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Microscopy and imaging science: practical approaches to applied research and education (A. Méndez-Vilas, Ed.)
Epi-fluorescence microscopy and 3D printing: An easily implemented
approach for producing accurate physical models of micro- and macroscopic biological samples
K.A. Holt1 and M.S. Savoian2,*
1
Institute of Agriculture and Environment, Massey University, Palmerston North, NZ
Institute of Fundamental Sciences, Massey University, Palmerston North, NZ
* Author for correspondence
2
A common difficulty experienced by students of the life sciences is the inability to visualise the three dimensional (3D)
nature of small (micrometer to millimetre-scale) complex biological specimens. Most representations are two dimensional
(2D) and cannot unambiguously convey complex 3D distributions or textures. Here we outline an approach for generating
tactile models through the combination of scanning confocal microscopy (SCM) and 3D thermoplastic printing. The
versatility of the approach is demonstrated using three highly divergent samples of different scales and geometries:
prophase nuclei (subcellular level), pollen shells (cellular level) and finally, fruit flies (small organism level).
Considerations for image acquisition, data handling and physical printing are discussed.
Keywords: Scanning confocal microscopy; 3D printing, scale models; pollen; Drosophila
1. Introduction
One of the most striking aspects of biology is the spectrum of morphological variation; differences in object shape and
texture can be observed from the subcellular level up to that of the tissue and beyond. Translating complex biological
architecture into a form that can be readily studied while remaining accurate in all three dimensions has proven
challenging. Simple observations through the microscope are grossly incomplete providing no information on texture.
Furthermore, confusion and misrepresentation regarding an object’s 3D distribution rapidly increase with sample
thickness or complexity. Traditional approaches to study thicker and larger specimens utilised physical serial
sectioning. However the sequential slicing of a sample is labour intensive, can lead to loss of information or artefacts
due to treatment or mechanical damage and further leaves the investigator with a collection of 2D sections and resultant
images that require meticulous alignment to generate a reconstruction.
Scanning confocal microscopy provides a comparatively non-invasive way to image complex 3D samples without
mechanical sectioning. Like other epi-fluorescence-based techniques, SCM relies on the excitation of molecules whose
emitted fluorescence is collected for visualisation. Objects or features of interest may be selectively labelled using
specific exogenous probes or depending on composition, may be autofluorescent and emit without additional dyes. In
contrast to the routinely used widefield epi-fluorescence systems found in most research and teaching laboratories,
scanning confocal microscopes employ a pinhole in front of the detector that excludes light from outside of the plane of
focus. The practical effect of this is sharp images with a high signal to noise ratio and a spatial resolution that when
using high numerical aperture (NA) lenses can approach the diffraction limit of light even in thick samples. The absence
of image blur and signal masking that normally occur due to contaminating light from adjacent planes promotes optical
sectioning with comparatively little loss of signal quality throughout the entire volume. Each captured focal plane or
image slice may be compiled and stacked in silico to reconstruct the sample. This virtual 3D model can be rotated on
any axis, zoomed to examine different features and re-sliced to reveal cross-sectional information.
Virtual reconstructions are not entirely interchangeable with physical models. At least in some disciplines that
require an intense understanding of spatial distribution such as anatomy, physical reconstructions appear to offer a
superior teaching experience over their 2D counterparts [1, 2]. Our anecdotal observations of undergraduate palynology
students also suggest that tangible materials facilitate a better understanding of sample geometry. Additionally, they are
more engaging to the broader biological community. This should not be surprising given the subjective interpretations
those with poorer spatial perception skills must make in the absence of tactile information about proportion, distribution
and texture.
In order to translate SCM’s high quality image data into physical models we have taken advantage of 3D printing
(3DP) and additive manufacturing approaches. The 3DP of image data is far more efficient than previous reconstruction
efforts. These relied e.g., on sequentially tracing each image onto a slice of deformable media such as clay or foam
which was then stacked together. In 3DP the model is generated through the precise deposition of material into a crosssectional area corresponding to each image data slice. The successive layering of these sections reconstructs the
protrusions and interstices of the data into a tangible form. There are several types of 3D printer available which vary in
construction method, materials, output quality and cost [3]. We employ Fused Deposition Modelling (FDM) also called
Fused Filament Fabrication (FFF) technology, which heats and extrudes a plastic wire through a fine aperture. Although
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these printers do not provide the highest resolution output available, their purchase and operating costs are relatively
low, traits making them popular and available to most institutions and even the home consumer market.
Here we outline an easily implemented approach for producing scale physical 3D models from scanning confocal
microscope data. Considerations about image acquisition, data modelling and export are presented and representative
3D printed outputs shown. The general applicability of the method is demonstrated using objects of diverse sizes and
complexities; animal cell nuclei (subcellular level), pollen shells (cellular level) and fruit flies (millimetre-sized small
organism level).
2. Scanning confocal microscopy considerations, sample preparation and imaging
The first step in the printing of biological sample models is acquiring data that accurately represents the specimen in
3D. Epi-fluorescence microscopy requires that both excitation and emission signals travel through the entire optical
pathway. Photons must move bi-directionally through the sample, the mountant, the coverslip and if using a high NA
lens, the immersion oil. Thus along with optimising the microscope, it is important to optimise the sample. For larger
specimens this may involve introducing a clearing step to reduce opacity and scattering [4]. Variations in refractive
index (RI) along the pathway scatter the light and diminish the image’s spatial resolution and brightness. In thinner
specimens such as most tissue culture cells, small mismatches do not preclude quality imaging. Conversely, samples 10
µm or more in thickness are increasingly sensitive to alterations in RI with details and object boundaries becoming less
distinct with depth. Therefore, the refractive index of the sample, mounting agent and imaging chamber should be
matched whenever possible. For larger objects like the pollen shells or insects studied here, we use 2,2’-Thiodiethanol
(TDE) for sample mounting. This water miscible media’s RI can be modulated based on its concentration. Its RI is 1.51
when 97% concentrated, a value that is the same as coverslip glass and standard immersion oil, making it well suited for
many thick specimen imaging tasks. However at concentrations of 80% and above TDE quenches the fluorescence of
the genetically encoded probe, enhanced green fluorescent protein [5]. When using this label lesser TDE concentrations
may be desired or the fluorescence may be supplemented by adding an exogenous dye with similar spectral properties.
Even with refractive index matching, thicker objects may experience a degradation of signal intensity. In these instances
it is important to carefully isolate and orient the sample to ensure the feature of interest is fully captured. Alternatively,
objects may be sequentially imaged from opposing directions if appropriate imaging chambers are used. After careful
alignment the two data sets may be combined into a common data stack.
2.1 Sample preparation and image acquisition for samples ranging in size from the subcellular to macroscopic
Towards establishing the broad applicability of our model making workflow we imaged three specimen types of unique
size and geometric characteristics; a prophase nucleus (diameter of ~12 µm), pollen shells ≥30 µm in size and fruit flies
~3 mm long. All imaging was performed using a Leica SP5 DM 6000B upright scanning confocal microscope. The
pinhole was kept at a default value of 1.0 airy units. In order to maximise object contrast without losing information
detector settings were empirically determined for each sample that allowed the signal intensities within the imaged
volume to approach but never reach saturation while the background was kept near null values. Details of each of the
specimens’ preparation and image acquisition are described below.
For the imaging of prophase nuclei, human tissue culture cells (HeLa) were grown in DMEM media supplemented
with 10% FBS and penicillin/streptomycin and maintained at 37°C under a 5% CO2 atmosphere. Prior to fixation cells
were plated onto No. 1.0 thickness coverslips and allowed to adhere for 1 hour. The media was removed and the cells
were immediately fixed with 4% paraformaldehyde in Phosphate Buffered Saline (PBS) for 15 minutes. Following PBS
washes the coverslips were mounted onto slides using Vectashield supplemented with the DNA staining dye DAPI
(Abacus ALS Ltd) (RI; 1.45). DAPI fluorescence was excited with a 405 nm laser line and its emission collected
between 415-505 nm. Samples were imaged with a 63X NA 1.4 lens. Optical sections were collected at 335 nm
intervals with a voxel size (x, y, z) of 80 nm x 80 nm x 335 nm.
Pollen from two species, Brachyglottis repanda and Fuscospora fusca were collected from fresh plant material. In
order to remove the cellular components samples were acetolysed using a modification of [6] as suggested by [7]. The
remaining pollen shells, or exines were mounted in 2,2’-Thiodiethanol (TDE) according to the method of [5, 7]. In
brief, samples were placed for 10 minutes each in the following concentrations of TDE diluted in PBS: 10%, 25% and
50% followed by two changes in 97% TDE. A third 97% TDE change (final RI; 1.51) was used to mount the pollen
onto slides beneath a No. 1.0 glass coverslip. Exine autofluorescence was excited with 561 nm laser light and the
emission collected from 597-748 nm. Pollen were imaged with a 63X NA 1.4 lens. Optical sections were taken every
340 nm. Each voxel (x, y, z) was 60 nm x 60 nm x 340 nm.
Wildtype Drosophila melanogaster were used as macroscopic samples. Cultures were maintained at 24°C under
standard conditions [8]. Selected adults were washed in PBS to remove adherent food. Whole animals were fixed by
immersion in Modified Karnovsky’s Fixative (2% paraformaldehyde, 3% glutaraldehyde in 0.1M Phosphate buffer)
overnight at 24°C. This fixative was selected as paraformaldehyde has been reported to increase arthropod cuticle
autofluorescence while glutaraldehyde promotes that of soft tissues [9]. For intact fly studies, cuticles were cleared of
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pigment using 30% H2O2. Similar to [10], after ~10 days the eyes and cuticles became colourless. Individuals were then
PBS washed and immersed in a graded TDE series as above. Whole animals or non-cleared, isolated heads were placed
within standard concave slides and mounted in 97% TDE beneath a No. 1.0 coverslip. Preparations were imaged with a
10x NA 0.4 lens. Tissue autofluorescence was excited across the spectrum using the following laser lines: 405 nm, 488
nm, 568 nm, 633 nm; the corresponding detection windows were: 415-505 nm, 493-600 nm, 580-630 nm, 640-800 nm.
For examinations of head morphology images were acquired at 5000 nm intervals with a voxel size (x, y, z) of 1515 nm
x 1515 nm x 5000nm. Images of intact flies were captured with a z step of 2979 nm and the voxel size (x, y, z) was
3033 nm x 3033 nm x 2979 nm.
3. 3D virtual model making and physical model printing
In order to translate scanning confocal images into 3D printed models the data must be converted into virtual
reconstructions and then output in a printer compatible file format. We perform all steps prior to printing with the public
domain programme ImageJ [11]. After import the quality of the image stack is determined by viewing the component
optical sections (Figs. 1A, 2A, A’). This identifies planes beyond the object of interest and facilitates their removal. It
further allows fluorescence signal intensities and noise to be assessed. Dim signals may be increased while noisy data
may be smoothed using a variety of filters. As illustrated by the prophase nucleus in Fig. 1B, the quality of the data may
be further evaluated by collapsing a stack into a maximum intensity projection that simultaneously displays all of an
object’s features without z-axis context.
Transformation of image stack data into virtual 3D models is performed with ImageJ’s 3D Viewer plugin [12]. We
find that the most important parameter in the conversion is the object threshold; this defines the intensity values to be
incorporated into the reconstruction. Altering the value to be more inclusive can lead to models that look “smooth”.
Conversely, making it more exclusive may cause features that are dim in the original image data to be absent in the
model. The threshold value needs to be empirically determined and optimised for each sample towards maximising
virtual model accuracy (Figs. 1C, 2B, B’, 3B). Despite its ability to generate 3D models, ImageJ lacks the tools for
editing reconstructions beyond modifying the source SCM data. To correct edges, fill in missing faces or otherwise alter
model features, alternative programmes must be utilised. There are many commercially available 3D modelling
packages as well as public domain equivalents that can be readily found on the internet. These should all read the .stl
format that the virtual model is exported as by the 3D Viewer plugin.
Our additive printing is performed using thermo-plastic based extrusion technology (UP Plus 2; 3D Printing systems
NZ). As represented by the model of the prophase nucleus in Fig. 1D, FDM/FFF printers deposit a scaffold at the
object’s base (arrowheads) as well as in some interstices (arrows) for support during manufacture. Its mechanical
removal is required in order to see the complete object (Fig. 1E). An unexpected biological feature identified when
manipulating the scaffold-free physical model was the limited occupancy of the nucleus. Unlike the chromosomes
which extensively contacted one another throughout much of the image data, the reconstruction revealed a complex
series of channels passing throughout the nuclear volume that was not easily discernible in individual sections or the
virtual reconstruction.
Upon closer inspection of the 3D printed nucleus the extruded plastic appeared as layers on some surfaces (Fig. 1F).
While the deposition process is the same for all of the reconstructions made with FDM/FFF printers, the texture is not
always apparent (compare with Figs. 2C, C’, 3C, D, F). Its conspicuousness is a function of the specimen’s small real
world size and object intricacy relative to the printer’s maximum z-resolution of 150 µm. The nucleus is ~12 µm in
diameter and contains chromosomes whose surface features are well below the SCM’s practical diffraction limited
spatial resolution limit (~0.3 µm and ~0.5 µm lateral and axial, respectively). They are therefore printed without
features beyond the extruded material’s texture. It is noteworthy that some thermoplastics may be chemically smoothed
to remove this printing artefact. Irrespective of the plastic’s identity, the orientation of the virtual model may be
changed within the print bed to alter the deposition pattern and the design and position of the scaffold.
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Fig 1. Translation of scanning confocal microscopy images of a prophase nucleus into a 3D printed model. A) Successive optical
sections from a stack of a nucleus. Numbers refer to optical section number. B) Maximum intensity projection of all 20 sections. C)
Virtual model of the scanning confocal data. D) 3D printed thermoplastic model of the same nucleus with the supporting scaffold
present at the base (arrowheads) and object interstices (arrows). E) The model after the scaffold has been removed. F) Close up of the
boxed region in E). A layered texture is visible in this contrast enhanced view of the model. Bars in B), E) and F) are 10 µm, 4 cm
and 1cm, respectively.
Fig. 2. Scanning confocal imaging and 3D printing of pollen exines. Pollen was isolated from the two indicated species.
A, A’) Optical sectioning reveals that exines are hollow structures with multiple entry apertures and unique surface
protrusions. B, B’) Virtual models made from the data stacks in A, A’). C, C’) The corresponding 3D printed
reconstructions of Brachyglottis and Fucospora pollen shells. Bars in A and A’ are 20 µm, C and C’ are 5 cm.
We next produced physical models of cell-sized objects using pollen exines as our specimens. Two round yet
morphologically distinct pollen species were selected, Brachyglottis repanda and Fuscospora fusca, each with a
diameter ≥30 µm. As illustrated by their individual optical sections (Figs. 2A, A’) and consistent with previous
observations, both exines are marked by a large central chamber and a characteristic number of openings or apertures.
Virtual reconstructions (Fig. 2B, B’) recapitulated the presence of the apertures and indicated that the surfaces of each
were textured with protrusions that are blunted but spike-like for Brachyglottis and much smaller bumps for Fucospora.
These features were tangibly demonstrated in our printed physical models which were ~10,000 times larger than the
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real object size (Fig. 2C, C’). Comparison with scanning electron micrographs revealed that the apices of the
protuberances in both species should be much finer than seen in our data and resultant models (unpublished
observations). The discrepancy is due to the limited spatial resolution of SCM. Its light-based restriction precludes it
from differentiating and displaying the same fine features that can be imaged using electron microscopy.
Finally, we applied our imaging and printing workflow to Drosophila melanogaster, a classic model system for
studying animal development and physiology. At adulthood the fruit fly is ~3 mm in length with well-defined body
segments (Fig. 3A). Initial investigations were made using isolated fly heads which are ~750 µm wide. As shown in
Fig. 3B, detailed virtual reconstructions of these near millimetre-sized objects could be generated using material that
has not undergone any special preparation beyond fixation and mounting. The resultant tactile models (Fig. 3C)
reproduced all of the salient morphological features of the fly’s head such as the multiple parts of the mouth, antennae
and eyes. Even when imaging at lower magnifications and resolutions, substantial detail could be seen and the
individual facets or ommatidia of the eye, which are ~20 µm wide in the native organism, were fully formed and clearly
defined. Noticeably absent however were the setae bristles of the head. These do not autofluoresce and appeared as
cavities in the virtual (Fig. 3B; arrowheads) and physical printed (Fig. 3C; arrowheads) models.
Intact and whole Drosophila adults were then examined. Due to the size of the organism relative to our image
capture area the fly’s posterior portion containing most of the abdomen was not included in our data sets. By exciting,
capturing and combining the autofluorescence signals from across the spectrum in these bleached and cleared
specimens we were able to image some features to a depth of ≤300 µm, or approaching half of the fly’s width. The
printed physical model presented in Fig. 3D reveals the same high level of detail observed for our head reconstructions
but along the entire animal. Individual eye facets were still visible along with the mouth parts. In addition, the delicate
hairs and bristles characteristic of the thorax, head and legs (not shown) were faithfully replicated. The veins of the
wing were also visible (arrowheads). Some samples experienced a slight compression due to their size and orientation
within the viewing chamber. This manifested as a featureless flat region on the physical reconstruction (Fig. 3D; arrow).
The optical sectioning capability of SCM further permitted us to document the fly’s internal soft tissues. No single
tissue type could be imaged fully independent of the cuticle using only autofluorescence. The most distinct among these
mixed signals was the musculature. In volumetric projections of the thoracic interior (Fig. 3E), the muscles appeared as
collections of darkened bands running parallel to and across the long axis (arrowheads). This complex tissue
arrangement was fully reproduced in our ~5 cm long thermoplastic reconstruction (Fig. 3F; arrowheads identify
structures corresponding to those in Fig. 3E). Because it is physically manipulable, the model conveys the segmented
and orthogonal arrangement of the muscles to a much great extent than the volumetric representation.
Fig 3. A series of virtual and physical reconstructions made from Drosophila. A) A living fruit fly. B) Virtual model of an isolated
head. Arrowheads denote several signal-free regions corresponding to the non-autofluorescent setae bristles. C) Different views of
the printed 3D model shown in B). Note the detailed eye facets and mouth parts. The setae’s lack of autofluorescent signals are
modelled as bristle-shaped cavities (arrowheads). D) 3D Printed model made from a cleared intact fly. Prominent fine features
include hairs along the head and thorax, individual facets on the eyes and wing veins (arrowheads). The area indicated by the arrow
identifies part of the source animal that flattened during sample preparation. E) Volumetric rendering of the internal thoracic region.
The muscles (arrowheads) are discreet darkened bands. F) 3D printed model of the internal thoracic anatomy. Arrowheads denote
corresponding muscles as in E). Bars in A, C, D and F are 2 mm, 10 cm, 10 cm and 5 cm, respectively.
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4. Summary
Here we have discussed an approach that combines scanning confocal microscopy with 3D printing to readily generate
high-quality tactile models. It utilises equipment and software available to most imaging facilities. Thus, it offers a
means for educators and researchers to easily translate their data into physical scale reconstructions for demonstration
and study. The method is robust, flexible and works on specimens ranging from the subcellular to macroscopic in scale
with highly diverse architectures. Our physical reconstructions were produced using FDM/FFF techniques. This
economical technology requires the construction of a removable support scaffold and sometimes suffers from a threaded
texture artefact. Both can be attenuated by altering the position of the virtual model in the print area. Texture may be
further altered by exposure to common solvent vapour. Models may also be detailed through dying or painting to
distinguish key features of interest.
We find that the greatest restriction on the quality of the output physical models comes from the source microscope
data. Like all conventional light microscopies, SCM is restricted to conveying information approaching the diffraction
limit of light. This precludes the resolving of especially fine features, such as details on the surface of prophase
chromosomes and the sharp apices at the end of pollen spike-like protrusions. The result is inaccurate object geometry,
a shortcoming that may be lessened using post-processing methods such as deconvolution [7, 13] or implementing nondiffraction limited “super-resolution” imaging technologies [14]. A further deficiency is the limited imaging penetration
depth. Innovations in sample clearing will undoubtedly allow for the optical sectioning of much thicker or challenging
materials without the loss of signal intensity. Such imaging advancements will enable the collection data from a variety
of specimens across the life sciences at never before seen quality. These can be collected into shared virtual libraries.
When combined with 3DP technologies they will allow the rapid and inexpensive generation of single or class-wide sets
of purpose-built, tactile models that promote teaching and facilitate understanding in exciting new ways.
Acknowledgements This work was supported by Massey University’s Institute of Agriculture and Environment Summer School
programme (K. A. H.) and the Institute of Fundamental Sciences (M. S. S.). All imaging was performed at Massey University’s
Manawatu Microscopy & Imaging Centre. We wish to thank O. Griewaldt and the Institute of Fundamental Sciences Engineering
Services for the 3D printing and T. K. Hale for supplying Hela cells.
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