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快速高分辨率三维成像的 Methods xxx (xxxx) xxx–xxx

膨胀生物标本与晶格光片
Contents lists available at ScienceDirect

Methods

显微镜 journal homepage: www.elsevier.com/locate/ymeth

Rapid high resolution 3D imaging of expanded biological specimens with

lattice light sheet microscopy
Yun-Chi Tsaia,1, Wei-Chun Tanga,1, Christine Siok Lan Lowb, Yen-Ting Liua, Jyun-Sian Wua,
⁎
Po-Yi Leea, Lindsay Quinn Chena, Yi-Ling Linc, Pakorn Kanchanawongb,d, Liang Gaoe,f, ,
⁎
Bi-Chang Chena,
a
Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan
b
Mechanobiology Institute, National University of Singapore, Singapore
c
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
d
Department of Biomedical Engineering, National University of Singapore, Singapore
e
School of Life Sciences, Westlake University, Hangzhou, Zhejiang Province, China
f
Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang Province, China

A R T I C LE I N FO A B S T R A C T

Keywords: Expansion microscopy was invented to surpass the optical diffraction limit by physically expanding biological
Lattice light sheet microscopy specimens with swellable polymers. Due to the large sizes of expanded specimens, 3D imaging techniques that
Expansion microscopy are capable to acquire large volumetric data rapidly at high spatial resolution are therefore required for ex-
Tiling light sheet pansion microscopy. Lattice light sheet microscopy (LLSM) was developed to image biological specimens rapidly
at high 3D spatial resolution by using a thin lattice light sheet for sample illumination. However, due to the
current limitations of LLSM mechanism and the optical design of LLS microscopes, it is challenging to image
large expanded specimens at isotropic high spatial resolution using LLSM. To address the problem, we first
optimized the sample preparation and expansion procedure for LLSM. Then, we implement a tiling lattice light
sheet method to minimize sample translation during imaging and achieve much faster 3D imaging speed at high
spatial resolution with more isotropic performance. Taken together, we report a general and improved 3D super-
resolution imaging method for expanded samples.

1. Introduction itself, better resolving ability is obtained by physically expanding the

Fluorescence microscopy is an essential technique used to image
fluorescently labeled biological specimens. The spatial resolution of
fluorescence microscopy is usually limited by the diffraction limit of the
light, which hinders detailed observations of sub-cellular structures.
Super-resolution fluorescence microscopy techniques, such as stimu-
lated emission depletion microscopy (STED) [1–3], structured illumi-
nation microscopy (SIM) [4–6], photoactivated localization microscopy
(PALM) [7–9], and stochastic optical reconstruction microscopy
(STORM) [10–12], extend the spatial resolution of fluorescence mi-
croscopy beyond the diffraction limit through the use of various optical
methods and photoactivatable/photoswitchable fluorescent molecules.
Expansion microscopy (ExM) solves the problem differently. Instead of
improving the achievable spatial resolution of the imaging technique
specimen with swellable chemical polymers [13–15]. With the latest
proExM technique [14], the sample was expanded roughly four times in
each dimension, which represents a ∼64 fold increase in volume, not to
mention that the sample could be further enlarged through an iterative
expansion process [15]. Thus, the ability to image expanded specimens
rapidly in 3D at high spatial resolution is required for expansion mi-
croscopy.
Lattice light sheet microscopy (LLSM) is one of the latest high speed,
high resolution 3D imaging techniques [16–21]. In LLSM, the specimen
is illumined by an optical lattice, i.e. an array of coherently interfering
Bessel Beams, which restricts sample illumination and the consequent
photobleaching to a thin slice near the detection focal plane. LLSM has
two operation modes, a high speed dithered mode and a super-resolu-
tion SIM mode. In the dithered mode, 3D imaging acquisition could be

⁎
Corresponding authors at: Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan, or School of Life Sciences, Westlake University, Hangzhou,
Zhejiang Province, China.
E-mail addresses: gaoliang@westlake.edu.cn (L. Gao), chenb10@gate.sinica.edu.tw (B.-C. Chen).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ymeth.2019.04.006
Received 25 January 2019; Received in revised form 2 April 2019; Accepted 3 April 2019
1046-2023/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Yun-Chi Tsai, et al., Methods, https://doi.org/10.1016/j.ymeth.2019.04.006
Y.-C. Tsai, et al.
carried out at a spatial resolution of ∼230 nm and ∼500 nm in the
lateral and axial direction respectively. In combination with SIM
[18,19], the 3D spatial resolution of LLSM is more isotropic, and is
extended beyond the diffraction limit of the light in the lattice scan
direction despite the imaging speed is usually five times slower than
that of the dithered mode.
Volumetric imaging in LLSM is conducted by either sample scan or
detection objective scan. Sample scan is more suited for imaging thin
and flat specimens. In sample scan, the sample is placed on a piezo
driven sample holder and scanned across the stationary lattice light
sheet (LLS) for volumetric imaging. The major advantage of sample
scan is that the LLS thickness stays at the minimum, with the LLS length
barely longer than the thinnest dimension of the specimen. This ensures
the maximum imaging quality since the optical sectioning ability of
LLSM deteriorates quickly as the length of the LLS increases. On the
contrary, objective scan is more suitable for imaging samples with si-
milar dimensions in all directions. Both the detection focal plane and
the excitation light sheet are scanned across the sample simultaneously.
In either case, a stack of 2D images are collected at a fixed step size
sequentially during scanning. The collected images are assembled into a
3D image of the specimen in the end.
Unfortunately, LLSM has not been optimized for imaging large
specimens despite the high imaging speed and spatial resolution can be
achieved on small specimens of dozens of microns [19,22,23]. There
are two major limitations. First, the 3D imaging ability of LLSM drops
substantially as the length of LLS increases in the light propagation
direction to illuminate the entire range of the specimen. Thus, large
samples must be translated to be imaged at high spatial resolution using
a short LLS [20,21]. Second, to maximize the photon collection effi-
ciency and spatial resolution of LLSM, two high numerical aperture
(NA) objectives are placed orthogonally next to the specimen. Such a
configuration leaves a very limited working space to move the specimen
freely for imaging [19,26]. Altogether, sample translation required to
image large specimens in LLSM significantly decreases the imaging
speed, increases the imaging complexity and introduces image regis-
tration problems after image acquisition. In addition, the limited space
between the two objectives and the sample also makes it difficult to
navigate the imaging plane across the specimen while preventing large
expanded samples from colliding with the objectives. These limitations
render LLSM a less than optimal tool for rapid high resolution imaging
of expanded specimens.
In this research, we first optimized the sample preparation and ex-
pansion procedure for LLSM and evaluated the imaging performance of
LLSM on expanded specimens. More importantly, we implemented a
tiling LLS method that enables LLSM to image large specimens more
rapidly at high spatial resolution [23–25]. Specifically, tiling LLSs are
generated by applying different binary phase maps to the binary spatial
light modulator (SLM) used in the LLS microscope. In this set up, a large
specimen can be imaged at high spatial resolution with minimum need
for sample translation, avoiding sample translation associated technical
limitations. Furthermore, due to the twin LLSs generated in the tiling
process, the imaging speed of LLSM is doubled, which significantly
reduces the imaging acquisition time. We extensively evaluated the
performance benefit of this new technique and show that the tiling LLS
method is a promising new tool for rapid imaging of expanded samples
at high spatial resolution.
2. Results and discussion
We conducted our research on a LLS microscope that is almost
identical to the LLS microscope reported previously (Fig. 1(A)) [19,26].
Specifically, a fast switching binary spatial light modulator (SLM)
conjugated to the image focal plane of the excitation objective is used to
generate the desired optical lattice for sample illumination. A pair of
galvanometer mirrors are used to scan the optical lattice in the X and Z
direction to create a LLS and scan the LLS in the axial direction for 3D
2
Methods xxx (xxxx) xxx–xxx
imaging respectively.
The imaging chamber of the microscope contains three high NA
water-immersion objectives (Fig. 1(B)), including an excitation objec-
tive used for LLS projection, a detection objective placed orthogonally
used for fluorescence light collection, and an epi-fluorescence objective
used to guide precise positioning of the specimen, so that the region of
interest on the specimen is placed at the common focus of the excitation
objective and the detection objective for 3D imaging with LLSM. The
detection objective is mounted on a piezo driven stage for 3D imaging
via detection objective scan. A round 5 mm coverslip is used as the
substrate to hold the sample due to the limited working space confined
by the three objectives. The coverslip is mounted on a sample holder
with the surface normal 32.8 and 57.2 degrees to the optical axes of the
excitation objective and the detection objective respectively to avoid
the interference of the illumination and detection light by the coverslip.
The sample holder is also mounted on a piezo driven stage attached to a
motorized 3D translation stage for volumetric imaging by sample scan
and for sample translation in 3D.
In the latest protein-retention expansion microscopy (proExM) [14],
antibody-recognized protein molecules are linked to the swellable gel,
and the gel undergoes 3D expansion of ∼4 times linearly in each di-
mension when immersed in water, by which the specimen is physically
expanded ∼64 times of its original size. The specimen can be further
expanded to ∼20 times in each dimension by repeating the expansion
process using iterative expansion microscopy (iExM) [15]. In compar-
ison with ExM sample preparation for conventional epi-illumination
fluorescence imaging techniques, two additional issues must be ad-
dressed to make the specimen more suited for 3D imaging with LLSM.
First, the thickness of the ExM gel must be carefully controlled in order
to mount the sample into the tight working space defined by the three
objectives as illustrated in Fig. 1(C). Second, although the ExM gel is
supposed to be aberration free in water environment, refractive index
mismatch between the gel and water was observed in our experiments.
Therefore, the specimen should be orientated upside down on the
coverslip as shown in Fig. 1(C) to avoid the optical aberrations in-
troduced by the gel. Both the excitation light and the detection light
pass through a thick layer of gel, which deteriorates the imaging quality
of LLSM, if the specimen is oriented in the other way as shown in
Fig. 1(D).
Accordingly, we modified the ExM sample preparation method for
LLSM as shown in Fig. 2(A). The cells cultured on a 5 mm round cov-
erslip were immunostained and AcX-treated. The coverslip was first
placed upside down onto a ∼3 μl gel droplet to form a sandwiched gel
structure. The sandwiched gel solution was put into a 3 cm petri dish
humidified with wet Kimwipe and the petri dish was sealed with a piece
of parafilm to prevent evaporation of the gel solution. Next, the sample
was expanded following the proExM protocol reported previously [14].
The expanded ExM gel is about ∼600 µm thick after expansion.
Fig. 2(B) shows a photo of two specimens prepared following the reg-
ular ExM sample preparation protocol and our modified protocol for
LLSM. The region of interest must be located first on a different mi-
croscope as shown in Fig. 2(B). It was conducted on a Leica DMi8 in-
verted fluorescence microscope in our experiments. The area of interest
on the ExM gel was cropped by a 3 mm round biopsy punch. It is im-
portant to keep the region of interest roughly centered on the cropped
gel pad, otherwise the area may not be able to be placed at the LLSM
focus due to the tight space between objectives. The cropped gel was
centered and mounted on another 5 mm cover glass (∼170 µm thick)
coated with CellTek tissue adhesive as shown in the bottom panel of
Fig. 2(B). Finally, the 5 mm coverslip was mounted on the LLS micro-
scope sample holder and transferred to the LLS microscope for 3D
imaging (Fig. 2(C)). The imaging chamber is filled with water as the
imaging buffer to maintain the swelling property of the ExM gel and
match the refractive index of the three water immersion objectives.
Fig. 2(D) show a zoom in view of the arrangement of the expanded
specimen with respect to the excitation and detection objectives. As
Y.-C. Tsai, et al.
Fig. 1. LLS microscope configuration. (A) Schematic diagram of the LLS microscope. (B) The imaging chamber of the LLS microscope, showing the arrangement of
three long working distance water-immersion objectives, including the excitation objective for LLSM sample illumination (EO), detection objective for LLSM
fluorescence collection (DO) and a sample finding epi objective (Epi). (C) Zoom in view of the preferred sample mounting orientation in LLSM. (D) Zoom in view of
the wrong sample orientation, as the gel introduces more optical aberrations.
emphasized, the specimen to be imaged is oriented towards the ex-
citation and detection objectives to minimize the optical aberrations
introduced by the ExM gel. Fig. 2(E) show the images of 100 nm
fluorescent particles embedded in ExM gel at different depth below the
gel surface collected with LLSM. Stronger optical aberrations were
observed on fluorescent particles at ∼200 μm depth below the gel
surface compared to those embedded near the gel surface.
We characterized the imaging performance of LLSM on expanded
specimens by imaging the same cell before and after expansion. We first
selected and imaged a group of fixed HeLa cells cultured on a 5 mm
coverslip with Cy3 labeled microtubules and DAPI labeled cell nuclei
using sample scan (Fig. 3(A) and 3(B)) [27]. The specimen was ex-
panded afterwards following the expansion protocol described above.
The selected cell was located and imaged again under the same imaging
conditions (Fig. 3(C)). Obviously, higher spatial resolution was ob-
tained from the expanded cells despite the actual size of the field of
view is much smaller due to the sample expansion. We compared the 3D
structure of both cell organelles before and after the expansion by re-
gistering the 3D images using Amira. Microtubule structures in the cell
were expanded isotropically for ∼3.6 times in all directions. However,
slightly different magnification ratios were observed for the cell nucleus
labelled by DAPI, possibility due to lacking of the interactive amine
groups in DNA that are required to crosslink to the swellable gel. The
expansion of DNA results from the integration between the gel structure
and nuclear proteins such as histones, which might result in the in-
consistent expansion ratio of the cell nucleus.
3
Methods xxx (xxxx) xxx–xxx
Due to the limited travel distance of the sample scan piezo stage and
the length of the LLS, we have to move an expanded cell via an addi-
tional 3D translational stage to image the whole cell volume. As shown
in Fig. 4, an expanded cell was translated three times, which is corre-
sponding to four subvolumes, to have the entire cell imaged. All sub-
volumes were imaged via sample scan with a ∼60 µm long LLS. A
∼20% overlap was maintained between adjacent subvolumes to ensure
accurate image registration. The acquired images of all subvolumes
were registered and merged together after imaging acquisition to obtain
the 3D image of the entire cell.
Although we can image a large specimen through sample transla-
tion, this strategy has several limitations. First, the specimen must be
translated to be imaged and a sufficient overlap is required between
each two adjacent subvolumes for image registration, which un-
avoidably decreases the imaging speed. Second, the image registration
and merging of different subvolumes are required afterwards, which
create a significant computation burden for data analysis. Furthermore,
it is particular difficult to position the specimen in 3D to have the entire
specimen imaged with LLSM due to the tight sample mounting space
and the difference between the sample translation coordinate and the
3D imaging coordinate of the LLS microscope. Obviously, all problems
become increasingly more difficult to deal with as the sample size in-
creases. Although the amount of sample translation and subvolumes
can be reduced by imaging using longer LLSs, the spatial resolution and
optical sectioning ability of LLSM decreases at the same time due to the
poor light confinement ability of long LLSs. Therefore, rapid high
Y.-C. Tsai, et al. Methods xxx (xxxx) xxx–xxx

Fig. 2. ExM sample preparation for LLSM. (A) ExM sample preparation procedure for LLSM. (B) The method to locate the region of interest on the expanded gel. The
bottom picture shows the gel thickness difference following the original ExM and our modified ExM protocol. (C) The arrangement of the sample holder with respect
to excitation objective and detection objective in the imaging chamber. (D) Zoom in view of marked area in (C). (E) The 3D maximum intensity projectionfiles of
100 nm fluorescent beads embedded in a blank gel pad near the top and bottom surfaces imaged with LLSM showing different level of optical aberrations.

resolution 3D imaging of large specimens using LLSM is challenging.
We implemented a recently developed tiling LLS method to address
these limitations [24]. Specifically, binary phase maps acquired from
off-center cross-sections of an optical lattice are applied to the binary
SLM in the LLS microscope, so that a thin LLS can be tiled to image
large specimens while maintaining the same spatial resolution and
optical sectioning ability in the entire field of view (FOV). There are
several advantages. First, complicated biological structures can be
imaged at high spatial resolution and SNR using short tiling LLSs as a
short LLS can be tiled to image a large specimen just by applying dif-
ferent binary phase maps to the SLM in the LLS microscope. Second, the
entire specimen can be imaged at the same spatial resolution without or
with minimal sample translation, which is only required when the
sample size is larger than the FOV of the detection camera. As a result,
the imaging speed could be much faster than that of imaging the whole
specimen through sample translation with conventional LLSM, since it

4
Y.-C. Tsai, et al.
Fig. 3. Comparison of the same cell imaged before and after expansion with LLSM. Microtubules (Cy3) shown in green; nuclei (DAPI) shown in magenta. (A) A group
of cells imaged with LLSM before expansion via sample scan. (B) Zoom in view of the selected cell in (A). (C) The same cell imaged with LLSM after expansion under
the same imaging conditions. (D-G) Cell nucleus and microtubules imaged before expansion. (H-K) Cell nucleus and microtubules imaged after expansion. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
takes at least dozens of milliseconds, and up to several seconds, to
translate the sample to a different position each time. Third, due to the
twin LLSs generated by the binary SLM, the imaging speed is improved
for another two-fold compared to that of the conventional LLSM [24].
In addition, the pixel values of the final image are assigned directly
from the collected raw images by using tiling LLSs. The image re-
construction is significantly easier compared to before which requires
extensive image registration and merging, that are computationally
heavy but must be performed whenever the sample is moved. Mean-
while, since the post-expanded samples are very fragile and jelly-like,
the sample often suffers from drifting when it is moved too fast, which
makes the image registration even harder.
We demonstrate the imaging ability of LLSM with tiling LLSs by
imaging an expanded anaphase HeLa cell with Alexa 488 labeled β-
tubulin. Since the structure of the mitotic spindle is extremely com-
plicated, it is important to maintain a good optical sectioning capability
of LLSM through the entire sample volume. A ∼15 µm long LLS was
therefore tiled to image the cell (Fig. 5(A)). Besides the initial tile im-
aged with the original LLS, the LLS was further tiled at six positions to
image the mitotic spindle by applying three different binary phase maps
to the binary SLM, which generated three pairs of twin LLSs (Fig. 5(B)-
5(D)). Images were acquired via objective scan without moving the
sample. The corresponding XZ projections are shown in Fig. 5(E)-5(H).
As uniform thin LLSs were used to image the entire specimen, nearly
isotropic 3D spatial resolution was obtained. Because of the fast LLS
tiling speed and the use of twin LLSs, the imaging procedure is simpler
and the imaging speed is much faster compared to that of imaging the
cell though sample translation. The selected areas of the collected
images were stitched together as shown in Fig. 5(I)-5(J). Fig. 5(K)-5(L)
show the maximum intensity projection of the reconstructed result in
the lateral and axial direction without deconvolution. Fig. 6 shows the
5
Methods xxx (xxxx) xxx–xxx
3D volume rendering of the reconstructs images before and after de-
convolution, and the selected image slices through the imaged cell
volumes. It is worth noting that the intensities in images from different
tiles were adjusted to reach a smooth transition between adjacent tiles
because the intensity of tiling LLSs varies slightly during the tiling
process.
3. Conclusions
In summary, we demonstrated several improvements in imaging
expanded specimens with LLSM. Specifically, we optimized the sample
expansion and preparation procedure, so that the expanded specimen
can be loaded into LLS microscopes more easily and the optical aber-
rations introduced by the swellable gel is minimized. We demonstrated
the imaging performance of LLSM on expanded specimens and com-
pared the expansion ratios of different cell organelles before and after
expansion. We also showed how to image large but thin specimens at
high spatial resolution through sample scan and sample translation.
Most importantly, we described the use of tiling LLSs to facilitate rapid
high resolution 3D imaging of expanded specimens with LLSM. In this
set up, a short LLS can be tiled to image large specimens without un-
necessary sample translation to obtain high spatial resolution with
more isotropic performance. This strategy also greatly improves ima-
ging speed and simplifies the post-imaging data processing.
4. Materials and methods
4.1. LLS microscope configuration
The schematic diagram of our LLS microscope is shown in Fig. 1.
The microscope is equipped with three laser sources with the
Y.-C. Tsai, et al.
Fig. 4. An expanded HeLa cell with microtubule (Alex488) imaged with LLSM via sample scan and sample translation. (A-D) The four subvolumes of the expanded
cell imaged via sample scan and sample translation. (E) The registered and merged result of the whole HeLa cell. (F-J) Five selected XY cross sections of the
reconstructed 3D image.
wavelength of 405 nm (300 mW, Oxxius, LBX-405-300-CIR-PP), 488 nm
(300 mW, Coherent, Sapphire 488 nm) and 561 nm (200 mW, Oxxius,
LMX-561S-200-COL-PP). Each laser beam is expanded to ∼4 mm dia-
meter by a pair of achromatic lenses (L1 = L2 = L3 = 8 mm FL, Thor-
labs, C240TME-A and L4 = L5 = L6 = 20 mm FL, Edmundoptics,
47–661). All laser beams are combined before entering an acousto-optic
tunable filter (AA Quanta Tech, AOTFnC-400.650-TN) for laser wave-
length selection and intensity control.
A pair of cylindrical lenses (cyL1 = 25 mm FL, Edmundoptics,
NT68-160 and cyL2 = 250 mm FL, Thorlabs, ACY254-250-A) are used
to expand the combined laser beam in the X direction after the AOTF.
The expanded beam is sent to a binary spatial light modulator (SLM)
assembly that consists of a polarized beam splitter cube (Newport,
10FC16PB.3), a half wave plate (Bolder Vision Optik, BVO AHWP3) and
a binary SLM (Forth Dimension, QXGA-3DM) for laser modulation. The
modulated laser beam is focused on a selected transparent annulus on a
quartz mask for beam filtering (L5 = 350 mm FL, Edmundoptics,
49–395). The transparent annulus is further conjugated to a pair of
galvanometer mirrors (Cambridge Technology, 6215HP-1HB) and to
the rear pupil of the excitation objective (Special Optics, 0.65 NA,
3.74 mm WD) through three pairs of relay lenses (L6 = 100 mm FL,
Thorlabs, AC254-100-A, L7 = 75 mm FL, Thorlabs, AC254-075-A,
L8 = L9 = 85 mm, Thorlabs, AC254-85-A, L10 = 125 mm FL, Thorlabs,
AC254-125-A, L11 = 400 mm, Thorlabs, AC254-400-A). LLSs of dif-
ferent geometries are generated and tiled by selecting different trans-
parent annuli on the quartz mask and applying different binary phase
maps to the binary SLM.
Fluorescence signal is collected by a water immersed detection ob-
jective (Nikon, CFI Apo LWD 25XW, 1.1 NA, 2 mm WD) placed ortho-
gonal to the excitation objective. The detection objective is mounted on
piezo stage (Physik Instrumente, P-726 PIFOC) for 3D imaging via
6
Methods xxx (xxxx) xxx–xxx
detection objective scan. The collected fluorescence signal is imaged
onto a sCMOS camera (Hamamatsu, Orca Flash 4.0 v3) with a 500 mm
tube lens (Edmundoptics, 49–290) through a multinotch filter
(Semrock, FF01-446/523/600/677). The sample holder is mounted on
a piezo stage (Physik Instrumente, P-621.1CD), which is attached to a
3D motorized translational stage for 3D imaging via sample scan and
sample translation.
4.2. Coverslip preparation
Prepare a 150 ml mixture solution of sodium hydroxide (NaOH) and
H2O in a 1:1 ratio by volume. Soak the coverslips in the solution and
place the solution in 70 °C water bath overnight. Rinse the coverslips
with 70% alcohol and water iteratively for at least three times. Spread
the coverslips on a paper towel under UV light. The cleaned coverslips
can be stored in sterile containers for more than a week.
4.3. Cell culture and immunostaining
HeLa cells were cultured in DMEM supplemented with 10% fetal
bovine serum (Life Technologies, US) on 5 mm diameter round cover-
slips cleaned following the protocol described above (Warner
Instruments, 64-0700). Cells were fixed with 4% paraformaldehyde
(Electron Microscopy Sciences, PA) pre-warmed in 37 °C water bath for
20 min. After fixation, free aldehydes were quenched with 1 mg/ml
sodium borohydride (Sigma, 452882) for 7 min. Wash the fixed cells
with PBS three times and permeate the cells with 0.3% Triton X-100 for
10 min at 37 °C. Wash the cells with PBS three times again and incubate
the cells with blocking solution (5% normal donkey serum
(JacksonImmunoResearch, 017-000-001) in PBS) for 1 h.
Microtubules were probed using cascade binding ability between
Y.-C. Tsai, et al. Methods xxx (xxxx) xxx–xxx

Fig. 5. An expanded anaphase mitotic HeLa cell with microtubule (Alex488) imaged with tiling LLSs. (A-D) The XY maximum intensity projections of the cell imaged
with a regular LLS (A), and twin tiling LLSs (B-D). (E-F) The corresponding YZ maximum intensity projections. (I) XY maximum intensity projection of the re-
constructed image stitched from the selected areas in (A-D) rendered in different colors. (J) YZ maximum intensity projection of the reconstructed image stitched
from the selected areas in (A-D) rendered in different colors. (K) XY maximum intensity projection of the reconstructed image. (L) YZ maximum intensity projection
of the reconstructed image.

the primary antibody (mouse anti-α-tubulin monoclonal antibody,
Sigma, T5168; or mouse anti-β-tubulin monoclonal antibody, Sigma,
T4026), Biotin-conjugated secondary antibody (biotin-conjugated Goat
anti-mouse IgG antibody, JacksonImmunoResearch, 115-065-166), and
Cy3-conjugated streptavidin (Jackson ImmunoResearch, 016-160-084).
Cells were treated with 0.1 mg/ml Acryloyl-X, SE (6-((acryloyl)amino)
hexanoic acid, succinimidyl ester; abbreviated AcX; Thermo-Fisher,
A20770) in PBS at 4 °C overnight according to the protein-retention
expansion microscopy (abbreviated proExM) reported by Tillberg et al.
[14].
For the fixed microtubule in Fig. 3, primary antibodies was a mix-
ture of mouse monoclonal anti-Alpha-Tubulin (Sigma Aldrich, T5168)
and mouse monoclonal anti-Beta-Tubulin antibody (Sigma-Aldrich,
T4026) both diluted 1:2500 in 1X PBS. Secondary antibody was Biotin-
SP (long spacer) AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Im-
munoResearch, 115-065-166) in 1:1000 dilution. The tertiary dye is
CyTM3 Streptavidin (Jackson ImmunoResearch, 016-160-084) in
1:1000 dilution. For fixed microtubule in Figs. 4 and 5, the primary
antibody was rabbit anti beta-tubulin monoclonal antibody (abcam
ab6046 rabbit Anti-beta Tubulin antibody) diluted 1:100 in 1X PBS.
The secondary antibody was Biotin-SP (long spacer) AffiniPure Goat
Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, 115-065-144) in
1:1000 dilution. The tertiary dye is Alexa Flour®488 Streptavidin
(Jackson ImmunoResearch, 016-540-084) in 1:1000 dilution. All sam-
ples were stained with DAPI (Roche, 10236276001) as indicated con-
centration.
4.4. ExM sample expansion
The ExM gel was prepared following a modified version of the
protocol reported previously by Tillberg et al. [14]. Put a piece of
parafilm in a humidified chamber. Add a ∼5 µl gel droplet on the
parafilm. Place a 5 mm coverslip with fixed and stained cells up-side
down on the droplet. Cover and seal the chamber carefully to prevent
evaporation. After gelation, remove the gel from the parafilm gently.
4.5. Sample mounting
Place the dilated gel on a petri dish, remove excess water. Use an
inverted fluorescence microscope to check the examined gel and locate
the region of interest. Use a 3-mm biopsy punch (Integra Miltex, US) to
pick the region of interest. Use a #15 Surgical Blades (Graham-Field,
US) with #4 Scalpel Handles (Key Surgical, US) to transfer the cropped
gel to a 5 mm coverslip coated with ∼2 μl CellTek (Corning, US).
Center the gel on the coverslip carefully with a tweezer. The cover slip
is clipped to the LLS microscope sample holder and loaded into the
microscope for imaging afterwards.
4.6. Image analysis and visualization
Images acquired with conventional LLSs and sample scan were
deskewed following the method described previously [19,26]. The in-
tensity of the raw images collected using tiling LLSs were adjusted ac-
cording to the overlapped areas between adjacent tiles. The intensity

7
Y.-C. Tsai, et al.
Fig. 6. 3D volume rendering of the reconstructed image result before (A) and after (B) deconvolution. (C-D) The selected image slices shown in (B). Scale bars: 10 µm
in all figures.
adjusted images were stitched together according to the length and
tiling positions of the tiling LLSs afterwards as described in previous
publications [23,24]. Subvolume 3D images acquired via sample
translation were registered and merged in Amira. All 3D images were
rendered for visualization in Amira (Thermo Fisher Scientific. Image
deconvolution was performed using Richardson-Lucy deconvolution
algorithm in MATLAB.
Acknowledgements
This work was supported by the Ministry of Science and
Technology, Taiwan (Project No. MOST 106-2627-M-001-006- and
105-2119-M-001-026-MY2 to C.B.C.). P.K. acknowledged the funding
support from the Singapore Ministry of Education Academic Research
Fund Tier 2 (MOE-T2-1-124) and the MBI seed grant. L.G. acknowl-
edges the support from the Westlake University. The control software of
the microscope was licensed from the Howard Hughes Medical
Institute, Janelia Research Campus. HeLa cells were provided by Dr. Yi-
Ling Lin.
Author contributions
B.C.C. and W.C.T. designed and constructed the lattice lightsheet
microscope. B.C.C. and W.C.T. performed imaging experiments. Y.C.T.,
S.L.L., L.Q.C. and Y.L.L. prepared the samples. Y.C.T developed the
sample mounting method. S.L.L. and P.K. shared the protocol of ex-
pansion microscopy. Y.C.T., Y.T.L., and P.Y.L. analyzed the data. Y.C.T.,
J.S.W., L.G. and B.C.C. wrote the manuscript.
8
Methods xxx (xxxx) xxx–xxx
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ymeth.2019.04.006.
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Another random document with
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trousers, a narghile, and a mosque slightly larger than the narghile)
could never cause to look like anything except a disguised bed. She
was curled up, pinching her ankle with one tired and nervous hand,
and reading a stimulating chapter of Laura Jean Libbey. Her shirt-
waist was open at the throat, and down her slim stocking was a
grievous run. She was so un-Juanita-like—an ash-blonde, pale and
lovely, with an ill-restrained passion in her blue eyes.
Nellie, a buxom jolly child, dark as a Jewess, was wearing a
frowsy dressing-gown. She was making coffee and narrating her
grievances against her employer, the pious dressmaker, while
Juanita paid no attention whatever.
The young men crept into the room without knocking.
“You devils—sneaking in like this, and us not dressed!” yelped
Nellie.
Jim sidled up to her, dragged her plump hand away from the
handle of the granite-ware coffee-pot, and giggled, “But aren’t you
glad to see us?”
“I don’t know whether I am or not! Now you quit! You behave, will
you?”
Rarely did Elmer seem more deft than Jim Lefferts. But now he
was feeling his command over women—certain sorts of women.
Silent, yearning at Juanita, commanding her with hot eyes, he sank
on the temporarily Oriental couch, touched her pale hand with his
broad finger-tips, and murmured, “Why, you poor kid, you look so
tired!”
“I am and— You hadn’t ought to of come here this afternoon.
Nell’s aunt threw a conniption fit the last time you were here.”
“Hurray for aunty! But you’re glad to see me?”
She would not answer.
“Aren’t you?”
Bold eyes on hers that turned uneasily away, looked back, and
sought the safety of the blank wall.
“Aren’t you?”
She would not answer.
 “Juanita! And I’ve longed for you something fierce, ever since I
saw you!” His fingers touched her throat, but softly. “Aren’t you a little
glad?”
As she turned her head, for a second she looked at him with
embarrassed confession. She sharply whispered, “No—don’t!” as he
caught her hand, but she moved nearer to him, leaned against his
shoulder.
“You’re so big and strong,” she sighed.
“But, golly, you don’t know how I need you! The president, old
Quarles—quarrels is right, by golly, ha, ha, ha!—’member I was
telling you about him?—he’s laying for me because he thinks it was
me and Jim that let the bats loose in chapel. And I get so sick of that
gosh-awful Weekly Bible Study—all about these holy old gazebos.
And then I think about you, and gosh, if you were just sitting on the
other side of the stove from me in my room there, with your cute lil
red slippers cocked up on the nickel rail—gee, how happy I’d be!
You don’t think I’m just a bonehead, do you?”
Jim and Nellie were at the stage now of nudging each other and
bawling, “Hey, quit, will yuh!” as they stood over the coffee.
“Say, you girls change your shirts and come on out and we’ll blow
you to dinner, and maybe we’ll dance a little,” proclaimed Jim.
“We can’t,” said Nellie. “Aunty’s sore as a pup because we was
up late at a dance night before last. We got to stay home, and you
boys got to beat it before she comes in.”
“Aw, come on!”
“No, we can’t!”
“Yuh, fat chance you girls staying home and knitting! You got
some fellows coming in and you want to get rid of us, that’s what’s
the trouble.”
“It is not, Mr. James Lefferts, and it wouldn’t be any of your
business if it was!”
While Jim and Nellie squabbled, Elmer slipped his hand about
Juanita’s shoulder, slowly pressed her against him. He believed with
terrible conviction that she was beautiful, that she was glorious, that
she was life. There was heaven in the softness of her curving
shoulder, and her pale flesh was living silk.
“Come on in the other room,” he pleaded.
“Oh—no—not now.”
He gripped her arm.
“Well—don’t come in for a minute,” she fluttered. Aloud, to the
others, “I’m going to do my hair. Looks just ter-ble!”
She slipped into the room beyond. A certain mature self-reliance
dropped from Elmer’s face, and he was like a round-faced big baby,
somewhat frightened. With efforts to appear careless, he fumbled
about the room and dusted a pink and gilt vase with his large
crumpled handkerchief. He was near the inner door.
He peeped at Jim and Nellie. They were holding hands, while the
coffee-pot was cheerfully boiling over. Elmer’s heart thumped. He
slipped through the door and closed it, whimpering, as in terror:
“Oh—Juanita—”

VI
They were gone, Elmer and Jim, before the return of Nellie’s
aunt. As they were not entertaining the girls, they dined on pork
chops, coffee, and apple pie at the Maginnis Lunch.
It has already been narrated that afterward, in the Old Home
Sample Room, Elmer became philosophical and misogynistic as he
reflected that Juanita was unworthy of his generous attention; it has
been admitted that he became drunk and pugnacious.
As he wavered through the sidewalk slush, on Jim’s arm, as his
head cleared, his rage increased against the bully who was about to
be encouraged to insult his goo’ frien’ and roommate. His shoulders
straightened, his fists clenched, and he began to look for the
scoundrel among the evening crowd of mechanics and coal-miners.
They came to the chief corner of the town. A little way down the
street, beside the red brick wall of the Congress Hotel, some one
was talking from the elevation of a box, surrounded by a jeering
gang.
 “What they picking on that fella that’s talking for? They better let
him alone!” rejoiced Elmer, throwing off Jim’s restraining hand,
dashing down the side street and into the crowd. He was in that most
blissful condition to which a powerful young man can attain—
unrighteous violence in a righteous cause. He pushed through the
audience, jabbed his elbow into the belly of a small weak man, and
guffawed at the cluck of distress. Then he came to a halt, unhappy
and doubting.
The heckled speaker was his chief detestation, Eddie Fislinger,
president of the Terwillinger College Y. M. C. A., that rusty-haired
gopher who had obscenely opposed his election as president.
With two other seniors who were also in training for the Baptist
ministry, Eddie had come over to Cato to save a few souls. At least,
if they saved no souls (and they never had saved any, in seventeen
street meetings) they would have handy training for their future jobs.
Eddie was a rasping and insistent speaker who got results by
hanging to a subject and worrying it, but he had no great boldness,
and now he was obviously afraid of his chief heckler, a large, blond,
pompadoured young baker, who bulked in front of Eddie’s rostrum
and asked questions. While Elmer stood listening, the baker
demanded:
“What makes you think you know all about religion?”
“I don’t pretend to know all about religion, my friend, but I do
know what a powerful influence it is for clean and noble living, and if
you’ll only be fair now, my friend, and give me a chance to tell these
other gentlemen what my experience of answers to prayer has been
—”
“Yuh, swell lot of experience you’ve had, by your looks!”
“See here, there are others who may want to hear—”
Though Elmer detested Eddie’s sappiness, though he might have
liked to share drinks with the lively young baker-heckler, there was
no really good unctuous violence to be had except by turning
champion of religion. The packed crowd excited him, and the
pressure of rough bodies, the smell of wet overcoats, the rumble of
mob voices. It was like a football line-up.
 “Here, you!” he roared at the baker. “Let the fellow speak! Give
him a chance. Whyn’t you pick on somebody your own size, you big
stiff!”
At his elbow, Jim Lefferts begged, “Let’s get out of this, Hell-cat.
Good Lord! You ain’t going to help a gospel-peddler!”
Elmer pushed him away and thrust his chest out toward the
baker, who was cackling, “Heh! I suppose you’re a Christer, too!”
“I would be, if I was worthy!” Elmer fully believed it, for that
delightful moment. “These boys are classmates of mine, and they’re
going to have a chance to speak!”
Eddie Fislinger bleated to his mates, “Oh, fellows, Elm Gantry!
Saved!”
Even this alarming interpretation of his motives could not keep
Elmer now from the holy zeal of fighting. He thrust aside the one
aged man who stood between him and the baker—bashing in the
aged one’s derby and making him telescope like a turtle’s neck—and
stood with his fist working like a connecting-rod by his side.
“If you’re looking for trouble—” the baker suggested, clumsily
wobbling his huge bleached fists.
“Not me,” observed Elmer and struck, once, very judiciously, just
at the point of the jaw.
The baker shook like a skyscraper in an earthquake and caved to
the earth.
One of the baker’s pals roared, “Come on, we’ll kill them guys
and—”
Elmer caught him on the left ear. It was a very cold ear, and the
pal staggered, extremely sick. Elmer looked pleased. But he did not
feel pleased. He was almost sober, and he realized that half a dozen
rejoicing young workmen were about to rush him. Though he had an
excellent opinion of himself, he had seen too much football, as
played by denominational colleges with the Christian
accompaniments of kneeing and gouging, to imagine that he could
beat half a dozen workmen at once.
 It is doubtful whether he would ever have been led to further
association with the Lord and Eddie Fislinger had not Providence
intervened in its characteristically mysterious way. The foremost of
the attackers was just reaching for Elmer when the mob shouted,
“Look out! The cops!”
The police force of Cato, all three of them, were wedging into the
crowd. They were lanky, mustached men with cold eyes.
“What’s all this row about?” demanded the chief.
He was looking at Elmer, who was three inches taller than any
one else in the assembly.
“Some of these fellows tried to stop a peaceable religious
assembly—why, they tried to rough-house the Reverend here—and I
was protecting him,” Elmer said.
“That’s right, Chief. Reg’lar outrage,” complained Jim.
“That’s true, Chief,” whistled Eddie Fislinger from his box.
“Well, you fellows cut it out now. What the hell! Ought to be
ashamed yourselves, bullyragging a Reverend! Go ahead,
Reverend!”
The baker had come to, and had been lifted to his feet. His
expression indicated that he had been wronged and that he wanted
to do something about it, if he could only find out what had
happened. His eyes were wild, his hair was a muddy chaos, and his
flat floury cheek was cut. He was too dizzy to realize that the chief of
police was before him, and his fuming mind stuck to the belief that
he was destroying all religion.
“Yah, so you’re one of them wishy-washy preachers, too!” he
screamed at Elmer—just as one of the lanky policemen reached out
an arm of incredible length and nipped him.
The attention of the crowd warmed Elmer, and he expanded in it,
rubbed his mental hands in its blaze.
“Maybe I ain’t a preacher! Maybe I’m not even a good Christian!”
he cried. “Maybe I’ve done a whole lot of things I hadn’t ought to of
done. But let me tell you, I respect religion—”
“Oh, amen, praise the Lord, brother,” from Eddie Fislinger.
 “—and I don’t propose to let anybody interfere with it. What else
have we got except religion to give us hope—”
“Praise the Lord, oh, bless his name!”
“—of ever leading decent lives, tell me that, will you, just tell me
that!”
Elmer was addressing the chief of police, who admitted:
“Yuh, I guess that’s right. Well now, we’ll let the meeting go on,
and if any more of you fellows interrupt—” This completed the chief’s
present ideas on religion and mob-violence. He looked sternly at
everybody within reach, and stalked through the crowd, to return to
the police station and resume his game of seven-up.
Eddie was soaring into enchanted eloquence:
“Oh, my brethren, now you see the power of the spirit of Christ to
stir up all that is noblest and best in us! You have heard the
testimony of our brother here, Brother Gantry, to the one and only
way to righteousness! When you get home I want each and every
one of you to dig out the Old Book and turn to the Song of Solomon,
where it tells about the love of the Savior for the Church—turn to the
Song of Solomon, the fourth chapter and the tenth verse, where it
says—where Christ is talking about the church, and he says—Song
of Solomon, the fourth chapter and the tenth verse—‘How fair is thy
love, my sister, my spouse! how much better is thy love than wine!’
“Oh, the unspeakable joy of finding the joys of salvation! You
have heard our brother’s testimony. We know of him as a man of
power, as a brother to all them that are oppressed, and now that he
has had his eyes opened and his ears unstopped, and he sees the
need of confession and of humble surrender before the throne
— Oh, this is a historic moment in the life of Hell-c—— of Elmer
Gantry! Oh, Brother, be not afraid! Come! Step up here beside me,
and give testimony—”
“God! We better get outa here quick!” panted Jim.
“Gee, yes!” Elmer groaned, and they edged back through the
crowd, while Eddie Fislinger’s piping pursued them like icy and
penetrating rain:
 “Don’t be afraid to acknowledge the leading of Jesus! Are you
boys going to show yourselves too cowardly to risk the sneers of the
ungodly?”
They were safely out of the crowd, walking with severe
countenances and great rapidity back to the Old Home Sample
Room.
“That was a dirty trick of Eddie’s!” said Jim.
“God, it certainly was! Trying to convert me! Right before those
muckers! If I ever hear another yip out of Eddie, I’ll knock his block
off! Nerve of him, trying to lead me up to any mourners’ bench! Fat
chance! I’ll fix him! Come on, show a little speed!” asserted the
brother to all them that were oppressed.
By the time for their late evening train, the sound conversation of
the bartender and the sound qualities of his Bourbon had caused
Elmer and Jim to forget Eddie Fislinger and the horrors of
undressing religion in public. They were the more shocked, then,
swaying in their seat in the smoker, to see Eddie standing by them,
Bible in hand, backed by his two beaming partners in evangelism.
Eddie bared his teeth, smiled all over his watery eyes, and
caroled:
“Oh, fellows, you don’t know how wonderful you were tonight!
But, oh, boys, now you’ve taken the first step, why do you put it off—
why do you hesitate—why do you keep the Savior suffering as he
waits for you, longs for you? He needs you boys, with your splendid
powers and intellects that we admire so—”
“This air,” observed Jim Lefferts, “is getting too thick for me. I
seem to smell a peculiar and a fishlike smell.” He slipped out of the
seat and marched toward the forward car.
Elmer sought to follow him, but Eddie had flopped into Jim’s
place and was blithely squeaking on, while the other two hung over
them with tender Y. M. C. A. smiles very discomforting to Elmer’s
queasy stomach as the train bumped on.
For all his brave words, Elmer had none of Jim’s resolute
contempt for the church. He was afraid of it. It connoted his
boyhood. . . . His mother, drained by early widowhood and drudgery,
finding her only emotion in hymns and the Bible, and weeping when
he failed to study his Sunday School lesson. The church, full thirty
dizzy feet up to its curiously carven rafters, and the preachers, so
overwhelming in their wallowing voices, so terrifying in their pictures
of little boys who stole watermelons or indulged in biological
experiments behind barns. The awe-oppressed moment of his
second conversion, at the age of eleven, when, weeping with
embarrassment and the prospect of losing so much fun, surrounded
by solemn and whiskered adult faces, he had signed a pledge
binding him to give up, forever, the joys of profanity, alcohol, cards,
dancing, and the theater.
These clouds hung behind and over him, for all his boldness.
Eddie Fislinger, the human being, he despised. He considered
him a grasshopper, and with satisfaction considered stepping on
him. But Eddie Fislinger, the gospeler, fortified with just such a
pebble-leather Bible (bookmarks of fringed silk and celluloid smirking
from the pages) as his Sunday School teachers had wielded when
they assured him that God was always creeping about to catch small
boys in their secret thoughts—this armored Eddie was an official,
and Elmer listened to him uneasily, never quite certain that he might
not yet find himself a dreadful person leading a pure and boresome
life in a clean frock coat.
“—and remember,” Eddie was wailing, “how terribly dangerous it
is to put off the hour of salvation! ‘Watch therefore for you know not
what hour your Lord doth come,’ it says. Suppose this train were
wrecked! Tonight!”
The train ungraciously took that second to lurch on a curve.
“You see? Where would you spend Eternity, Hell-cat? Do you
think that any sportin’ round is fun enough to burn in hell for?”
“Oh, cut it out. I know all that stuff. There’s a lot of arguments
— You wait’ll I get Jim to tell you what Bob Ingersoll said about hell!”
“Yes! Sure! And you remember that on his death-bed Ingersoll
called his son to him and repented and begged his son to hurry and
be saved and burn all his wicked writings!”
 “Well— Thunder— I don’t feel like talking religion tonight. Cut it
out.”
But Eddie did feel like talking religion, very much so. He waved
his Bible enthusiastically and found ever so many uncomfortable
texts. Elmer listened as little as possible, but he was too feeble to
make threats.
It was a golden relief when the train bumped to a stop at
Gritzmacher Springs. The station was a greasy wooden box, the
platform was thick with slush, under the kerosene lights. But Jim was
awaiting him, a refuge from confusing theological questions, and with
a furious “G’night!” to Eddie he staggered off.
“Why didn’t you make him shut his trap?” demanded Jim.
“I did! Whadja take a sneak for? I told him to shut up and he shut
up and I snoozed all the way back and— Ow! My head! Don’t walk
so fast!”
 CHAPTER II

I
for years the state of sin in which dwelt Elmer Gantry and Jim
Lefferts had produced fascinated despair in the Christian hearts of
Terwillinger College. No revival but had flung its sulphur-soaked
arrows at them—usually in their absence. No prayer at the Y. M. C.
A. meetings but had worried over their staggering folly.
Elmer had been known to wince when President the Rev. Dr.
Willoughby Quarles was especially gifted with messages at morning
chapel, but Jim had held him firm in the faith of unfaith.
Now, Eddie Fislinger, like a prairie seraph, sped from room to
room of the elect with the astounding news that Elmer had publicly
professed religion, and that he had endured thirty-nine minutes of
private adjuration on the train. Instantly started a holy plotting against
the miserable sacrificial lamb, and all over Gritzmacher Springs, in
the studies of ministerial professors, in the rooms of students, in the
small prayer-meeting room behind the chapel auditorium, joyous
souls conspired with the Lord against Elmer’s serene and zealous
sinning. Everywhere, through the snowstorm, you could hear
murmurs of “There is more rejoicing over one sinner who repenteth
—”
Even collegians not particularly esteemed for their piety,
suspected of playing cards and secret smoking, were stirred to
ecstasy—or it may have been snickering. The football center, in
unregenerate days a companion of Elmer and Jim but now engaged
to marry a large and sanctified Swedish co-ed from Chanute, rose
voluntarily in Y. M. C. A. and promised God to help him win Elmer’s
favor.
The spirit waxed most fervent in the abode of Eddie Fislinger,
who was now recognized as a future prophet, likely, some day, to
have under his inspiration one of the larger Baptist churches in
Wichita or even Kansas City.
 He organized an all-day and all-night prayer-meeting on Elmer’s
behalf, and it was attended by the more ardent, even at the risk of
receiving cuts and uncivil remarks from instructors. On the bare floor
of Eddie’s room, over Knute Halvorsted’s paint-shop, from three to
sixteen young men knelt at a time, and no 1800 revival saw more
successful wrestling with the harassed Satan. In fact one man,
suspected of Holy Roller sympathies, managed to have the jerks,
and while they felt that this was carrying things farther than the Lord
and the Baptist association would care to see, it added excitement to
praying at three o’clock in the morning, particularly as they were all
of them extraordinarily drunk on coffee and eloquence.
By morning they felt sure that they had persuaded God to attend
to Elmer, and though it is true that Elmer himself had slept quite
soundly all night, unaware of the prayer-meeting or of divine
influences, it was but an example of the patience of the heavenly
powers. And immediately after those powers began to move.
To Elmer’s misery and Jim’s stilled fury, their sacred room was
invaded by hordes of men with uncombed locks on their foreheads,
ecstasy in their eyes, and Bibles under their arms. Elmer was safe
nowhere. No sooner had he disposed of one disciple, by the use of
spirited and blasphemous arguments patiently taught to him by Jim,
than another would pop out from behind a tree and fall on him.
At his boarding-house—Mother Metzger’s, over on Beech Street
—a Y. M. C. A. dervish crowed as he passed the bread to Elmer,
“Jever study a kernel of wheat? Swonnerful! Think a wonnerful
intricate thing like that created itself? Somebody must have created
it. Who? God! Anybody that don’t reconize God in Nature—and
acknowledge him in repentance—is dumm. That’s what he is!”
Instructors who had watched Elmer’s entrance to classrooms with
nervous fury now smirked on him and with tenderness heard the
statement that he wasn’t quite prepared to recite. The president
himself stopped Elmer on the street and called him My Boy, and
shook his hand with an affection which, Elmer anxiously assured
himself, he certainly had done nothing to merit.
He kept assuring Jim that he was in no danger, but Jim was
alarmed, and Elmer himself more alarmed with each hour, each new
greeting of: “We need you with us, old boy—the world needs you!”
Jim did well to dread. Elmer had always been in danger of giving
up his favorite diversions—not exactly giving them up, perhaps, but
of sweating in agony after enjoying them. But for Jim and his
remarks about co-eds who prayed in public and drew their hair back
rebukingly from egg-like foreheads, one of these sirens of morality
might have snared the easy-going pangynistic Elmer by proximity.
A dreadful young woman from Mexico, Missouri, used to coax
Jim to “tell his funny ideas about religion,” and go off in neighs of
pious laughter, while she choked, “Oh, you’re just too cute! You don’t
mean a word you say. You simply want to show off!” She had a
deceptive sidelong look which actually promised nothing whatever
this side of the altar, and she might, but for Jim’s struggles, have led
Elmer into an engagement.
The church and Sunday School at Elmer’s village, Paris, Kansas,
a settlement of nine hundred evangelical Germans and Vermonters,
had nurtured in him a fear of religious machinery which he could
never lose, which restrained him from such reasonable acts as
butchering Eddie Fislinger. That small pasty-white Baptist church
had been the center of all his emotions, aside from hell-raising,
hunger, sleepiness, and love. And even these emotions were
represented in the House of the Lord, in the way of tacks in pew-
cushions, Missionary Suppers with chicken pie and angel’s-food
cake, soporific sermons, and the proximity of flexible little girls in thin
muslin. But the arts and the sentiments and the sentimentalities—
they were for Elmer perpetually associated only with the church.
Except for circus bands, Fourth of July parades, and the singing
of “Columbia, the Gem of the Ocean” and “Jingle Bells” in school, all
the music which the boy Elmer had ever heard was in church.
The church provided his only oratory, except for campaign
speeches by politicians ardent about Jefferson and the price of
binding-twine; it provided all his painting and sculpture, except for
the portraits of Lincoln, Longfellow, and Emerson in the school-
building, and the two china statuettes of pink ladies with gilt flower-
baskets which stood on his mother’s bureau. From the church came
all his profounder philosophy, except the teachers’ admonitions that
little boys who let garter-snakes loose in school were certain to be
licked now and hanged later, and his mother’s stream of opinions on
hanging up his overcoat, wiping his feet, eating fried potatoes with
his fingers, and taking the name of the Lord in vain.
If he had sources of literary inspiration outside the church—in
McGuffey’s Reader he encountered the boy who stood on the
burning deck, and he had a very pretty knowledge of the Nick Carter
Series and the exploits of Cole Younger and the James Boys—yet
here too the church had guided him. In Bible stories, in the words of
the great hymns, in the anecdotes which the various preachers
quoted, he had his only knowledge of literature—
The story of Little Lame Tom who shamed the wicked rich man
that owned the handsome team of grays and the pot hat and led him
to Jesus. The ship’s captain who in the storm took counsel with the
orphaned but righteous child of missionaries in Zomballa. The
Faithful Dog who saved his master during a terrific conflagration
(only sometimes it was a snowstorm, or an attack by Indians) and
roused him to give up horse-racing, rum, and playing the harmonica.
How familiar they were, how thrilling, how explanatory to Elmer of
the purposes of life, how preparatory for his future usefulness and
charm.
The church, the Sunday School, the evangelistic orgy, choir-
practise, raising the mortgage, the delights of funerals, the snickers
in back pews or in the other room at weddings—they were as
natural, as inescapable a mold of manners to Elmer as Catholic
processionals to a street gamin in Naples.
The Baptist Church of Paris, Kansas! A thousand blurred but
indestructible pictures.
Hymns! Elmer’s voice was made for hymns. He rolled them out
like a negro. The organ-thunder of “Nicæa”:
Holy, holy, holy! all the saints adore thee,
Casting down their golden crowns around the glassy sea.
The splendid rumble of the Doxology. “Throw Out the Lifeline,”
with its picture of a wreck pounded in the darkness by surf which the
prairie child imagined as a hundred feet high. “Onward, Christian
Soldiers,” to which you could without rebuke stamp your feet.
Sunday School picnics! Lemonade and four-legged races and the
ride on the hay-rack, singing “Seeing Nelly Home.”
Sunday School text cards! True, they were chiefly a medium of
gambling, but as Elmer usually won the game (he was the first boy in
Paris to own a genuine pair of loaded dice) he had plenty of them in
his gallery, and they gave him a taste for gaudy robes, for marble
columns and the purple-broidered palaces of kings, which was later
to be of value in quickly habituating himself to the more decorative
homes of vice. The three kings bearing caskets of ruby and
sardonyx. King Zedekiah in gold and scarlet, kneeling on a carpet of
sapphire-blue, while his men-at-arms came fleeing and blood-
stained, red blood on glancing steel, with tidings of the bannered
host of Nebuchadnezzar, great king of Babylon. And all his life Elmer
remembered, in moments of ardor, during oratorios in huge
churches, during sunset at sea, a black-bearded David standing
against raw red cliffs—a figure heroic and summoning to ambition, to
power, to domination.
Sunday School Christmas Eve! The exhilaration of staying up,
and publicly, till nine-thirty. The tree, incredibly tall, also incredibly
inflammable, flashing with silver cords, with silver stars, with cotton-
batting snow. The two round stoves red-hot. Lights and lights and
lights. Pails of candy, and for every child in the school a present—
usually a book, very pleasant, with colored pictures of lambs and
volcanoes. The Santa Claus—he couldn’t possibly be Lorenzo
Nickerson, the house-painter, so bearded was he, and red-cheeked,
and so witty in his comment on each child as it marched up for its
present. The enchantment, sheer magic, of the Ladies’ Quartette
singing of shepherds who watched their flocks by nights . . . brown
secret hilltops under one vast star.
And the devastating morning when the preacher himself, the Rev.
Wilson Hinckley Skaggs, caught Elmer matching for Sunday School
contribution pennies on the front steps, and led him up the aisle for
all to giggle at, with a sharp and not very clean ministerial thumb-nail
gouging his ear-lobe.
 And the other passing preachers: Brother Organdy, who got you
to saw his wood free; Brother Blunt, who sneaked behind barns to
catch you on Halloween; Brother Ingle, who was zealous but young
and actually human, and who made whistles from willow branches
for you.
And the morning when Elmer concealed an alarm clock behind
the organ and it went off, magnificently, just as the superintendent
(Dr. Prouty, the dentist) was whimpering, “Now let us all be
particularly quiet as Sister Holbrick leads us in prayer.”
And always the three chairs that stood behind the pulpit, the
intimidating stiff chairs of yellow plush and carved oak borders,
which, he was uneasily sure, were waiting for the Father, the Son,
and the Holy Ghost.
He had, in fact, got everything from the church and Sunday
School, except, perhaps, any longing whatever for decency and
kindness and reason.

II
Even had Elmer not known the church by habit, he would have
been led to it by his mother. Aside from his friendship for Jim
Lefferts, Elmer’s only authentic affection was for his mother, and she
was owned by the church.
She was a small woman, energetic, nagging but kindly, once
given to passionate caresses and now to passionate prayer, and she
had unusual courage. Early left a widow by Logan Gantry, dealer in
feed, flour, lumber, and agricultural implements, a large and
agreeable man given to debts and whisky, she had supported herself
and Elmer by sewing, trimming hats, baking bread, and selling milk.
She had her own millinery and dressmaking shop now, narrow and
dim but proudly set right on Main Street, and she was able to give
Elmer the three hundred dollars a year which, with his summer
earnings in harvest field and lumber-yard, was enough to support
him—in Terwillinger, in 1902.
She had always wanted Elmer to be a preacher. She was jolly
enough, and no fool about pennies in making change, but for a
preacher standing up on a platform in a long-tailed coat she had
gaping awe.
Elmer had since the age of sixteen been a member in good
standing of the Baptist Church—he had been most satisfactorily
immersed in the Kayooska River. Large though Elmer was, the
evangelist had been a powerful man and had not only ducked him
but, in sacred enthusiasm, held him under, so that he came up
sputtering, in a state of grace and muddiness. He had also been
saved several times, and once, when he had pneumonia, he had
been esteemed by the pastor and all visiting ladies as rapidly
growing in grace.
But he had resisted his mother’s desire that he become a
preacher. He would have to give up his entertaining vices, and with
wide-eyed and panting happiness he was discovering more of them
every year. Equally he felt lumbering and shamed whenever he tried
to stand up before his tittering gang in Paris and appear pious.
It was hard even in college days to withstand his mother. Though
she came only to his shoulder, such was her bustling vigor, her swift
shrewdness of tongue, such the gallantry of her long care for him,
that he was afraid of her as he was afraid of Jim Lefferts’ scorn. He
never dared honestly to confess his infidelity, but he grumbled, “Oh,
gee, Ma, I don’t know. Trouble is, fellow don’t make much money
preaching. Gee, there’s no hurry. Don’t have to decide yet.”
And she knew now that he was likely to become a lawyer. Well,
that wasn’t so bad, she felt; some day he might go to Congress and
reform the whole nation into a pleasing likeness of Kansas. But if he
could only have become part of the mysteries that hovered about the
communion table—
She had talked him over with Eddie Fislinger. Eddie came from a
town twelve miles from Paris. Though it might be years before he
was finally ordained as a minister, Eddie had by his home
congregation been given a License to Preach as early as his
Sophomore year in Terwillinger, and for a month, one summer (while
Elmer was out in the harvest fields or the swimming hole or robbing
orchards), Eddie had earnestly supplied the Baptist pulpit in Paris.
 Mrs. Gantry consulted him, and Eddie instructed her with the
dignity of nineteen.
Oh, yes, Brother Elmer was a fine young man—so strong—they
all admired him—a little too much tempted by the vain gauds of This
World, but that was because he was young. Oh, yes, some day
Elmer would settle down and be a fine Christian husband and father
and business man. But as to the ministry—no. Mrs. Gantry must not
too greatly meddle with these mysteries. It was up to God. A fellow
had to have a Call before he felt his vocation for the ministry; a real
overwhelming mysterious knock-down Call, such as Eddie himself
had ecstatically experienced, one evening in a cabbage patch. No,
not think of that. Their task now was to get Elmer into a real state of
grace and that, Eddie assured her, looked to him like a good deal of
a job.
Undoubtedly, Eddie explained, when Elmer had been baptized, at
sixteen, he had felt conviction, he had felt the invitation, and the
burden of his sins had been lifted. But he had not, Eddie doubted,
entirely experienced salvation. He was not really in a state of grace.
He might almost be called unconverted.
Eddie diagnosed the case completely, with all the proper
pathological terms. Whatever difficulties he may have had with
philosophy, Latin, and calculus, there had never been a time since
the age of twelve when Eddie Fislinger had had difficulty in
understanding what the Lord God Almighty wanted, and why, all
through history, he had acted thus or thus.
“I should be the last to condemn athaletics,” said Eddie. “We
must have strong bodies to endure the burden and the sweat of
carrying the Gospel to the world. But at the same time, it seems to
me that football tends to detract from religion. I’m a little afraid that
just at present Elmer is not in a state of grace. But, oh, Sister, don’t
let us worry and travail! Let us trust the Lord. I’ll go to Elmer myself,
and see what I can do.”
That must have been the time—it certainly was during that
vacation between their Sophomore and Junior years—when Eddie
walked out to the farm where Elmer was working, and looked at
Elmer, bulky and hayseedy in a sleeveless undershirt, and spoke
reasonably of the weather, and walked back again. . . .
Whenever Elmer was at home, though he tried affectionately to
live out his mother’s plan of life for him, though without very much
grumbling he went to bed at nine-thirty, whitewashed the henhouse,
and accompanied her to church, yet Mrs. Gantry suspected that
sometimes he drank beer and doubted about Jonah, and uneasily
Elmer heard her sobbing as she knelt by her high-swelling, white-
counterpaned, old-fashioned bed.

III
With alarmed evangelistic zeal, Jim Lefferts struggled to keep
Elmer true to the faith, after his exposure to religion in defending
Eddie at Cato.
He was, on the whole, rather more zealous and fatiguing than
Eddie.
Nights, when Elmer longed to go to sleep, Jim argued; mornings,
when Elmer should have been preparing his history, Jim read aloud
from Ingersoll and Thomas Paine.
“How you going to explain a thing like this—how you going to
explain it?” begged Jim. “It says here in Deuteronomy that God
chased these Yids around in the desert for forty years and their
shoes didn’t even wear out. That’s what it says, right in the Bible.
You believe a thing like that? And do you believe that Samson lost all
his strength just because his gal cut off his hair? Do you, eh? Think
hair had anything to do with his strength?”
Jim raced up and down the stuffy room, kicking at chairs, his
normally bland eyes feverish, his forefinger shaken in wrath, while
Elmer sat humped on the edge of the bed, his forehead in his hands,
rather enjoying having his soul fought for.
To prove that he was still a sound and freethinking stalwart,
Elmer went out with Jim one evening and at considerable effort they
carried off a small outhouse and placed it on the steps of the
Administration Building.
 Elmer almost forgot to worry after the affair of Eddie and Dr.
Lefferts.
Jim’s father was a medical practitioner in an adjoining village. He
was a plump, bearded, bookish, merry man, very proud of his
atheism. It was he who had trained Jim in the faith and in his choice
of liquor; he had sent Jim to this denominational college partly
because it was cheap and partly because it tickled his humor to
watch his son stir up the fretful complacency of the saints. He
dropped in and found Elmer and Jim agitatedly awaiting the arrival of
Eddie.
“Eddie said,” wailed Elmer, “he said he was coming up to see me,
and he’ll haul out some more of these proofs that I’m going straight
to hell. Gosh, Doctor, I don’t know what’s got into me. You better
examine me. I must have anemics or something. Why, one time, if
Eddie Fislinger had smiled at me, damn him, think of him daring to
smile at me!—if he’d said he was coming to my room, I’d of told him,
‘Like hell you will!’ and I’d of kicked him in the shins.”
Dr. Lefferts purred in his beard. His eyes were bright.
“I’ll give your friend Fislinger a run for his money. And for the
inconsequential sake of the non-existent Heaven, Jim, try not to look
surprised when you find your respectable father being pious.”
When Eddie arrived, he was introduced to a silkily cordial Dr.
Lefferts, who shook his hand with that lengthiness and painfulness
common to politicians, salesmen, and the godly. The doctor rejoiced:
“Brother Fislinger, my boy here and Elmer tell me that you’ve
been trying to help them see the true Bible religion.”
“I’ve been seeking to.”
“It warms my soul to hear you say that, Brother Fislinger! You
can’t know what a grief it is to an old man tottering to the grave, to
one whose only solace now is prayer and Bible-reading”—Dr.
Lefferts had sat up till four a. m., three nights ago, playing poker and
discussing biology with his cronies, the probate judge and the
English stock-breeder—“what a grief it is to him that his only son,
James Blaine Lefferts, is not a believer. But perhaps you can do