows that for routine practice it might be
helpful to develop a special fibre treatment device in order
to make the specimen preparation easier and to
overcome problems in specimen drying during changing
the preparation capillaries. Moreover, it might also be useful
to miniaturise the mounting adapter so that in a new version
of the 2p-tilting device the external stepper motor can be
omitted.
Another possibility how to make the fibre preparation
easier is to increase the fibre radius. This requires either lower
magnification (lower sampling frequency) which is not
acceptable in FISH imaging or a larger CCD resolution. For
example, a CCD chip containing 2048 £ 2048 pixels (instead
of currently used 1300 £ 1030 pixels) would yield
L ¼ 200 mm, R # 102 mm at P ¼ 30 mm (Fig. 4). This
would allow us to use fibres with 200 mm diameter which are
easier to handle. The increased volume of view would also be
better for handling the fibre axis drift. Unfortunately, the
CCD chips with large numbers of pixels are usually not
cooled or produce more readout noise, which is not desirable
in low-light FISH imaging. Nevertheless, new CCD cameras
(such as Photometrics Quantix 6303E) might resolve this
problem. Also the acquisition time could be considerably (at
least 2 times) reduced with state-of-the-art CCD cameras that
presently reach very high quantum efficiency (up to 70%).
Despite several approaches to further optimise micro-
axial tomography, it is so far a useful device in fluorescence
microscopy for the analysis of the 3D-architecture of cell
nuclei (Cremer et al., 2000), where for statistical reasons
large amounts of nuclei have to be imaged and evaluated
under optimum microscopic conditions. With the develop-
ment of appropriate software for 3D image reconstruction
and analysis microaxial tomography is going to become a
routine technique in our laboratories for applications in
human and mouse cytogenetics.
Acknowledgements
This research was supported by the Volkswagen Stiftung
(Grant No. I/75 946) and by the Ministry of Education of the
Czech Republic (Grant No. MSM-143300002). M.H.
acknowledges the financial support by a NCI/CCR Intra-
mural Research Award to Siegfried Janz, NIH, Bethesda,
USA. The authors thank Pavel Matula and Dr Gregor Kreth
for their help in preparing the figures and all colleagues who
read the draft of this manuscript for useful comments. The
authors are indebted to Prof. C. Cremer, University of
Heidelberg, for his continuous support of the joint project.
Finally the authors thank the mechanic workshop of the
Kirchhoff-Institute of Physics, Heidelberg, and the glass
workshop of the Physical Institute, Heidelberg, for their
production of the experimental set-up.
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