# Fast Micron-Scale 3D Printing with a Resonant-Scanning Two-Photon Microscope

Benjamin W. Pearre (1),
Christos Michas (2),
Jean-Marc Tsang (2),
Timothy J. Gardner (1,2),
Timothy M. Otchy (1)
((1) Dept of Biology, Boston University, Boston, MA, USA,
(2) Dept of Biomedical Engineering, Boston University, Boston, MA, USA)

###### Abstract

3D printing allows rapid fabrication of complex objects from digital designs.
One 3D-printing process, direct laser writing, polymerises a light-sensitive
material by steering a focused laser beam through the shape of the object to
be created. The highest-resolution direct laser writing systems use a
femtosecond laser to effect two-photon polymerisation. The focal
(polymerisation) point is steered over the shape of the desired object with
mechanised stages or galvanometer-controlled mirrors. Here we report a new
high-resolution direct laser writing system that employs a resonant mirror
scanner to achieve a significant increase in printing speed over galvanometer-
or piezo-based methods while maintaining resolution on the order of a micron.
This printer is based on a software modification to a commerically available
resonant-scanning two-photon microscope. We demonstrate the complete process
chain from hardware configuration and control software to the printing of
objects of approximately $400\times 400\times 350\;\mu$m, and validate
performance with objective benchmarks. Released under an open-source license,
this work makes micro-scale 3D printing available the large community of two-
photon microscope users, and paves the way toward widespread availability of
precision-printed devices.

Comments: Corresponding author: BWP (bwpearre@bu.edu). TJG and TMO contributed
equally to this work. Conflict-of-Interest statement: TJG is an employee of
Neuralink Inc. Keywords: 3d printing, additive manufacturing, lithography,
direct laser writing, DLW, two-photon microscopy, resonant scanning.

##  1 Introduction

Direct laser writing (DLW) lithography Maruo et al. [1997] is a 3D-printing
technology that can be used to fabricate small-scale objects with complex
geometries by programmatically exposing a light-sensitive material to a
focused laser beam Atwater et al. [2011], Buckmann et al. [2012], Cumpston et
al. [1999], Farsari and Chichkov [2009], Gissibl et al. [2016b]. Using
femtosecond laser pulses and two-photon polymerisation processes to write a
solid object structure into a photoresist, DLW enables on-demand fabrication
of complex 3D objects with micron-scale features Gissibl et al. [2016a],
Malinauskas et al. [2010, 2013], Niesler and Tanguy [2016], Farsari and
Chichkov [2009], Kabouraki et al. [2015], Gattass and Mazur [2008], Sun and
Kawata [2004], Skylar-Scott et al. [2017], Gottmann [2009]. While DLW achieves
diffraction-limited resolution, the printing speed of DLW is slow, practically
limiting the size of printed objects to millimetres. This speed limitation is
changing rapidly, with a number of advancements reported. The slowest DLW
printers use piezo stages, at speeds ranging from $\sim$ 0.1–30 millimetres
per second Straub and Gu [2002], Ovsianikov et al. [2011]. Galvanometer-based
printers can bring the laser scan rate up to tens or hundreds of mm/s Thiel et
al. [2010], Maruo and Ikuta [2000], Farsari et al. [2006], Obata et al.
[2013], Gottmann [2009]. Recent reports include raster-scanned printing with
high-speed galvanometers that achieve up to 400 mm/s Skylar-Scott et al.
[2016] by operating the scan mirror near its resonant frequency. Here we
extent this trend by incorporating a resonant mirror operating at 8 kHz, which
allows printing at speeds up to $\sim 8000$ mm/s.

In order to increase not only the speed of this technology but also its
flexibility and availability, we present a raster-scanning DLW (rDLW) system
built on a standard resonant-scanning two-photon microscope and open-source
control software that are common equipment in many physical and life science
laboratories. Open design and standard commercial components offer easy
modification and adaptation to accomodate new materials, object sizes, and
techniques.

We demonstrate the capabilities of our resonant rDLW printer in the
fabrication of micron-scale objects. The instantiation reported here is
capable of fabricating objects that are up to $\sim 400\times 400\times
350\;\mu\textrm{m}$ with minimum feature sizes of $\sim 4\times 1\times
2\;\mu$m (X, Y, and Z, respectively), and with finer X-axis features available
through the use of the microscope’s zoom. (We note that much larger objects
may be constructed by stitching together overlapping pieces of this size,
although a full discussion of this topic is beyond the scope of this work.) We
show that the use of a resonant scanner allows our system to print an object
of this size and arbitrary geometric complexity in about 20 seconds—a
significant increase in printing speed over fast galvanometer-based systems.
We evaluate performance with objective metrics assessed using IP-Dip
(Nanoscribe, GmbH), a proprietary refraction-index–matched resist developed
specifically for rapid, high-resolution DLW, although we expect that a wide
variety of photoresists may be used.

Finally, we release our application software under an open-source license.
Taken together, this work provides a new platform for innovation in DLW and
makes this technology more easily accessible to the community of two-photon
microscope users.

Figure 1: Overview of the resonant rDLW printer. (a) Schematic of the optical
path from laser source to printed object. (b) The raster scanner rapidly
sweeps the laser focus across the X axis of the printing workspace. (c) Top:
laser power is modulated above (red line) and below (grey dotted line) the
polymerisation threshold (green dashed line) throughout the X-axis sweep.
Bottom: by applying this pattern of laser modulation across the workspace,
solid features can be built up line by line and layer by layer. (d) SEM
micrograph of a Charles Darwin statuette printed with our rDLW printer.

##  2 Results

The rDLW system we report fabricates objects by raster-scanning the focal
point of a femtosecond laser through a volume of photoresist, defining the
object structure line by line. The device schematic in Fig. 1a depicts the
hardware configuration tested and reported here—essentially, a standard two-
photon microscope with a resonant raster scanner and high-speed/high-
extinction-ratio laser power modulator. This schematic need not be taken as
prescriptive, as one of the benefits to having an open system is the ability
to modify components to meet the requirements of new applications.

###  2.1 A 3D printer built on a two-photon microscope

Our printer was built around a commercial two-photon microscope platform. A
resonant+galvanometer scan module controls the laser’s X-Y focal point within
the printable workspace. (Throughout the manuscript, we use Cartesian
coordinates to refer to directions and dimensions in the printing workspace.
Following this nomenclature, X and Y are the perpendicular axes spanning a
single focal plane of the orthogonal Z direction. In keeping with this, X
denotes the direction of the high-speed (7.91 kHz) raster scanner’s sweeps,
and Y identifies the slow galvanometer-controlled row index (Fig. 1b)). An
immersion objective lens ($25\times$ magnification; numerical aperture (NA) of
0.8) with a refraction compensation ring was used for both printing and
imaging. A piezo scanner enabled fast, precise Z-axis positioning of the
objective lens (and hence the focal plane) during printing. A photomultiplier
camera allowed imaging of the workspace and printed objects.

A tunable Ti-Sapphire laser system ($\sim$120 fs pulse duration, 80 MHz
repetition) provided the light for both polymerisation and visualisation of
the photoresist and printed objects. We used pump laser powers in the range of
6–10 W, resulting in a $\sim$600–1000-mW mode-locked output beam at the
polymerisation half-wavelength (tunable, but typically 780 nm). Beam intensity
was modulated by a Pockels cell (ConOptics 350-80 cell and 302RM voltage
amplifier) interfaced with a 3.33-MHz DAC (we note that this Pockels cell and
driver are not rated for 3-MHz use, but the nonlinearity of the polymerisation
reaction allows us to control printing voxelisation at a frequency higher than
that for which the Pockels cell is rated. Nonetheless, we recommend that users
work with a faster Pockels cell and driver in order to improve small-feature
accuracy). Laser intensity was continuously monitored by sampling the passing
beam. To flatten the profile and improve collimation, the beam was routed
through a $2\times$ Galilean beam expander before entering the microscopy
optics (Fig. 1a).

All components were interfaced with the control computer via a dedicated data
acquisition system. Vibration due to floor movements was minimised by building
the rDLW system on an air-shock isolation table.

###  2.2 PrintImage: a resonant-rDLW control application

Because the printer is built on a two-photon microscope, we chose to use a
popular open-source microscopy software package, ScanImage (Vidrio
Technologies; Version $\geq$ 5.2.3) Pologruto et al. [2003], as the basis for
system control. To implement printer functionality, we developed a custom
MATLAB application, PrintImage, that runs alongside ScanImage to control print
object voxelisation, calculate the laser power modulation sequence, and manage
the printing-specific parts of the imaging process.

Print objects may be designed using any computer-aided-design or engineering
(CAD/CAE) software capable of exporting Stereolithography (STL) files. STL
files, which define the unstructured triangulated surface of the object by the
unit normal and vertices of the triangles using a 3D Cartesian coordinate
system, are transformed into a “watertight” solid object of specific
dimensions that is mapped onto the predefined set of printer positions via a
mesh voxelisation routine. Voxel Y and Z positions are determined by the
number of scan lines and vertical slices specified by the user; X positions
are computed as described below.

Once the object is voxelised, the series of filled and empty voxels along the
X direction of each Y row (blue arrows, Fig. 1b) is converted into a vector of
supra- and sub-polymerisation-threshold laser powers (Fig. 1c) that defines
the geometry (for each Y row) of the printed object. Repeating this
translation for each Y row in every Z plane, the required laser power at every
point within the printer’s workspace is computed before the volume print scan
is initiated. Power correction factors (see below) are applied to compensate
for variable beam speed, spherical aberrations in the objective lens, or other
nonuniformities. During printing, ScanImage executes a volume scan (as is
typically performed for volumetric two-photon calcium imaging) using the laser
power sequence precomputed by PrintImage (Fig. 1c), thus creating the printed
object (Fig. 1d).

###  2.3 Calibration

To achieve maximum precision, calibration of imaging and printing parameters
is necessary. We accomplish this by calibrating ScanImage’s optical workspace
size parameters, using two methods: (1) producing fluorescent objects of known
dimensions and imaging them with the rDLW printer, and (2) printing objects
with the rDLW printer and measuring them on a calibrated device.

To create objects of precisely known dimensions, we used a commercial DLW
printer to print rulers with IP-Dip photoresist (Fig. 2a; see Methods), and
confirmed the dimensions of the rulers with SEM micrographs (the 10-$\mu$m
ruler tics measured $9.93\;\mu$m with a SEM two-pixel error $\pm 0.25\;\mu$m).
We imaged these rulers with our rDLW printer and adjusted ScanImage’s optical
scaling parameters accordingly. We note that fluorescent rulers may be created
without a calibrated DLW system Khan and Brumberg [2014].

Figure 2: Rulers for calibrating the rDLW system. (a) Ruler for measuring X-
and Y-workspace dimensions. (b) Cubes used to calibrate object size and
uniformity of power delivery. The cubes shown have widths 300, 200, and 100
$\mu$m. The printing parameters were 2.2$\times$ (i.e., 302 $\times$ 302
$\mu$m FOV), 3.3$\times$ and 6.6$\times$ magnification (zoom), respectively.
Each X-Y plane was built with $152\times 1024$ voxels, and the vertical
spacing between the planes was 0.5 $\mu$m for all three cubes. (c) Ruler for
Y-axis calibration. The printing parameters are the same as for the 300-$\mu$m
cube in (b). The horizontal line spacing on the ruler is 5 $\mu$m. (d)
Vertical ruler for Z-axis calibration. Each row along the X axis contains 11
steps with 1-$\mu$m height difference. Adjacent steps along the Y axis have
10-$\mu$m height difference. The total height of the ruler is 300 $\mu$m. The
printing parameters were the same as for the 300-$\mu$m cube in (b).

To calibrate the X-Y plane, we printed calibration cubes (Fig. 2b) and a
calibration ruler (Fig. 2c) with the rDLW printer, measured with SEM
micrographs the discrepancy between desired and actual object dimensions, and
adjusted ScanImage’s workspace size parameters to null the difference.
Calibrating the Z print scale required printing a vertical calibration ruler
(Fig. 2d) with regularly spaced Z planes that could be precisely measured
using an optical surface profiler.

As the resonant scanner sweeps the laser’s focal point back and forth across
the X axis of the printer’s workspace, the beam moves through the photoresist
with sinusoidally varying velocity (Fig. 3a). If we define the centre of the
sweep as $t=0$, the oscillation frequency as $F_{r}$, and the maximum beam
excursion as $\xi$, the focal point’s position $x$ at time $t$ is given by
$x=\xi\sin(2\pi tF_{r})$ (Fig. 3b, blue line). Beam velocity, $\delta x/\delta
t$, rapidly approaches zero at the sweep extremes, so ScanImage restricts the
usable portion of the raster scan to a central fraction, $D$, of the scan
line, resulting in a printing workspace of width $2\xi D$. From the equation
above, one sweep from $-\xi$ to $\xi$ will take time $t=1/(2F_{r})$, so the
beam will traverse the subsection spanning $D$ in $t=2\arcsin(D)/(2\pi
F_{r})$. If laser power (controlled by the Pockels cell) has a modulation
frequency of $F_{p}$, then power can be updated every $1/F_{p}$ seconds; this
update rate enables $r_{x}=2F_{p}\arcsin(D)/(2\pi F_{r})$ potential changes in
laser power level (i.e., printing voxels) during a single X-axis scan. In our
instantiation, the resonant scanner frequency ($F_{r}\approx 8$ kHz), the
Pockels cell update rate ($F_{p}\approx 3.33$ MHz), and ScanImage’s workspace
restriction ($D=0.9$), result in a maximum of 152 print voxels along the X
axis.

Figure 3: Sinusoidal laser velocity over the X axis results in nonuniform
voxel size. Both that and optical nonuniformities such as vignetting require
corrective laser power compensation. (a) Laser focal point velocity as the
resonant scanner sweeps across the X axis. (b) Laser focus position varies
sinusoidally with time (blue line). The active scanning region is restricted
to a portion $D$ of the sweep, with X-axis voxel positions shown as black
horizontal dashes. For clarity, we show where voxels would be defined for an
8-kHz resonant scanner with a 1-MHz control system, which yields only 45
voxels. In order to maintain uniform energy deposition across the workspace,
laser power is modulated by two factors: it is scaled along the X axis by the
focal point’s speed $\cos(t)$, and along the X-Y plane by a learned model of
the inverse of optical darkening due to polymerisation. (c) Cross-section of
the power compensation along X, in which
$y=0,\;\;x\in[-208,208]\;\mu\textrm{m}$ ($1.6\times$ zoom on our rDLW system).
(d–g) $400\times 400\times 100\textrm{-}\mu\textrm{m}$ bricks used to measure
and calibrate energy deposition. The upper image shows the print power mask
over the $208\times 208\textrm{-}\mu$m workspace; the middle image shows an
actual printed object (normalised using a baseline fluorescence image); and
the bottom image shows brightness data gathered by sweeping the object over
the lens so that the same set of pixels in the imaging system may be used for
each measurement in order to bypass optical nonuniformities therein. Shown:
(d) constant power (note that (1) at this zoom optical vignetting comes close
to compensating for X-axis nonuniformity due to varying beam speed; and (2)
this image was printed at lower nominal power than the others in order to
avoid boiling; for the other images, the speed compensation appropriately
reduces power); (e) only (X-sinusoidal) speed power compensation; (f–g) two
iterations of adaptive power compensation over the visual field (see text).
The images and data were obtained with ScanImage on our rDLW system.

Resonant-scanner–based control results in higher resolution near the edges of
its sweep than in the centre, but allows higher resolution as workspace size
decreases. For example, on our rDLW system, printing at 1.3$\times$ zoom
yields a $512\times 512\textrm{-}\mu\textrm{m}$ X-Y workspace. On the X axis,
voxels are spaced on average every $V/r_{x}$ for a workspace of span $V$, so
at this zoom our rDLW printer is expected to have a 3.4-$\mu$m mean voxel size
along the X axis. At 2.6$\times$ zoom, the mean voxel size along X is expected
to be 1.7 $\mu$m over the $256\times 256\textrm{-}\mu\textrm{m}$ workspace.

The use of a resonant scanner leads to significant variation about this mean,
since laser power can be changed only at locations specified by the position
function ($\xi\sin(2\pi tF_{r})$; Fig. 3b, black ticks) at a frequency equal
to the laser power modulation rate, $F_{p}$. Thus, actual voxel size should be
nonuniform across the X axis, with smaller voxels at the edges of the
workspace than near the center, proportional to $\cos(\arcsin x)$ for
$x\in[-D\xi\ldots D\xi]$ scaled and centred over $V$. As zoom level reduces
workspace size $V$, expected voxel sizes over the X axis decrease linearly
until they become limited by optics or photon wavelength (see Section 2.6).

###  2.4 Varying the laser power to ensure uniform printing

Given sinusoidally varying scan velocity (Fig. 3a) and constant laser power at
the focal point, the photoresist will experience different light exposure
conditions as the beam accelerates from the start of a raster line until it
reaches peak velocity at the centre of the sweep and then deccelerates as it
approaches the end of a line. Under these conditions, the photoresist will not
polymerise evenly, and may vaporise or boil in overexposed regions. Thus the
baseline power of the polymerising laser must be corrected by a factor of
$\cos(t)=\cos(\arcsin x)$—proportional to the focal point’s speed—to maintain
constant exposure.

Another source of variability in the laser energy available for polymerisation
is attenuation of the beam due to inhomogenieties in laser intensity over the
workspace. This may be due to vignetting, which attenuates laser power toward
the edges of the workspace, or to other effects such as those resulting from
imperfect alignment of optical components. Falloff due to vignetting is
complex, depending on the angles at which the laser enters and exits each lens
in the system, relative alignments of all optical components, the shape of the
laser beam, partial occlusions throughout the optical path, and possibly
attenuation of the laser beam (although this should be minimal in its near
field). Furthermore, some of these factors may change frequently in a
developing multipurpose tool such as a two-photon microscope in a research
setting.

Due to the difficulty of modeling these factors precisely, we use a simple
adaptive approach to compensate for nonuniform optical fields. Given a model
$M=f:\;x,y\rightarrow$ falloff, power may may be boosted by $1/M$ to
compensate. The liquid photoresist used in these assays (IP-Dip) fluoresces
when exposed to 390-nm light (i.e., two near-simultaneous 780-nm photons), and
its refractive index and transparency are functions of the degree of
polymerisation. Thus $M$ may be approximated by measuring the reduction in
fluorescence of polymerised photoresist over a uniform printed object
(Methods). From these data we fit a curve such that falloff at any point may
be interpolated (in Fig. 3 we use fourth-order polynomials in X and Y,
although other functions may also be suitable). Due to the nonlinear
relationships between applied laser power and degree of polymerisation Mueller
et al. [2014], Sun et al. [2003] and between degree of polymerisation and
reduced fluorescence of the polymerised photoresist, this will not yield a
perfect compensation model in one step, so the process may be iterated until
sufficiently uniform energy deposition is achieved (Fig. 3f–g).

We assayed the uniformity of energy deposition across the workspace by
printing $400\times 400\times 100$-$\mu$m solid bricks and measuring the
fluorescence variation across the printed objects (Fig. 3d–g) (see Methods).
Simple beam speed power compensation—i.e., reducing power at the extrema of
the X axis where the beam moves more slowly—was effective for producing even
power deposition over small objects, but resulted in nonuniformities at
$\lesssim 2.5\times$ zoom: the extreme edges of the X axis fluoresced more
brightly than at the centre, indicating a lower degree of polymerisation. A
compensation function fitted to the measured fluorescence variation increased
the power at both the X and Y extrema, compensating for vignetting and other
optical irregularities and resulting in nearly uniform polymerisation.

These two forms of power compensation—for focal-point speed and for optical
inhomogenieties—are important for uniform printing, but beyond that they
demonstrate the ease with which polymerisation may be arbitrarily controlled
on a per-voxel basis throughout printed objects, potentially allowing for easy
development of techniques that take advantage of nonuniform polymerisation.

Figure 4: Complex geometric objects printed with our rDLW printer. (a-c)
Woodpile structure with design dimensions $60\times 60\times
60\;\mu\textrm{m}$. Along the X axis, bar thickness was 2 voxels ($0.8\;\mu$m)
and bar spacing 4 voxels ($1.6\;\mu$m). Bar thickness and spacing on the Y
axis were 13 and 26 voxels respectively in order to be the same size as the
X-axis beams, and on the Z axis bars are 1 voxel thick with $6$-$\mu$m
spacing. The focal plane resolution was $152\times 1024$ voxels, and the focal
plane (Z) spacing was $0.2\;\mu$m. (d) A torus knot design printed at
$100\times 100\times 150\;\mu$m (top right) and $50\times 50\times 75\;\mu$m
(bottom left). The inset shows details within the circumscribed region of the
bottom left structure. Both knots were printed with focal plane resolution
$152\times 512$ voxels and focal plane spacing $0.2\;\mu$m.

###  2.5 Accuracy

We estimated the accuracy of our rDLW system by printing simple geometric
shapes (Fig. 2b–d) and comparing the final object dimensions with those of the
original print model (measured with SEM micrographs for X and Y, and the
surface profiler for Z). We found that print errors were not identical across
the three dimensions, but instead varied by the print axis. Given that the
laser focal position in 3D space is controlled by three distinct mechanisms (X
axis: resonant scanning mirror; Y axis: galvanometer mirror; Z axis: piezo
objective scanner), and that size is calibrated independently for each
dimension, this is expected. For $300\times 300\textrm{-}\mu\textrm{m}$ cubes
printed at $2.2\times$ zoom, we found the errors in the size of the cube to be
-$5.6\pm 1.2\;\mu$m ($-1.9\pm 0.4$%) on the X axis and $6.5\pm 1.0\;\mu$m
($2.2\pm 0.33\%$) on the Y axis ($\pm x$ indicates SEM pixel size $x/2$).
Z-axis accuracy was measured using the staircase ruler shown in Fig. 2d. Since
our printing process leads to small variations (a few microns) in the starting
height of the print, we measured Z accuracy at each step of the staircase (we
leave more accurate automatic detection of substrate height for future work).
The steps had a nominal height of 10 $\mu$m, and an actual mean height of
10.0316 $\mu$m—an error of $\sim 0.32\%$, well within the surface profiler’s
claimed accuracy of $<0.75\%$.

All measurements were made following immersion of the printed objects in
solvent to remove excess/unpolymerised resist (see Methods), and thus our
estimates from SEM micrographs include some degree of post-processing–related
object shrinkage. Achieving maximum printing accuracy—with this or any other
DLW system—requires careful calibration, high-precision components, and
control of post-processing deformation. Disentangling the contribution of each
to our accuracy estimates is beyond the scope of this work.

###  2.6 Resolution

The minimum feature resolution of a two-photon polymerisation process is a
nonlinear function of the precision of laser focal point control, laser power,
and the chemical kinetics of the photoresist LaFratta et al. [2007]. This
complexity makes it challenging to predict the effective printing resolution
of any DLW system, and thus each hardware configuration and photoresist
combination must be verified experimentally.

One constraint on minimum feature size is the size of the laser’s focal point,
as photon density is sufficiently high to initiate the polymerising reaction
throughout this region. The laser focal point radial (i.e., along the X- and
Y-axes) and axial (along the Z-axis) dimensions are functions of the laser’s
wavelength, $\lambda$, and the numerical aperture, NA, of the objective lens.
Assuming an ideal (i.e., Gaussian) beam profile, the full-width half-maximum
size of the point spread function is $\lambda/(2\cdot\textrm{NA})$ (radial)
and $\lambda/(2\cdot\textrm{NA}^{2})$ (axial) Urey [2004]. With our operating
wavelength (780) nm and objective NA (0.8), the theoretical focal point
dimensions are 488 and 609 nm, respectively. Other factors affect the
effective size of the focal point—for example, if the laser beam incompletely
fills the back of the objective, the effective NA will be lower, whereas
changing the laser’s power will control the portion of the point spread
function that crosses the polymerisation threshold Kawata et al. [2001].

We treat the location of the centre of the focal point as the voxel location,
since the degree to which it can be controlled defines another constraint on
feature size. While the Y and Z positioning of the focal point are addressable
with sub-micron accuracy via analogue control of the galvanometer mirror and
the objective-lens scanner, respectively, the continuous sinusoidal motion of
the resonant scanline along the X axis precludes direct control of position.
Instead, X-axis voxel positions and sizes are defined by the rate at which the
laser beam power can be modulated across the polymerisation threshold. Given
the Pockels cell update frequency and the resonant scanner sweep rate, we
estimate the X-axis resolution to be $\sim$ 2.5–5.6 $\mu$m (at the edge and
centre of the resonant sweep, respectively) at 1$\times$ zoom, with minimum
feature size decreasing linearly with increasing magnification—for example,
the 300-$\mu$m scale used for the resolution tests in Fig. 5 should have X
voxel widths of $\sim$ 1.1–2.5 $\mu$m (edge and centre, respectively). In the
following discussion we report only worst-case resolution—that at the centre
of the X sweep.

Figure 5: Objects with one- and two-voxel features printed on our rDLW system.
(a) Object used to estimate minimum voxel size on the Y and Z axes. All
bridges have single-voxel height (Z), and increasing width on the Y axis. The
bottom bridge has one-voxel width; thus, it gives an idea of the thinnest
suspended structure that can achieved with the used parameters and
photoresist. The object was printed with 2.2$\times$ magnification for a
302$\times$302 $\mu$m field of view. The resolution of each focal plane is
$152\times 1024$ voxels, and the vertical distance between Z planes is 0.5
$\mu$m. (b) Object used to estimate the voxel size on the X axis. The printing
parameters are the same as in (a). The bridges were designed to be two voxels
wide on the X axis, so their size follows a sinusoidal distribution due to the
cosinusoidal speed profile of the laser beam. (c) Top view of the central
bridge of (b), which represents the largest value in the workspace of double-
voxel X resolution at this zoom level. (d) Top view of the lowest bridge of
(a). (e) View of the lowest bridge of (a) at 60∘ from the top view.

Given these theoretical estimates of printer performance, we quantified real
performance by printing objects with thin single-voxel features (Fig. 5a,b)
and measuring feature dimensions using SEM micrographs. During polymerisation,
the forming polymer tends to shrink due to both the emerging binding forces
and polymerisation quenching from molecules present in the liquid solution.
These effects are reduced when the forming polymer is attached to a solid
object (e.g., a previously polymerised structure). We report the feature size
as it is measured in features attached to a larger solid structure, but we
also note the sizes of isolated features. We printed structures that consisted
of channels containing single-voxel bridges, at 2.2$\times$ zoom (an FOV of
$302\times 302\;\mu$m). Because single-X-voxel bridges often broke during
postprocessing, we also report 2-X-voxel–wide bridges. We estimated resolution
by measuring the size of the bridges where they are attached to the supporting
walls, and also estimated minimum size of suspended features—giving an idea of
possible shrinkage—by measuring the bridges at their centre. Results are shown
in Table 1.

| Voxel | Attached | Isolated
---|---|---|---
| dimension ($\mu$m) | ($\mu$m) | ($\mu$m)
X | 2.5/1.1 | 1.09 $\pm$0.14 | 0.3 $\pm$0.14
2X | 5.0/2.2 | 2.73 $\pm$0.06 | 1.26 $\pm$0.06
Y | 0.3 | 1.25 $\pm$0.03 | 0.35 $\pm$0.03
Z | 0.5 | 2.10 $\pm$0.05 | 0.45 $\pm$0.05
Table 1: Printing resolution estimates. Resolution was estimated from SEM
micrographs of the single- and double-voxel bridges in the objects shown in
Fig. 5. Measurements of attached features were made proximal to the wall of
the support structure; isolated-feature sizes were measured at the bridge
centres. “Voxel dimension” is defined by the cell sizes used for voxelisation.
We list theoretical voxel dimension for the X axis as two numbers: at the
centre and edges of the resonant scanner’s sweep, respectively. We report
measurements of X feature size at the centre of the resonant sweep—the region
of the workspace in which we expect the largest minimum feature sizes.
Discrepancies are expected due to the nonzero size and anisotropic shape of
the focal point, postprocessing deformation, and a possible difference between
rise and fall response times of our Pockels system.

The reported resolutions can be used to build complex thin-feature structures,
such as the 60-$\mu$m woodpile shown in Fig. 4a–c and the hollow torus knot
structure in Fig. 4d. Though these minimum feature sizes will be sufficient
for some applications, further improvement is possible. As the X-axis voxel
resolution is in part defined by the rate of laser power modulation, upgrading
the Pockels cell and/or control hardware may produce a substantial reduction
in feature size across the whole X axis. Improvement in all axes could be
achieved by reducing the focal point size (e.g., by increasing the objective
lens aperture, flattening the beam profile, or reducing power Kawata et al.
[2001]), or using photoresists with higher polymerisation thresholds or
reduced spatial expansion factors.

###  2.7 Speed

A key aim for our rDLW design was to increase fabrication speed through the
introduction of a resonant scanner. As a first approximation, fabrication time
is governed by two parameters: the speed with which the beam moves through the
resist and the linear distance that the beam must traverse Malinauskas et al.
[2013]. DLW systems typically use some combination of stage-based (i.e., using
motorised stages to move the printed object relative to a stationary laser
focus) and mirror-based (i.e., using mirrors to move the laser focus relative
to a objective’s stationary object) methods for polymerising the desired
location. Each has its advantages, making direct comparisons challenging, but
mirror-based scanning is capable of realising significantly higher scanning
speeds while maintaining micro- and nanoscale feature sizes (Table 2).

Many DLW systems realise significant time savings by optimising the laser path
such that travel distance is minimised. For printed objects with small fill
ratios, this strategy can produce substantial improvements in fabrication
speed. Other strategies, such as the core-and-shell printing process Thiel et
al. [2016], can reduce fabrication time for objects with low surface-
area/volume ratios. Our approach achieves uniform fabrication times across
fill ratios by using a resonant scanner to sweep the beam over every point in
the printing workspace (Fig. 1b), maximising travel distance but at a higher
mean speed than in many previously described systems (Table 2).

Positioning | Scanning | Nominal Feature |
---|---|---|---
Mechanism | Speed (mm/s) | Size ($\mu$m) | Reference
Stepper motor | 10 | 1 | Kumi et al. [2010]
stage |  |  |
Piezo stage | 0.03–0.09 | 0.28–1.5 | Straub and Gu [2002]
| 0.06 | 0.065 | Haske et al. [2007]
| 10–30 | 1.5 | Ovsianikov et al. [2011]
Galvo-galvo | 0.005–0.2 | 0.085–1.5 | Thiel et al. [2010]
mirror | 0.01 | 1.3 | Maruo and Ikuta [2000]
| 7 | 0.78–1 | Farsari et al. [2006]
| 0.4–200 | 0.2–1.2 | Obata et al. [2013]
| 21–103 | 0.086–0.43 | Gottmann [2009]
| 400 | 1–10 | Skylar-Scott et al. [2016]
Rotating | 7200 | 1 | Rensch et al. [1989]
polygon-galvo |  |  |
mirror |  |  |
Resonant-galvo | 3300–8200 | 1–4 | Present work
mirror |  |  |
Table 2: Representative DLW laser scanning speeds and nominal minimum feature
sizes reported in recent literature. “Present work” gives scan speed with the
printer configured as described for most of the examples in this paper
(1.6$\times$ zoom yielding a $416\times 416$-$\mu\textrm{m}$ workspace, and
printing during only the left-to-right sweep of the resonant scanner) and the
maximum speed that we’ve used (bidirectional printing at 1.3$\times$ zoom).
Minimum feature size and scan speed covary as described in the text.

In a resonant-scanner–based system with a resonant frequency of $F_{r}$ and
useable workspace dimensions of $2\xi D$ along the scanning dimension, the
average beam speed is $2\xi DF_{r}$. For example, at 1.6$\times$ zoom, our
system’s useable workspace along the X axis is approximately 412 $\mu$m,
resulting in an effective mean beam speed of 3.3 m/s. Note that this estimate
assumes printing only in one direction of the laser scan (Fig. 1b); bi-
directional printing effectively doubles beam speed, although any misalignment
of the two scan directions leads to inferior results. Note also that
decreasing magnification will increase the distance that the beam travels
while commensurately increasing beam speed, leaving print time unchanged
(provided that the laser can supply sufficient power to polymerise resist at
the higher speed).

For our rDLW system, we can estimate fabrication time for an object from the
linear printing distance (i.e., length of a scan line $2\xi D$ times the
number of scan lines per layer $S_{y}$ times the number of layers $S_{z}$)
times the mean beam speed. For the large block in Fig. 2b, this results in an
estimated fabrication time of $\sim 19$ s, which comports well with our actual
print time of $\sim 25$ s. DLW systems vary widely and there are no
established benchmarks, making general comparisons of writing speed and
printing time difficult LaFratta et al. [2007], Sun and Kawata [2004].
Galvanometer-based two-photon microscopes are typically an order of magnitude
slower than resonant-scan microscopes. For example, at $512\times 512$ pixels,
resonant-scan microscopes typically achieve 30-Hz frame rates while typical
galvo-based systems achieve $\sim$1–2-Hz frame rates at the same scan angle
and resolution Jonkman and Brown [2015]. A pure galvanometer beam control
system designed for calcium imaging might see a beam speed of 200 mm/s Obata
et al. [2013]. If such a system were used to write the simple block in Fig.
2b, fabrication would take about 3.8 minutes.

We emphasise that these calculations are for an object with a fill ratio of 1
(i.e., 100% of the total object volume is polymerised), so these estimates
represents a worst-case fabrication time for an object of this size. Objects
with smaller fill fractions—as would be likely for most objects of
interest—would see reduced fabrication times on galvanometer- or stage-based
systems that optimise beam path to reduce total travel distance. As with
estimates of accuracy and resolution, our estimates of printing speed are
highly dependent on our choice of optical components, printing parameters, and
photoresist. Significant improvements or diminishments in all assayed metrics
can be realised with a different choice of hardware, laser power, or row/layer
density.

##  3 Discussion and Conclusion

We reported on rDLW: a 3D printer based on a standard two-photon microscope
with a resonant raster scanner and our custom PrintImage control application.
The rDLW system provides several key features including full access to
fabrication parameters, high printing speeds, and ease of extensibility.
Building on the widely-used open-source ScanImage microscopy package, this
work provides a platform for future modifications and customisations.

Because our rDLW printer exposes all process parameters, and indeed all
control software, to the user, our system is easily adaptable to experiments
with novel fabrication techniques that take advantage of the unique feature of
voxel-by-voxel modulation of laser power. This fine-grained power control
proved useful in compensating for nonuniform optical effects such as
vignetting, and could further be used to take advantage of intermediate states
of polymerisation and the material properties that so arise (i.e., refractive
index, rigidity, or fluorescence) Gissibl et al. [2016b]. In addition, laser
power is nonlinearly correlated with minimum feature resolution Kawata et al.
[2001], so per-voxel power modulation could provide additional control of the
sizes of different single-voxel features in a single print process.

The use of a tunable femtosecond laser adds significant cost to our system,
and could be replaced by fixed-wavelength fibre-based femtosecond sources.
However, since tunable femtosecond lasers are common components of two-photon
microscopes, we suggest that this more flexible laser may open up new material
choices for polymerisation at a range of wavelengths. We have demonstrated the
capabilities of the system using IP-Dip, a proprietary refraction-
index–matched photoresist designed for high-resolution two-photon
polymerisation. However, the wide tunable range of modern two-photon laser
sources (for our laser, 700–1050 nm), or the ease with which another laser can
be added to the beam path, makes possible printing with commercial or custom
resists having significantly different absorption spectrum peaks. This
capability would simplify fabricating compound structures composed of multiple
photoresists, each with different mechanical or optical properties Zeng et al.
[2015].

A limitation of existing DLW techniques—which not infrequently influences
printed object design—is the need to add structural supports under suspended
features, lest gravity and movement of the photoresist during printing
displace the incomplete features before they are anchored to the body of the
object being printed. An unexpected benefit of the rDLW system is that the
high speed of printing allows, to some degree, the printing of unsupported,
suspended features in viscous liquid photoresists (like IP-dip). In addition
to streamlining object design, the ability to print without the need of
support structure potentially enables the fabrication of previously
unrealisable objects.

Though the maximum print size of the described rDLW system ($\sim 400\times
400\times 350\;\mu\textrm{m}$) is suitable for many micro-scale applications,
there are use cases (e.g., tissue culture scaffolding) that require larger
object sizes while maintaining micron resolution. Several photoresists,
including IP-Dip, Ormocer, and SU-8, allow newly polymerised material to bond
directly onto previously polymerised material without mechanical defect. This
allows an object to be built by stitching together several overlapping
sections, each of which we refer to as a metavoxel. Additionally, whereas for
a single metavoxel the zoom setting controls both X-axis resolution and
maximum object size, stitching allows decoupling of these two parameters by
printing a single piece as multiple smaller overlapping pieces at higher
magnification. When stitching multiple metavoxels together, a stage with
absolute linear accuracy on the order of the desired resolution is required.
While a discussion of stitching is outside the scope of this paper, we note
that, as of this writing, PrintImage allows stitching using either the
microscopy stage or a commercial hexapod system, thus allowing fast printing
of arbitrarily large objects, and it can easily be extended to use other
hardware.

While resonant scanners have been previously used in 2D laser printing
Schermer and Dowd [1990], Takeshi and Kaoru [1995], a more common approach for
raster-scan printing uses a multi-sided mirror rotating at constant speed to
sweep the across the workspace Takizawa and Kataoka [1997]. Replacing the
resonant scanner in our rDLW printer with such a raster-scan mirror would
triple print speed by eliminating the flyback and near-zero-speed portions of
the beam path. It would allow nearly linear beam speed, providing uniform
voxel size and obviating sinusoidal power compensation. Though this would
remove the resonant rDLW’s capacity to increase print resolution without a
concomitant reduction in printing speed (i.e., zoom printing), a similar
effect may be achievable by incorporating a zoom lens. Conversely, a change in
mirror rotation speed would allow changes in X-axis resolution without
affecting workspace size.

##  4 Materials and Methods

###  4.1 Programming and analysis

All programming, modeling and analysis was done in MATLAB (The Mathworks,
Framingham, MA) running under Windows 10 on a desktop computer with an Intel
i7 processor and 16 GB of RAM. The PrintImage software is available at
https://github.com/gardner-lab/printimage. Its documentation lists its
software dependencies.

###  4.2 Design of calibration objects and print models

All custom benchmarking and example objects described here were created with
Solidworks2016 (Dassault Systèmes, Concord, MA) and exported using the native
STL converter. Calibration objects not printed on our rDLW system (see Figure
2a) were printed using a Nanoscribe Photonic Professional GT (Nanoscribe GmbH,
Stutensee, Germany). STL files for the Darwin Bust (Fig. 1d) and Torus Knot
(Fig. 4c) were obtained from the Museum of Applied Arts and Sciences in
Sydney, Australia and Tadej Skofic, respectively.

###  4.3 Photoresists and Post-Processing

A key step in developing a DLW solution is identifying photoresists that are
compatible with both the specifications of the printing process (e.g., two-
photon polymerisation, laser wavelength and power output, printing speed,
etc.) and the requirements of the application (e.g., hardness, adhesion,
biocompatibility, optical clarity, etc.). Though multiple photoresist
formulations have been described, the majority used for two-photon DLW consist
of soluble organic monomers or oligomers (typically acrylate derivatives) that
are cross-linked, and thus made insoluble, by free radicals or cations produce
by the exposure of a photoinitiator or photoacid generator Fourkas [2015]. The
use of a tunable laser in the described rDLW system offers the possibility of
printing with a variety of commercially available (e.g., Ormocer, KMPR, SU-8,
etc.), custom, or proprietary photoresists.

In an effort to ensure that our assays were representative of the limits of
our rDLW system’s performance, all objects reported here for illustration or
benchmark measurements were printed with a high-performance photoresist, IP-
Dip (Nanoscribe GmbH, Stutensee, Germany). IP-Dip is a proprietary liquid
photoresist that is refraction-index–matched to glass to minimise optical
distortion and enable rapid, fine-resolution two-photon polymerisation. IP-Dip
polymerises under 390-nm light (i.e., the two-photon effective wavelength of
our 780-nm source), producing solid, semi-transparent acrylic objects that
have been used in biomedical, optical, and microfluidic applications.
Following printing, residual un-polymerised resist was removed by submerging
the substrate and printed element in a solvent, propylene glycol methyl ether
acetate (PGMEA), for approximately 20 min. The prints were then rinsed in
methoxy-nonafluorobutane to remove trace PGMEA residue.

###  4.4 Scanning Electron Microscopy

Measurements of printed objects were made using SEM micrographs. To enhance
sample conductivity, the samples were sputter-coated with gold prior to
imaging. The samples were placed 3 cm under the gold target and were coated
for 1 min at 0.05 mbar and 20 mA. The samples were imaged at 6-mm working
distance with the secondary electron sensor, 3-kV accelerating voltage and
30-$\mu$m aperture size.

###  4.5 Energy Deposition Analysis

When IP-Dip polymerises, its fluorescent intensity changes, allowing printed
objects to be imaged by exposing them to the laser at a power that causes
fluorescence but is below the polymerisation threshold. The imaged fluorescent
intensity is inversely proportional to the degree of polymerisation.

The energy deposition profile was quantified by measuring the fluorescent
intensity of printed cubes at a depth of 5 $\mu$m below their top surfaces.
Images were made using the ScanImage software at 1$\times$ zoom, and were
analysed in MATLAB. As vignetting at the extreme corners of the workspace
reduces the laser’s intensity beyond our ability to compensate during
printing, we restricted our analysis to objects up to $400\times
400\;\mu\textrm{m}$ ($1.6\times$ zoom). To eliminate the effects of spatial
nonuniformity (such as vignetting) in our imaging system, rather than
photographing still images and measuring brightness values over the imaging
plane, we instead moved the printed objects under the lens (at 200 $\mu$m/s
along the X axis) using our stitching stage and recorded over time (at 15.21
Hz) the brightness values over the 1-pixel-by-10-$\mu$m XY region of the
printed cube at the centre of the camera’s reference frame at each X-axis
step. This was repeated for 15 equally spaced lines covering the Y axis. In
order to compensate for the non–vignetting-corrected imaging laser power, a
control image of the field of view without any objects was captured, and
intensity values of the images of each test object were divided by the control
image’s intensity.

##  5 Author Contributions

TJG and JMT conceptualised the project and implemented the initial proof of
concept. BWP wrote the software for controlling the print process and
integrated it with existing ScanImage routines. TMO and JMT designed the
microscope modifications to enable the new printing process. CM developed test
print objects, created calibration protocols, made all SEM micrographs, and
performed quantitative testing. BWP, TMO, and CM jointly contributed to device
and process refinement. TJG and TMO supervised the project. BWP, CM, amd TMO
drafted the manuscript with input from all other authors.

##  6 Acknowledgments

We wish to thank Alice White (Boston University) for use of her Nanoscribe
Photonic Professional GT 3D printing system and her expertise with
microfabrication processes; Jacob Franklin (Vidrio Technologies) for his
assistance with ScanImage; Tadej Skofic for designing the Torus Knot shown in
Fig. 4; and the Museum of Applied Arts and Sciences in Sydney, Australia for
providing the bust of Darwin shown in Fig. 1. We also wish to thank Alberto
Cruz-Martin, Todd Blute, Ian Davison, Jeff Gavornik, and L. Nathan Perkins for
their helpful comments on earlier drafts of this manuscript. Furthermore, we
are grateful for the packages on the MATLAB File Exchange from Alex A (Mesh
voxelisation), Eric Johnson (STL File Reader), Pau Micó (stlTools), and Teck
Por Lim (Significant Figures). This research was supported by NIH grants
(U01NS090454 and R01NS089679) and a sponsored research agreement with
GlaxoSmithKline.

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