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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