Three-Dimensional Imaging by Confocal

Scanning Fluorescence Microscopy"

G. J. BRAKENHOFF, H. T. M. VAN DER VOORT,
E. A. VAN SPRONSEN, AND N. N A N N I N G A
Department of Electron Microscopy and Molecular Cytology
University of Amsterdam
Plantage Muidergracht 14
I018 TV Amsterdam. The Netherlands

INTRODUCTION

The decision to pursue confocal microscopy was born from dissatisfaction with the
possibility of electron microscopy to acquire reliable data about the live morphology of
biological specimens in the submicron range. This possibility is strongly restricted by
the way various specimen-preparation methods affect the apparent structure in the
material. The required steps in the preparation (chemical fixation, dehydration and
thin sectioning) each introduce their own type of effect. For instance, a volume
shrinkage of bacteria has been observed with values up to 50%.' The resulting interest
in imaging techniques with a better resolution than standard light microscopy, but
where the specimen still could be observed live in its natural environment, led us to the
application in light microscopy of the confocal principle. With this approach, which
has been used before in acoustic microscopy: the resolution limitations of standard
light microscopy could be expected to be surpassed if optics of high numerical aperture
(N.A. = 1.3-1.4) were used. The actual demonstration of this fact in transmission
confocal microscopy by our group3 resulted in observed point responses of 196 nm at a
wavelength of 633 nm and 130-140 nm at wavelengths of 442 and 325 nm.
The confocal principle is also applicable in fluorescence microscopy where, actually
due to the incoherence of the fluorescence light, an even higher resolution may r e ~ u l t . ~
But even under conditions where practical considerations exclude these resolutions (see
below), there still remains the so-called sectioning effect in this mode by which
fluorescence contributions from off-focus layers in the specimen are prevented from
contributing to the image formation. Such contributions lead in normal fluorescence to
a strong reduction of the available contrast.
The serial way in which the data are produced in this type of microscope, together
with the sectioning effect, makes the confocal scanning laser microscope (CSLM)
particularly suitable for coupling to a computer system. Thus an apparatus results
which permits three-dimensional studies of biological specimens at high resolution
with relatively simple i n s t r ~ m e n t a t i o n . ~After
- ~ a description of the scanning micro-
scope (optical part, instrument control, computer system, processing algorithms used)
we present a measurement of the spatial point response and a number of applications of
three-dimensional imaging in biology. Finally, we will discuss some of the merits and
limitations of the various forms of scanning microscopy presently under development.

'Supported by the Stichting Technische Wetenschappen (STW) and the Foundation for
Fundamental Biological Research (BION/ZWO).

405
406 ANNALS NEW YORK ACADEMY OF SCIENCES

THE CONFOCAL SCANNING LASER MICROSCOPE

Principle

Characteristic for confocal microscopy is that one and the same point in the
specimen is both optimally illuminated from a point light source as well as optimally
imaged on a point detector. If the optics used are diffraction-limited, it can be shown
that at a given numerical aperture the point response of a confocal system will be
narrower by a factor of 1.4 in comparison to the conventional one. The demonstration
that this property, expected from a theoretical treatment in the paraxial approxima-
tion,* can be realized in practice with immersion optics of N.A. = 1.33 is important,
because only at those N.A. can an absolute gain with respect to conventional
microscopy be realized. The narrowing of the response transverse to the optical axis is
accompanied by a reduction of the depth of field along the axis by the same factor. So,
finally, in confocal microscopy the volume element in the specimen that contributes to
the image formation is smaller by a factor of (1.4)’ = 3 as compared to ordinary
microscopy.
The confocal principle also holds in fluorescence, though with two modifications:
the longer wavelength of the fluorescence radiation will lead to some reduction of the
expected resolution, while the incoherence of the fluorescence radiation may improve
the imaging4 When a not very small or point detector is used, as is often necessary in
fluorescence for reasons of detection efficiency, the effective instrument resolution is
determined by the dimensions of the diffraction-limited illumination spot. The
illumination-determined resolution in a fluorescence CSLM with a “large” detection
pinhole is in any case better than normal fluorescence microscopy because of the
wavelength-dependence of the resolution. In addition, the CSLM in fluoresence with
such a ‘‘large’’ (but not too large) detection pinhole still possesses the sectioning
property. In the normal fluorescence microscope all the radiation generated a t
different levels reaches the image plane, disturbing and reducing the contrast of the
image of the in-focus level in the specimen. In scanning fluorescence microscopy the
pinhole used, even if somewhat larger, will suppress quite effectively the out-of-focus
contributions, resulting in an image which is only related to the in-focus image
plane-a property which we call optical sectioning.

Instrument

In our CSLM we scan the specimen mechanically through the optically defined
confocal point. Some aspects of this choice will be discussed later. In FIGURE1 we show
the microscope in the fluorescence mode and give a description of the image formation.
The instrument is also equipped with a spectrograph, so that we can select a specific
wavelength band for fluorescence imaging. The system contains two computer systems;
one small, relatively slow, 8-bits system which takes care of the various instrument-
control functions and a faster 16-bits system with a 68000 processor dedicated to image
collection, processing and display functions. This second system can perform data
operations like stretching, inverting, filtering (gaussian, median) and local contrast
enhancement.s In addition, we generate from this system a continuous 60 Hz repetition
rate viewing image independent of the mechanical scan.
The most useful of the various programs presently available is the one for the
generation of three-dimensional images. This program consists of two units; the first
automatically acquires a set of images at various heights in the specimen according to
BRAKENHOFF et al.: CONFOCAL SCANNING FLUORESCENCE MICROSCOPY 407

the parameters set by the operator. The second unit takes care of the actual
three-dimensional stereoscopic image operation by a procedure as described in FIGURE
2. The stereoscopic viewing angles, as determined by the factors s, and s2, can be
chosen in such a way that the desired three-dimensional structure is optimally
presented.

mechanical scan control

! il,bminotion
,,
pin hole dichroic
mirror

r L i
laser

. +filter blocking

computer

t
- direct
display
computer
display
pin hole
-spectrograph

Instrument Response

In FIGURE3 a measurement of the spatial response characteristics of the

instrument in fluorescence (under typical operating conditions) is presented. For this
measurement we have used the above-described routine to obtain the set of through-
focus images through the bead. As can be seen from FIGURE3, the transverse
resolution (width at half intensity) can be estimated to be 220 nm, while the resolution
along the optical axis is about 730 nm. The size of the detection pinhole used during
408 ANNALS NEW YORK ACADEMY OF SCIENCES

this measurement was, backprojected in the specimen plane, 400 nrn. To put these
values in perspective, we estimate the dimensions of the illumination spot using
relations which are strictly speaking only true in the paraxial approximation; the actual
dimensions a t high N.A. may be expected to be somewhat higher. The width of the
illumination distribution transverse to the optical axis is about equal to the value of the
diffraction-limited resolution R of a lens system.

R = 0.61. Xo/N.A. (1)

To estimate the width of the illumination distribution along the optical axis we can use

section N

section 2

section 1

section 0

FIGURE 2. In the algorithm for generation of stereoscopic image pairs we assign the maximum
value of the pixel values encountered along “viewing” line L and R to the corresponding pixels in
the stereoscopic image pair, i.e. a pixed value Lij (ij: O..255) will be the maximum of the pixel
values Ai+slk,j+sk,k(k: O..N - 1, N number of optical sections) where A,,+,denotes a pixel of optical
section k with coordinates i j . In cases where s, of s2 are not integers, the compared value is the
result of a linear interpolation between the pixels nearest to the coordinate (i+s,k, j+s,k).

the relations (adapted from REFERENCE

9):
a (N.A.)~
I (u) = ((sin u)/u)’/I (0) with u = Z
2 n X,
(z: coordinate along optical axis, n: refractive index immersion medium, A,: vacuum
wave length light used). We can calculate from Equations 1 and 2 for A, = 483 nm and
N.A. = 1.3 a transverse width of the illumination distribution of 227 nm and along the
optical axis a width of 763 nm. We may therefore conclude that the observed
 X

--I +-.,
ItL
-Y
Fluorescence: X > 520 nm
Excitation: X = 488 nm

-2
)----.I Fluorescence: X > 520 nm
IP Excitation: X = 488 nm
FIGURE 3. CSLM instrument resolution in fluorescence as determined by a set of computer-
stored images of a 90-nm fluorescent bead taken at mutual distances of 100 nm. Excitation was at
h = 488 nm and fluorescence light detection at wave lengths above 520 nm. Top: Transverse or
X-Y resolution (width at half intensity) can be estimated to be 220 nm. Bottom: From the set of
through-focus images in the frame store 4 X-Z images are generated at Y positions close together
within the 90-nm bead. These 4 images were set side by side by the computer in the image
presented, and from the line plots through these images a Z-resolution of about 730 nm can be
inferred.
410 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 4. Chloroplast of a living green alga Spirogyra in water. (a) CSLM autofluorescence
image. Excitation illumination X = 483 nm. Image generated by the algorithm described in
FIGURE 3. Inset shows one section of the image set during this reconstruction. Image is taken with
a lOOX immersion objective of N.A. = 1.3. Note the large image field made possible by the
mechanical scanning approach. (b) Comparison image as obtained by normal transmission
microscopy (N.A. = 0.65). Bur = 20 pm.

fluorescent point resolution in our instrument is determined by the illuminating

excitation radiation and not by the emerging fluorescent radiation. From the fluores-
cent imaging we would have expected a transverse resolution of about 400 nm, as
determined by the size of the backprojected pinhole.

APPLICATION TO BIOLOGICAL SPECIMENS

We present some of the results acquired in our microscope which are chosen to
illustrate its capabilities. FIGURE 4(a) shows an image of a living green alga as
produced by the above-described three-dimensional procedure. We present just one
image (with s, = s2= 0) to show that by addition of the images acquired a t the different
height positions, which have a very short depth of field (see inset), we can in fact
produce a high-resolution image with a very large depth of j e l d . An image from
normal transmission microscopy is also given for comparison (FIGURE4b). Also note
the large field of view in this image acquired in our microscope operating in this and all
subsequent images with a N.A. = 1.3 lOOX oil immersion objective.
The cytoskeleton of a rat hepatoma cell stained with antibodies directed specifi-
cally against the keratin is depicted in FIGURE5. The stereoscopic images clearly show
the cytoskeleton with the location of the unstained nucleus visible as a black hole inside
this structure, illustrating the point that specific biochemical substances can be
localized and visualized in three-dimensions. As a third example we show in FIGURE 6
BRAKENHOFF ei al.: CONFOCAL SCANNING FLUORESCENCE MICROSCOPY 41 1

the mitochondria1 network inside a budding yeast cell. This high-resolution image,
with image-structural details going down to about 200 nm, presents information which
otherwise could only be obtained through normal sectioning techniques (with subse-
quent microscopy, either light or electron). The specimen imaged is still alive and we
expect to be able to follow such a specimen during its subsequent development in time.
Finally, we show in FIGURE 7 in the CSLM image of a spore how, by simply tuning the
spectrographic element, one can select either a reflection or two different fluorescence
images, representing cell components fluorescing a t differen; wavelengths.

COMMENTS AND CONCLUSIONS

We have presented above the mode of operation and some of the results of our
scanning microscope based on mechanical scanning of the specimen through the focal
point. This type of scanning, which we may call on-axis, is to be distinguished from
other types, which we call of-axis and which are characterized by the fact that the
information-collecting light beam is moved over the stationary specimen. Image points
are addressed which are located away from the optical axis of the instrument, hence
the name. The latter type of microscope occurs in two forms: in the first one a fine spot,
derived from a laser light source, is scanned rapidly over the specimen in the transverse
directions by a mirror (or a rapidly rotating polygon mirror system) placed in the
illumination path. Confocal conditions can in principle be realized if the emerging light
signal traverses the same light path. The second form is the tandem reflection scanning
light microscope (TRSLM) where actually an array of mercury arc lamp-illuminated

-
FIGURE 5. The cytoskeleton of a rat hepatoma (cancer) cell stained with fluorescently labeled
antibodies directed against one of the constituent proteins (keratin). Bar = 10 pm. Excitation X =
483 nm. Fluorescence detected X > 510 nm.
-
FIGURE 6. Living cell of the budding yeast Saccharomyces cerevisiae stained with the
fluorescent dye DASPMI in order to reveal the structure of the mitochondria1 network. DASPMI
is a vital dye, which can be taken up in a living cell without causing this cell to stop functioning.
Bar = 3 pm. Excitation X = 476 nm. Fluorescence detected X > 510 nm.

FIGURE 7. Images of one optical section through a field of Polytrichum commune spores taken
as a function of wavelength with the computer-controlled spectrograph. Excitation wavelength
X = 483 nm. (a) Reflection image detection a t A = 483 nm. (b) Fluorescence image detection at
X = 550 nm. (c) Idem detection at h = 620 nm. All images bandwidth X = 30 nm. (a) shows the
outer shell of the spore in reflection, (b) the green-yellow fluorescing cytoplasm and (c) the red
fluorescent chloroplast organelles inside the spores.
BRAKENHOFF et al.: CONFOCAL SCANNING FLUORESCENCE MICROSCOPY 413

holes in a rotating plate (Nipkov disc) is imaged on the specimen and the emerging
signal is imaged on the image plane after passing a corresponding set of holes." The
resolution to be achieved in both types of off-axis scanning microscopes depends
basically on two factors; the size of pinholes and the quality of off-axis imaging of the
lenses used.
In laser-based systems (both on-axis and off-axis) sufficient light is available to use
very small pinholes, so that the illumination distribution is set by the diffraction
limitations of the lenses used. The detection pinhole can in principle be made
sufficiently small so that the available confocal resolution gain can be r e a l i ~ e d . ~
However, for reasons of light efficiency, often a somewhat larger pinhole is used. In
this paper we show in fact that in our instrument of the two possible basic imaging
improvements in confocal fluorescence microscopy-(a) resolution determined by
shorter wavelength excitation radiation and (b) the full confocal resolution available
when very small pinholes are used-at least the first one has been realized. In the
TRSLM, however, due to the size of the holes used in Nipkov disc, the illumination
distribution is not diffraction-limited. The limitation on the hole size is connected with
possibilities of manufacturing precision and the requirement that sufficient light be
available. The result is that in this scanning approach it will be very difficult to realize
conditions in practice where, apart from the sectioning effect, a basic imaging
improvement with respect to normal microscopy is present.
A fundamental advantage of the on-axis technique is that, due to the mechanical
scan, for each point in the image the imaging conditions are identical. This is an aspect
which is very important for image processing, because one is able to use for the various
operations algorithms which are not dependent on pixel position. The mechanical scan
may prove limiting for the acquisition of images from large scan fields at high image
repetition rates. For small image frames with a reduced number of scan lines spaced a t
distances of the system resolution, the image repetition rate can, however, be made
rather high. For instance, 5 to 10 full resolution images per second of a field of 10 x 10
pm seem to be well realizable. In on-axis scanning microscopy the aspect of aberrations
introduced by the imaging of object points away from the optical axis does not play a
role. For the same reason the optics used can be considerably simpler (fewer lens
elements, higher transparency). In addition, in on-axis microscopy the image fields are
not limited by the field of view of the optics used, but can be much higher, being
determined by the amplitude of the mechanical scan. All the laser systems, either
on-axis or off-axis, have in common that the laser spot, if of sufficient power, can be
used after positioning on a specific point in a live specimen to interact actively with this
specimen.
In conclusion we may say that we have realized in the microscope of the on-axis
type presented here the capability of generating two- and three-dimensional images a t
high resolution. We believe that this capability will be invaluable for the study of live
biological specimens, especially during their development, not only because of the
images generated, but also because the processes observed can be described quantita-
tively.

ACKNOWLEDGMENTS

We would like to thank F.H.A. Aalst, J.A.W. Kalwij, 0. Peters and W .

Takkenberg for their technical assistance and V . Sarafis for providing the Polytrichum
commune specimen.
414 ANNALS NEW YORK ACADEMY OF SCIENCES

REFERENCES

1 WOLDRINGH, C. L., M. DE JONG, W. VAN DEN BERG& L. KOPPES.1976. Morphological

analysis of the division cycle of two Escherichia coli substrains during slow growth. J.
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2. LEMONS, R. A. & C. F. QUATE.1970. Acoustic microscopy-scanning version. Appl. Phys.
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with high aperture immersion lenses. J. Microsc. 117: 219-232.
4. Cox, 1. J., C. J . R. SHEPPARD & T. WILSON.1982. Super resolution by confocal fluorescent
microscopy. Optik 6 6 391-396.
5. VAN DER VOORT,H. T. M., G. J . BRAKENHOFF, J. A. C. VALKENBURG & N. NANNINGA.
1985 Design and use of a computer-controlled confocal microscope. Scanning 7: 66-78.
6. WIJNAENDTS V A N RESANDT, R. W., H. J. B. MARSMAN, R. KAPLAN,J. DAVOURT, E. H. K.
STELZER & R. STRICKER. 1985. Optical fluorescence microscopy in three dimensions:
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7. BRAKENHOFF, G. J., H . T. M. VAN DERVOORT, E. A . VAN SPRONSEN, W. A. M . L~NNEMANS
& N . NANNINGA. 1985. Three-dimensional chromatin distribution in neuroblastoma
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microscope. Scanning 7: 97-108.

DISCUSSION OF THE PAPER

F. SACHS (State University of New York, Buffalo, New York):How do you do the
scanning?
BRAKENHOFF: Very simply. We just have a scan table which is driven from this side
and the other side. The scan table just rests on a ruby, which scans over a hard metal
plate. There is a small spring that keeps it to the hard metal plate.
SACHS:What’s the pusher?
BRAKENHOFF: This is just something derived from the voice coil of a loud speaker.
There’s a sensor, of course, on the movement, and you have a feedback system.
D. A. ACARD (University of California Medical Center, Sun Frantisco, Califor-
nia): How many points per second?
BRAKENHOFF: The computer images were acquired a t 256 points per scan line and
it takes 0.01 of a second to complete a scan line, so that gives an indication. The scan
table is scanned a t 50 Hz but you only use the movement in one direction, hence the
0.01 second. When you photograph the image directly during the actual scan, the
resolution is determined by the C R T (cathode ray tube) in which it is displayed.
Incidentally, by varying the scan frequencies and the number of scan lines per image,
you can push the microscope to 5 or 10 images per second with small image fields like
10 x 10 pM, and have within these images still the full resolution of the confocal
microscope.
A. BOYDE(University College London, London. England): The whole family of
confocal microscopes has allowed us to overcome a serious problem with light
microscopy, that you can’t get a high-resolution stereo view with a conventional optical