Beruflich Dokumente
Kultur Dokumente
Correspondence
lippincottschwartzj@janelia.hhmi.org
(J.L.-S.),
betzige@janelia.hhmi.org (E.B.),
lidong@ibp.ac.cn (D.L.)
In Brief
A new approach for visualizing dynamic
processes within cells offers insight into
membrane-membrane contact
interactions and microtubule function.
Highlights
d Super-resolution live-cell imaging up to 266 fps at 97-nm
resolution
Resource
94305, USA
9Lead Contact
SUMMARY berg-Bord et al., 2016; Phillips and Voeltz, 2016; Prinz, 2014).
Organelle interactions occur at contact sites, where proteins
In eukaryotic cells, organelles and the cytoskeleton physically bridge their respective membranes and bind them
undergo highly dynamic yet organized interactions together. Such interactions are increasingly recognized to be
capable of orchestrating complex cellular functions. pivotal for diverse cellular functions (Bonifacino and Neefjes,
Visualizing these interactions requires noninvasive, 2017; Friedman et al., 2011; Rowland et al., 2014). Among the
long-duration imaging of the intracellular environ- various subcellular organelles, the largest is the endoplasmic re-
ticulum (ER), which spans the cytoplasm in an intricate network.
ment at high spatiotemporal resolution and low
Its importance is underscored by its role in lipid and protein
background. To achieve these normally opposing
biosynthesis; protein modification, translocation, and secretion;
goals, we developed grazing incidence structured and calcium handling. In addition, the cortical ER (cER) (i.e., the
illumination microscopy (GI-SIM) that is capable of portion lying in close apposition to the basal plasma membrane)
imaging dynamic events near the basal cell cortex is increasingly viewed as a hub for organelle interaction (Valm
at 97-nm resolution and 266 frames/s over thou- et al., 2017). ER-mitochondrion contact sites have been shown
sands of time points. We employed multi-color GI- to be important for calcium signaling, lipid transfer, mitochon-
SIM to characterize the fast dynamic interactions of drial fission (Friedman et al., 2011), and the synchronization of
diverse organelles and the cytoskeleton, shedding mitochondrial DNA synthesis (Lewis et al., 2016). Similarly, ER-
new light on the complex behaviors of these struc- endosome contact sites define the position and timing of endo-
tures. Precise measurements of microtubule growth somal fission (Rowland et al., 2014), and they help to connect
late endosomes (LEs) to the molecular motors that transport
or shrinkage events helped distinguish among
them (Raiborg et al., 2015).
models of microtubule dynamic instability. Analysis
These discoveries were only possible with the advent of imag-
of endoplasmic reticulum (ER) interactions with other
ing technologies that allowed the dynamic reorganization of or-
organelles or microtubules uncovered new ER re- ganelles and the cytoskeleton to be studied. Even so, limitations
modeling mechanisms, such as hitchhiking of the in resolution, speed, and z-depth of imaging have affected what
ER on motile organelles. Finally, ER-mitochondria is possible to observe. What has been missing is an imaging
contact sites were found to promote both mitochon- technique with high spatiotemporal resolution yet low propensity
drial fission and fusion. for photobleaching and phototoxicity to noninvasively follow
minute and rapid rearrangements of specific organelles like the
INTRODUCTION ER, over the entirety of their interactions with other compart-
ments. While conventional wide-field fluorescence imaging is
Organelles interact with one another and the cytoskeleton to sufficiently fast, it suffers from out-of-focus background, which
synergistically execute various physiological functions (Eisen- can easily obscure events happening on the scale of organelle
contacts. Total internal reflection fluorescence (TIRF) micro- the cER network at 97-nm resolution and 266 frames/s for thou-
scopy eliminates this background, but it can only image within sands of frames, and thereby we gained new insights into the
100 nm from the basal membrane (Steyer and Almers, 2001), mechanisms of MT dynamic instability and tubular ER genera-
which is too small a distance to span many organelles and their tion, and we examined dynamic subdomain segregation along
contact sites. Spinning-disk confocal microscopy (SDCM) elim- ER tubules. Furthermore, multi-color GI-SIM imaging enabled
inates both out-of-focus background and the restriction to the us to investigate the spatiotemporal coordination among organ-
basal membrane, but at the price of substantial photobleaching elles, such as the association of mitochondrial fission and fusion
and reduced speed: in previous SDCM studies of organelle inter- with ER-mitochondrion (ER-Mito) contacts and LE or lysosome
action dynamics (Friedman et al., 2011; Rowland et al., 2014), (Lyso) translocation with respect to ER-LE or Lyso contacts.
the imaging speed was limited to 2–5 s/frame for an 2-min Finally, we identified instances where organelles were moved
duration. Another issue common to all these methods is that or deformed by tethering onto other moving organelles.
they are limited by diffraction to 200-nm lateral resolution. To
visualize potential organelle contacts with greater clarity, we RESULTS
must turn to super-resolution (SR) fluorescence microscopy.
Of the various forms of super-resolution, structured illumina- Development and Characterization of GI-SIM
tion microscopy (SIM) is best suited to combining high resolution By illuminating the sample-substrate interface through an objec-
with long-duration high-speed multi-color imaging, due to its low tive lens at a highly oblique angle (Figure 1A, bottom), TIRF
excitation intensity, use of conventional fluorescent labels, and microscopy creates an excitation light field that extends only a
the need for only nine two-dimensional (2D) raw images to recon- subwavelength distance beyond the basal surface of the cell
struct an super-resolution one. Although wide-field 2D SIM (Figure 1A, middle) to provide high-contrast, background-free
(Gustafsson, 2000) is susceptible to out-of-focus background images that are ideal for SIM reconstruction. However, TIRF-
(which quickly leads to noisy and/or artifact-filled reconstruc- SIM is incapable of visualizing the entirety of organelles, such
tion), live-cell TIRF-SIM has been used to image microtubule as the ER that extends beyond this distance (Figure 1A, top).
(MT) dynamics in artificially flattened S2 cells at 112-nm lateral At the other extreme, excitation through the objective at normal
resolution and 3.7 frames/s (fps) (Kner et al., 2009) and cla- incidence, such as in wide-field microscopy (Figure 1B), illumi-
thrin-mediated endocytosis and actomyosin contraction with nates the entire cell and its contents, but at the cost of substan-
84-nm resolution at 3 s/frame (Li et al., 2015). However, as noted tial out-of-focus background that reduces contrast, introduces
above, most organelle interactions lie outside the range of TIRF noise, and accelerates photobleaching.
illumination. To find an optimum illumination depth between these ex-
To overcome this depth limitation of TIRF, we developed tremes, we repeatedly imaged COS-7 cells expressing markers
grazing incidence structured illumination microscopy (GI-SIM), for the ER or MT network, and we plotted the signal-to-back-
where the illumination entering the objective rear pupil is ground ratio (SBR) as we gradually reduced the numerical aper-
launched just inside the critical angle for TIRF, creating an illumi- ture (NA) of the excitation (and hence its angle of incidence at the
nation field parallel to the substrate that is comparable in interface) from NA = 1.55 to 1.31 (Figure S1). The maximum SBR
thickness to the objective depth-of-focus and, therefore, thin occurred at NA = 1.37, or just below the critical angle for TIRF at
enough to eliminate out-of-focus background yet thick enough the expected refractive index of the cell. Performing a similar
to encompass many organelles and their interactions with one experiment with a 4-mm-diameter bead in water, we determined
another or the cytoskeleton. With GI-SIM, we were able to image that, at this pre-critical NA, the illumination travels nearly parallel
A B C
D E F
G H
Figure 2. GI-SIM Reveals Fast Dynamics of Dynamic Instability of MTs and the ER Network
(A) GI-SIM image of MTs at one time point from a video of MT dynamic instability in a COS-7 cell (Video S1, part I).
(B) Comparison of MT end displacement plots analyzed using data obtained at temporal resolutions of 9.2 frames/s (lower curve) and down-sampled by 8-fold to
1.15 frames/s from the same dataset (upper curve). Green, blue, and red curves indicate the growth, shortening, and pause or fluctuation phases, respectively.
(C) Comparison of MT end detection with deconvolved GI (left) and GI-SIM (right) images.
(D and E) The distributions of MT growth (D) and shortening velocity (E) versus displacement.
(F) Catastrophe and rescue transition frequency computed at different imaging speed.
(G) GI-SIM (right) and GI (left) images of the ER network acquired at 266 frames/s in live COS-7 cells expressing mEmerald-KDEL (Video S1, part II).
(H) Tubular ER skeleton image color coded according to the local oscillation frequency and superimposed onto the fluorescence.
(I) Time-lapse GI-SIM images showing time-dependent variations in subdomains along an ER tubule. Cyan and orange arrows indicate the formation and
disappearance of two constriction sites. At 7.5 ms, these subdomains are well resolved in the GI-SIM image (left), but they cannot be observed clearly in the
diffraction-limited GI image (right). Scale bars, 2 mm (A and G) and 1 mm (C, H, and I).
to the interface in a sheet similar in width to the 1-mm depth-of- GI-SIM Investigation of MT Dynamic Instability
focus of the objective (STAR Methods; Figures S1K–S1M). We A fast process demanding high spatiotemporal resolution imag-
therefore term this condition GI illumination. It not only achieves ing is the dynamic instability of MTs. The spatiotemporal regula-
the twin goals of low background and complete illumination of tion of MTs is important in many aspects of cell biology (Akhma-
organelles near the basal membrane (Figure 1C) but also in- nova and Steinmetz, 2008), and dynamic instability is an
creases the signal 10-fold compared to TIRF illumination at important component of this regulation (Mitchison and Kirsch-
NA = 1.55. ner, 1984). To image MTs at unprecedented speeds and at su-
GI illumination is conceptually similar to the highly inclined and per-resolution for extended periods of time, we transfected
laminated optical sheet (HILO) approach used for single-mole- COS-7 cells with the bright and photostable MT-associated
cule imaging in whole cells (Tokunaga et al., 2008), yet GI con- fusion protein 33 mEmerald-ensconsin (Gierke et al., 2010) (Fig-
fines the light even closer to the critical angle to eliminate nearly ure 2A), which co-localizes well with a direct tubulin marker but
all of out-of-focus excitation. In HILO, the out-of-focus fluores- produces far less cytosolic background (Figures S2A and
cence background can be computationally removed using a S2B). GI-SIM imaging at 97-nm resolution (Figures S2E–S2K)
structured illumination pattern at a period of twice the diffraction and 9.2 frames/s for hundreds of frames (Video S1, part I), al-
limit (sHILO; Fiolka, 2016), but the lateral resolution extension is lowed us to quantify MT dynamic instability on a much finer
then limited to 1.53, rather than 23 in GI-SIM, and the potential spatiotemporal scale than what was achieved in previous
for photobleaching and phototoxicity contributed by the out-of- studies (Akhmanova and Steinmetz, 2008; Gierke et al., 2010).
focus excitation remains. For example, sampling the data at full temporal resolution
showed brief transitions between MT growth and shortening and energy sources that can drive such oscillations (Nixon-Abell
quick fluctuations when the growth or shortening of MT ends et al., 2016).
paused temporally (Video S1, part I). These transient processes The high spatiotemporal resolution of GI-SIM also revealed
cannot be captured if the sampling frequency drops by 8-fold to rapid changes in the thickness of the ER lumen along various tu-
1.15 Hz (Figure 2B). Likewise, the high spatial resolution of GI- bules (Figure 2I), which were not apparent with GI alone. In
SIM ensures detection of densely packed MTs undetectable to particular, we observed constrictions (arrows, Figure 2I) that
diffraction-limited methods (Figure 2C). Therefore, the loss of formed in as fast as 10 ms, with mEmerald-KDEL displaced
either spatial or temporal resolution was sufficient to degrade to either side (7.5- to 18.8-ms time points of Figure 2I). The total
the precision of MT dynamic characterization. fluorescence intensity during these events was constant to
We measured MT growth and shortening rates of 20.5 ± within 5% (Figures S2P and S2Q), suggesting that the lumen
11.6 mm/min (N = 178 from 15 cells) and 23.8 ± 13.9 mm/min contents were conserved during constriction and displacement.
(N = 143 from 15 cells), respectively, consistent with earlier re- Similar constrictions and bulges in ER tubules have long been
sults obtained using tubulin or EB1 markers (Matov et al., observed by electron microscopy (Behnke and Moe, 1964;
2010). Furthermore, when analyzing these rates as a function Friedman and Voeltz, 2011; Nixon-Abell et al., 2016), but here
of the displacement of each growth or shortening event (Figures we see that these features remodel on the timescale of tens of
2D and 2E), we found that long displacing events correlated with milliseconds.
slow rates of growth or shortening. There was a broad range of
rates during short displacing events, leading to diverse fluctua- Mechanisms of Tubular ER Formation
tions in MT ends. Transition frequencies of catastrophe and In mammalian cells, ER network rearrangements strongly
rescue were measured to be 0.59 ± 0.48 s1 and 0.67 ± depend on interactions with the MT cytoskeleton. New ER
0.51 s1, respectively (Figure 2F, left column), which are fast tubules can be pulled out of the existing ER membrane by asso-
enough to require 9.2 frames/s measurement to achieve accu- ciating with motor proteins and then extending along MTs (i.e.,
rate results (Figure 2F, middle and right columns). The scattered the sliding mechanism) or by attaching to the tips of polymer-
distribution of catastrophe and rescue transition might result izing MTs (i.e., tip attachment complex [TAC] mechanism)
from the heterogeneous microenvironments around the plus (Waterman-Storer and Salmon, 1998). We applied GI-SIM to
ends of different MTs, such as the availability of guanosine characterize ER-MT dynamic interactions and investigate
triphosphate (GTP)-tubulin dimers. These observations of the whether cells employ other mechanisms to reshape the ER
switch-like behavior of MT ends support the dynamic cap model network. COS-7 or U2OS cells were co-transfected with 33
(Howard and Hyman, 2009). This theory attributes MT dynamic mEmerald-ensconsin and mCherry-KDEL to label the MTs
instability to variations in the length of the GTP-tubulin cap at and ER simultaneously. A representative GI-SIM two-color im-
MT ends, which depend on the randomness of GTP-tubulin di- age frame from a movie of the ER and MTs (Figure 3A; Video
mers binding to or dissociating from proto-filaments. S2, part I) shows that ER-reshaping activities, such as ER ring
rearrangement and ER tubule branching, mostly occurred along
GI-SIM Investigation of ER Network Dynamics MTs. ER tubule-branching activities included instances of the
The ER is notable not only because it is the largest intracellular sliding and TAC mechanisms (Figures 3B and 3C; Video S2,
organelle but also because it continuously and rapidly remodels parts II and III). In addition, however, we observed that new
itself (Hu et al., 2011; Nixon-Abell et al., 2016). To investigate ER ER tubules could be pulled out by attaching to the plus ends
dynamics using GI-SIM, we transfected COS-7 cells with the ER of depolymerizing MTs (Figure 3D; Video S2, parts II and III).
luminal marker mEmerald-KDEL. GI-SIM, at 97-nm resolution, Because the plus-end-binding proteins of polymerizing and
resolved the ER with much finer detail than GI illumination alone depolymerizing MTs are very different (Akhmanova and Stein-
(Figures 2G and 2I; Video S1, part II). As we reported previously metz, 2008), this depolymerizing TAC (dTAC) mechanism may
(Nixon-Abell et al., 2016), continuous sheet-like structures be mediated by molecules other than the STIM1 and EB1 me-
observed in the peripheral ER under diffraction-limited GI illumi- diators of the polymerizing TAC (pTAC) mechanism (Grigoriev
nation were found to be riddled with fenestrations when viewed et al., 2008).
with GI-SIM (Figure 2G). Furthermore, due to the bright signals In addition to these three mechanisms, where growing ER tu-
afforded by GI-SIM and the use of interleaved reconstruction bules interact directly with MTs, we also discovered a fourth
(Ma et al., 2018), we were able to image these fenestrations at mechanism wherein new ER tubules were generated by hitchhik-
266 frames/s for 4,500 frames, which confirmed that most of ing on highly motile organelles, e.g., LEs or Lysos that were in
them consisted of tightly packed tubular matrices that constantly turn being transported along MTs by molecular motors (Fig-
remodeled themselves (Video S1, part II). By tracking all tubules ure 3E; Video S2, parts II and III). Although the molecules that
with a skeletonization algorithm, we created a spatial map of tether the ER membrane to the vehicle LE or Lyso are unknown,
their oscillations (Figure 2H). While the median amplitude and this tether may transiently break to either allow the growing ER
frequency were 144 nm peak to peak at 10.1 Hz, there was tubule to fuse with an existing ER tubule or to retract back to
considerable spatial heterogeneity in these oscillations (Figures the branching point in the event that the tether breaks in the mid-
S2L–S2O), with the fastest oscillations often occurring at tightly dle of ER tubule extension.
clustered tubules (Figure 2H). This may be because shorter tu- Finally, as a fifth mechanism, we observe that new ER tubule
bules, like shorter strings, are expected to have higher reso- branches can appear without apparent direct or indirect involve-
nance frequencies, or it may reflect the spatial variability of the ment of MTs. This process appears as de novo budding of the ER
A B C
D E F G
tubule from an existing ER tubule (Figure 3F; Video S2, parts II hitchhiking events were also comparable, and together they
and III), although the existence of an unseen mediator cannot comprised nearly 60% of the total events. In contrast, ER tubule
be ruled out. growth rates were significantly different for the two types of MT
Next, we characterized the relative proportions (Figure 3G) end-associated mechanisms: 8.3 ± 3.3 mm/min for pTAC versus
and growth rates (Figure 3H) of these five different types of 15.6 ± 3.6 mm/min for dTAC. In addition, pTAC events occurred
ER-branching events. We examined 16 cells having 529 events more frequently than dTAC ones. These differences further sug-
in which each ER tubule branch extended at least 1 mm. Sliding gest that pTAC and dTAC mechanisms for ER movement rely on
and hitchhiking events had similar growth rates of 40.5 ± different molecular machineries. Finally, the fifth mechanism,
16.2 mm/min and 43.0 ± 14.0 mm/min, respectively, presumably MT-independent de novo ER budding, occurred comparatively
because they both rely on MT-based molecular motors to rarely (8 events from 30 cells) and more slowly (5.5 ± 1.6 mm/min)
generate their driving force. The proportions of sliding and than the other four.
To investigate the dependence of these ER-branching mech- ER Interactions Regulate Transport of LEs or Lysos
anisms on MT dynamics, we treated COS-7 cells with Nocoda- The movements of intracellular organelles often correlate with
zole for 20 min (Figure S3). Consistent with previous studies cellular functions (Bonifacino and Neefjes, 2017). Emerging
(Friedman et al., 2010; Lu et al., 2009), most tubular ER evidence indicates that the ER plays an important role in
collapsed, and the pTAC and dTAC events, which depend on transporting and controlling the local concentration of intracel-
dynamic MT polymerization and depolymerization, ceased, lular organelles, such as LEs or Lysos (Raiborg et al., 2015). To
whereas sliding (Figure S3E) and hitchhiking events still occurred investigate how the ER contact affects the transport of LEs or
along MTs stabilized by acetylation (Friedman et al., 2010), albeit Lysos, we triply transfected COS-7 cells with 33 mEmerald-
less frequently. De novo budding was not affected (Figure S3F; enconsin, mCherry-KDEL, and Halo-LAMP1 labeled with the
9 events from 30 cells), proving it is an MT-independent JF642 ligand to visualize the MTs, ER, and LAMP1-positive
mechanism. LEs or Lysos by GI-SIM. Consistent with a previous study
(Rowland et al., 2014), we found that >90% of LEs or Lysos
Tubular ER’s Role in Mitochondrial Fission and Fusion were associated with the ER (Figure 5A; Video S4). We also
Recently, ER-Mito contact sites were found to define the posi- found that ER-LE or Lyso interaction was facilitated by corrals
tions of mitochondrial fission (Friedman et al., 2011). Consistent of ER tubules that shrank from a large polygon shape to a
with this, we observed that ER-Mito contact sites marked con- much smaller one that tightly surrounded the LE or Lyso (Fig-
stricted loci in mitochondria (arrows in Figures 4A and 4B), ure 5B). Afterward, the motion of the LE or Lyso was in step
where the majority of fission events occurred (84%, N = 100 with its surrounding ER polygon (88 to 238 s in Figure 5B;
from 97 cells, Figure 4F). In addition, we noticed that ER-Mito Video S5, part I).
contacts were retained through the entire division process A recent study found that ER-LE contacts mediate the transfer
(Video S3, parts I and II) and the two compartments defined of molecular motors onto motor adaptors on the LE membrane,
by the ER tubule writhed back and forth (Figure 4C; Video S3, prompting the translocation of LEs (Raiborg et al., 2015). We
part II). Interestingly, we found that during the writhing process confirmed that if an ER-associated LE or Lyso was close to
the area of one compartment slightly increased while the area of MTs, it tended to be motile, especially when the LE or Lyso
the other compartment decreased (Figure S4). Moreover, we was tightly encompassed by an ER polygon (Figure 5C). More-
observed that, in some fission events (e.g., upper zoom-in over, the contact between the ER and the leading edge of the
example in Video S3, part I), one compartment of the dividing moving LE or Lyso was preferentially maintained during the
mitochondrion was driven to move away from the other movement (zoom-in examples in Video S4), which prompted
compartment, which preferentially associated with the ER- the long-distance transportation of LE or Lyso structures. In
Mito contact and remained relatively static. The mitochondrial contrast, if the LE or Lyso was not associated with the ER, it
membrane at the fission site was elongated into tubular inter- generally underwent diffusive motion rather than directional
mediates until division occurred to give two independent mito- transport along MTs, even though the LE or Lyso was already
chondria. These observations suggest that ER-Mito contacts in contact with MTs (Video S4, part II).
not only mark the sites of mitochondrial division but also play Furthermore, we noted that the diffusive movement was small
a role in stabilizing the mitochondrion, thereby facilitating the di- for the LEs or Lysos tightly confined by ER corrals. We therefore
vision process. asked whether ER interactions assisted in stabilizing LEs or Ly-
Given that the ER usually participates in mitochondrial fission, sos before they docked onto MTs. To prevent directional trans-
an intriguing question is whether it can also serve to facilitate port of LEs or Lysos along MTs, we simultaneously knocked
mitochondrial fusion. We scrutinized 134 cases of mitochondrial down the adaptor proteins FYCO1 and RILP (Figures S5A and
fusion from 120 cells, and we found that ER-Mito contact was S5B), which are responsible for recruiting the molecular motors
involved in 59% of these events (Figure 4F). During such kinesin and dynein, respectively, onto LEs or Lysos (Phillips
events (Figure 4D; Video S3, parts I and III), one mitochondrion and Voeltz, 2016). As a result, the role of MTs in affecting LEs
first contacts the ER tubule lying between two opposing or Lysos’ movements could be eliminated. We found LEs or
mitochondria. As the two mitochondria come into contact, the Lysos tightly confined by ER corrals had smaller mean square
local fluorescence intensity of the outer membrane protein, displacements (MSDs) than those more loosely confined by
mEmerald-Tomm20, is transiently enhanced at the established the ER (Figure S5C). Therefore, our data suggest that ER-LE or
ER-Mito contact site (10 and 12 s in Figure 4D). The disappear- Lyso interactions play a role in stabilizing LEs or Lysos.
ance of the enhanced fluorescence indicates the coalescence On rare occasions, we observed ER fission events (Fig-
of outer membranes, followed by the relaxation of the constric- ure 5D; Video S5, part II) wherein an LE or Lyso (white arrow)
tion at the fusion site. We quantified the duration of all the fusion moving along an MT track bent an ER tubule in its path into a
events by measuring the time from the first contact of the two highly curved arc (12 and 14 s in Figure 5D) before eventually
mitochondria until complete relaxation of the post-fusion rupturing at an adjacent three-way junction (18 s in Figure 5D)
constriction. We found that mitochondrial fusion events without or in the middle of the ER tubule (example 2 in Video S5, part
ER involvement (Figure 4E; Video S3, part IV) on average took II). After passage of the LE or Lyso, a free end of the ruptured
longer to complete membrane coalescence compared to the ER tubule could either fuse with an adjacent ER tubule (32 and
ones with ER-Mito contact (12.5 versus 21.9 s; Figure 4G), indi- 36 s in Figure 5D), or the two free ends could fuse with each
cating that the ER accelerates membrane coalescence after the other to restore the original connectivity (example 2 in Video
initial contact. S5, part II).
Figure 4. The Association of Mitochondrial Fission and Fusion with the Tubular ER
(A) Contacts between mitochondria (green) and the ER (magenta) shown in a live COS-7 cell.
(B) Mitochondrial channel only. White arrows in (A) and (B) indicate constricted loci in mitochondria associated with ER-Mito contact sites, where mitochondrial
fission occurs (Video S3, upper panel of part I).
(C) Time-lapse images of a typical mitochondrial fission event at an ER-Mito contact site (Video S3, part II).
(D) Time-lapse images of a typical mitochondrial fusion event at an ER-Mito contact site (Video S3, lower panel of part I).
(E) Time-lapse images of a typical mitochondrial fusion event without ER-Mito contact (Video S3, part IV). White arrows in (E) indicate that the ER is absent at the
contact site of the two mitochondria.
(F) The proportion of mitochondrial fission or fusion events occurring with and without ER-Mito contacts.
(G) Comparison of the duration of mitochondrial fusion events with and without ER-Mito contacts.
Data are shown as mean ± SEM. **p < 0.01 (one-way ANOVA). Scale bars, 3 mm (A and B) and 1 mm (C–E).
GI-SIM Reveals Hitchhiking Interactions between molecular motors, rather than by directly coupling with molecular
Organelles motors themselves. However, it has not been confirmed whether
In the canonical mode of MT-based intracellular transport, the hitchhiking paradigm exists in mammalian cells (Salogiannis
cargo-specific adaptor molecules recruit molecular motors, and Reck-Peterson, 2017) and, if so, what its functions might be.
which then directly drive the cargo along the MT track. Recently, Hitchhiking is mediated by membrane contact sites that tether
a new paradigm termed hitchhiking was discovered in fungi (Gui- the hitchhiker to the vehicle organelles. For example, we found
maraes et al., 2015), in which some cargos achieve MT-depen- that ER tubules could anchor onto a moving LE or Lyso and a
dent motility by tethering onto other cargos that have recruited new ER tubule could be pulled out and elongate as it followed
the trajectory of the LE or Lyso (Figure 3E). We also observed that why such free-ended ER tubules have been observed previously
ER tubules that adhered to moving mitochondria could elongate (Friedman et al., 2011; Nixon-Abell et al., 2016; Rowland et al.,
(Figure 6A; Video S6, part I). In either case, the leading edge of 2014), although it was generally thought that they were unstable
the hitchhiking ER tubule was always attached to the rear of (Wang et al., 2016). Taken together, these results demonstrate
the moving organelle. that ER-organelle contacts can not only influence organelle func-
During this hitchhiking process, the extending ER tubule may tion but also play a role in rearranging the ER network.
fuse with existing ER tubules to form a new three-way junction Recent studies reported that mitochondria and Lyso or vacu-
(Video S6, part I). Alternatively, we observed vehicle organelles oles commonly contact each other (Elbaz-Alon et al., 2014;
that stopped while pulling a hitchhiking ER tubule, leaving the Wong et al., 2018). We also observed abundant dynamic Mito-
tethered end free in the cytoplasm (Figure S6). This explains LE or Lyso interactions using GI-SIM (Figure 6B; Video S6,
B Characterization of the irradiation depth of the GI illu- Bonifacino, J.S., and Neefjes, J. (2017). Moving and positioning the endolyso-
mination mode somal system. Curr. Opin. Cell Biol. 47, 1–8.
B RNAi transfection and real-time PCR Bosch, M., Castro, J., Saneyoshi, T., Matsuno, H., Sur, M., and Hayashi, Y.
B Nocodazole treatment and imaging (2014). Structural and molecular remodeling of dendritic spine substructures
during long-term potentiation. Neuron 82, 444–459.
B GI-SIM imaging for Drosophila embryo
d QUANTIFICATION AND STATISTICAL ANALYSIS Boulanger, J., Gueudry, C., Münch, D., Cinquin, B., Paul-Gilloteaux, P., Bardin,
S., Guérin, C., Senger, F., Blanchoin, L., and Salamero, J. (2014). Fast high-
B Tubular ER oscillation analysis
resolution 3D total internal reflection fluorescence microscopy by incidence
B Characterization of MT dynamic instability
angle scanning and azimuthal averaging. Proc. Natl. Acad. Sci. USA 111,
B Tubular ER growth analysis 17164–17169.
B Association of mitochondrial fission/fusion with ER- Cutrale, F., Trivedi, V., Trinh, L.A., Chiu, C.L., Choi, J.M., Artiga, M.S., and
Mito contacts Fraser, S.E. (2017). Hyperspectral phasor analysis enables multiplexed 5D
in vivo imaging. Nat. Methods 14, 149–152.
SUPPLEMENTAL INFORMATION de Brito, O.M., and Scorrano, L. (2008). Mitofusin 2 tethers endoplasmic retic-
ulum to mitochondria. Nature 456, 605–610.
Supplemental Information includes seven figures and seven videos and can be Demmerle, J., Wegel, E., Schermelleh, L., and Dobbie, I.M. (2015). Assessing
found with this article online at https://doi.org/10.1016/j.cell.2018.09.057. resolution in super-resolution imaging. Methods 88, 3–10.
Eisenberg-Bord, M., Shai, N., Schuldiner, M., and Bohnert, M. (2016). A Tether
ACKNOWLEDGMENTS Is a Tether Is a Tether: Tethering at Membrane Contact Sites. Dev. Cell 39,
395–409.
This work was supported by grants to Dong Li from the Chinese Ministry of Sci- Elbaz-Alon, Y., Rosenfeld-Gur, E., Shinder, V., Futerman, A.H., Geiger, T., and
ence and Technology (MOST; 2017YFA0505301 and 2016YFA0500203), the Schuldiner, M. (2014). A dynamic interface between vacuoles and mitochon-
National Natural Science Foundation of China (NSFC; 91754202 and dria in yeast. Dev. Cell 30, 95–102.
31770930), the Chinese Academy of Sciences (CAS; XDB19040101 and
Fiolka, R. (2016). Clearer view for TIRF and oblique illumination microscopy.
YZ201651), the CAS Pioneer Hundred Talents Program, the Thousand Young
Opt. Express 24, 29556–29567.
Talents Program of China, and the Joint Program between CAS and Peking
University and to J.-J.L. from MOST (2016YFA0500100) and NSFC Friedman, J.R., and Voeltz, G.K. (2011). The ER in 3D: a multifunctional dy-
(31530039). J.L.-S. and E.B. are funded by the Howard Hughes Medical Insti- namic membrane network. Trends Cell Biol. 21, 709–717.
tute (HHMI). Friedman, J.R., Webster, B.M., Mastronarde, D.N., Verhey, K.J., and Voeltz,
G.K. (2010). ER sliding dynamics and ER-mitochondrial contacts occur on
acetylated microtubules. J. Cell Biol. 190, 363–375.
AUTHOR CONTRIBUTIONS
Friedman, J.R., Lackner, L.L., West, M., DiBenedetto, J.R., Nunnari, J., and
Dong Li and E.B. conceived the idea. Dong Li designed the experiments and Voeltz, G.K. (2011). ER tubules mark sites of mitochondrial division. Science
built the microscope at Janelia under the supervision of E.B. Di Li and S.Z. built 334, 358–362.
and improved the microscope system at the Institute of Biophysics under the Gierke, S., Kumar, P., and Wittmann, T. (2010). Analysis of microtubule poly-
supervision of Dong Li. J.-J.L. designed the dendritic spine plasticity experi- merization dynamics in live cells. In Microtubules: In Vivo, L. Cassimeris and
ment, and D.P.K. designed the Drosophila dorsal closure experiment. Y.G., P. Tran, eds. (San Diego: Elsevier Academic Press Inc), pp. 15–33.
Di Li, Y.Y., R.P.M., and U.S.T. performed the experiments. D.E.M. wrote instru- Grigoriev, I., Gouveia, S.M., van der Vaart, B., Demmers, J., Smyth, J.T., Hon-
mentation control code. Y.G., Di Li, X.W., and C.L. analyzed the data with the nappa, S., Splinter, D., Steinmetz, M.O., Putney, J.W., Jr., Hoogenraad, C.C.,
conceptual advice from Dong Li, E.B., J.L.-S., and J.H. Di Li and Y.G. and Akhmanova, A. (2008). STIM1 is a MT-plus-end-tracking protein involved
composed the figures and videos under the supervision of Dong Li. Dong Li, in remodeling of the ER. Curr. Biol. 18, 177–182.
E.B., and J.L.-S. wrote the manuscript with input from all the authors. All au-
Grimm, J.B., English, B.P., Chen, J., Slaughter, J.P., Zhang, Z., Revyakin, A.,
thors discussed the results and commented on the manuscript.
Patel, R., Macklin, J.J., Normanno, D., Singer, R.H., et al. (2015). A general
method to improve fluorophores for live-cell and single-molecule microscopy.
DECLARATION OF INTERESTS Nat. Methods 12, 244–250.
Guimaraes, S.C., Schuster, M., Bielska, E., Dagdas, G., Kilaru, S., Meadows,
The GI-SIM technology described herein is covered by Chinese provisional
B.R.A., Schrader, M., and Steinberg, G. (2015). Peroxisomes, lipid droplets,
patent application 201710296751.6 filed by Dong Li and assigned to the Insti-
and endoplasmic reticulum ‘‘hitchhike’’ on motile early endosomes. J. Cell
tute of Biophysics.
Biol. 211, 945–954.
Gustafsson, M.G.L. (2000). Surpassing the lateral resolution limit by a factor of
Received: May 2, 2018
two using structured illumination microscopy. J. Microsc. 198, 82–87.
Revised: July 21, 2018
Accepted: September 26, 2018 Gustafsson, M.G.L., Shao, L., Carlton, P.M., Wang, C.J.R., Golubovskaya,
Published: October 25, 2018 I.N., Cande, W.Z., Agard, D.A., and Sedat, J.W. (2008). Three-dimensional res-
olution doubling in wide-field fluorescence microscopy by structured illumina-
tion. Biophys. J. 94, 4957–4970.
REFERENCES
Helenius, J., Brouhard, G., Kalaidzidis, Y., Diez, S., and Howard, J. (2006). The
Akhmanova, A., and Steinmetz, M.O. (2008). Tracking the ends: a dynamic depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtu-
protein network controls the fate of microtubule tips. Nat. Rev. Mol. Cell bule ends. Nature 441, 115–119.
Biol. 9, 309–322. Helle, S.C.J., Feng, Q., Aebersold, M.J., Hirt, L., Grüter, R.R., Vahid, A., Sir-
c
ianni, A., Mostowy, S., Snedeker, J.G., Sari , A., et al. (2017). Mechanical force
Behnke, O., and Moe, H. (1964). AN ELECTRON MICROSCOPE STUDY OF
MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY induces mitochondrial fission. eLife 6, e30292.
OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES. J. Cell Biol. 22, Hirabayashi, Y., Kwon, S.K., Paek, H., Pernice, W.M., Paul, M.A., Lee, J.,
633–652. Erfani, P., Raczkowski, A., Petrey, D.S., Pon, L.A., and Polleux, F. (2017).
ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mamma- Rowland, A.A., Chitwood, P.J., Phillips, M.J., and Voeltz, G.K. (2014). ER con-
lian neurons. Science 358, 623–630. tact sites define the position and timing of endosome fission. Cell 159,
Howard, J., and Hyman, A.A. (2009). Growth, fluctuation and switching at 1027–1041.
microtubule plus ends. Nat. Rev. Mol. Cell Biol. 10, 569–574. Salogiannis, J., and Reck-Peterson, S.L. (2017). Hitchhiking: A Non-Canonical
Mode of Microtubule-Based Transport. Trends Cell Biol. 27, 141–150.
Hu, J., Prinz, W.A., and Rapoport, T.A. (2011). Weaving the web of ER tubules.
Cell 147, 1226–1231. Self, S.A. (1983). Focusing of spherical Gaussian beams. Appl. Opt. 22,
658–661.
Kiehart, D.P., Montague, R.A., Rickoll, W.L., Foard, D., and Thomas, G.H.
(1994). High-resolution microscopic methods for the analysis of cellular move- Simunovic, M., Manneville, J.B., Renard, H.F., Evergren, E., Raghunathan, K.,
ments in Drosophila embryos. Methods Cell Biol. 44, 507–532. Bhatia, D., Kenworthy, A.K., Voth, G.A., Prost, J., McMahon, H.T., et al. (2017).
Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Pro-
Kiehart, D.P., Galbraith, C.G., Edwards, K.A., Rickoll, W.L., and Montague, teins. Cell 170, 172–184.e11.
R.A. (2000). Multiple forces contribute to cell sheet morphogenesis for dorsal
Steyer, J.A., and Almers, W. (2001). A real-time view of life within 100 nm of the
closure in Drosophila. J. Cell Biol. 149, 471–490.
plasma membrane. Nat. Rev. Mol. Cell Biol. 2, 268–275.
Kner, P., Chhun, B.B., Griffis, E.R., Winoto, L., and Gustafsson, M.G.L. (2009).
Sullivan, W., Ashburner, M., and Hawley, R. (2000). Drosophila Protocols (Cold
Super-resolution video microscopy of live cells by structured illumination. Nat.
Spring Harbor, NY: Cold Spring Harbor Laboratory Press).
Methods 6, 339–342.
Tokunaga, M., Imamoto, N., and Sakata-Sogawa, K. (2008). Highly inclined
Lewis, S.C., Uchiyama, L.F., and Nunnari, J. (2016). ER-mitochondria contacts thin illumination enables clear single-molecule imaging in cells. Nat. Methods
couple mtDNA synthesis with mitochondrial division in human cells. Science 5, 159–161.
353, aaf5549.
Tortarolo, G., Castello, M., Diaspro, A., Koho, S., and Vicidomini, G. (2018).
Li, D., Shao, L., Chen, B.C., Zhang, X., Zhang, M., Moses, B., Milkie, D.E., Evaluating image resolution in stimulated emission depletion microscopy. Op-
Beach, J.R., Hammer, J.A., 3rd, Pasham, M., et al. (2015). ADVANCED tica 5, 32–35.
IMAGING. Extended-resolution structured illumination imaging of endocytic
Valm, A.M., Cohen, S., Legant, W.R., Melunis, J., Hershberg, U., Wait, E., Co-
and cytoskeletal dynamics. Science 349, aab3500.
hen, A.R., Davidson, M.W., Betzig, E., and Lippincott-Schwartz, J. (2017).
Lu, L., Ladinsky, M.S., and Kirchhausen, T. (2009). Cisternal organization of the Applying systems-level spectral imaging and analysis to reveal the organelle
endoplasmic reticulum during mitosis. Mol. Biol. Cell 20, 3471–3480. interactome. Nature 546, 162–167.
Ma, Y., Li, D., Smith, Z.J., Li, D., and Chu, K. (2018). Structured illumination Wagner, W., Brenowitz, S.D., and Hammer, J.A., 3rd. (2011). Myosin-Va trans-
microscopy with interleaved reconstruction (SIMILR). J. Biophotonics 11, ports the endoplasmic reticulum into the dendritic spines of Purkinje neurons.
e201700090. Nat. Cell Biol. 13, 40–48.
Matov, A., Applegate, K., Kumar, P., Thoma, C., Krek, W., Danuser, G., and Wang, C., Du, W., Su, Q.P., Zhu, M., Feng, P., Li, Y., Zhou, Y., Mi, N., Zhu, Y.,
Wittmann, T. (2010). Analysis of microtubule dynamic instability using a plus- Jiang, D., et al. (2015). Dynamic tubulation of mitochondria drives mitochon-
end growth marker. Nat. Methods 7, 761–768. drial network formation. Cell Res. 25, 1108–1120.
Mitchison, T., and Kirschner, M. (1984). Dynamic instability of microtubule Wang, S., Tukachinsky, H., Romano, F.B., and Rapoport, T.A. (2016). Cooper-
growth. Nature 312, 237–242. ation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate
a tubular membrane network. eLife 5, 209.
Nixon-Abell, J., Obara, C.J., Weigel, A.V., Li, D., Legant, W.R., Xu, C.S., Pa-
solli, H.A., Harvey, K., Hess, H.F., Betzig, E., et al. (2016). Increased spatiotem- Wang, C., Taki, M., Sato, Y., Fukazawa, A., Higashiyama, T., and Yamagu-
poral resolution reveals highly dynamic dense tubular matrices in the periph- chi, S. (2017). Super-Photostable Phosphole-Based Dye for Multiple-Acqui-
eral ER. Science 354, aaf3928. sition Stimulated Emission Depletion Imaging. J. Am. Chem. Soc. 139,
10374–10381.
Phillips, M.J., and Voeltz, G.K. (2016). Structure and function of ER membrane
Waterman-Storer, C.M., and Salmon, E.D. (1998). Endoplasmic reticulum
contact sites with other organelles. Nat. Rev. Mol. Cell Biol. 17, 69–82.
membrane tubules are distributed by microtubules in living cells using three
Prinz, W.A. (2014). Bridging the gap: membrane contact sites in signaling, distinct mechanisms. Curr. Biol. 8, 798–806.
metabolism, and organelle dynamics. J. Cell Biol. 205, 759–769.
Wong, Y.C., Ysselstein, D., and Krainc, D. (2018). Mitochondria-lysosome con-
Raiborg, C., Wenzel, E.M., Pedersen, N.M., Olsvik, H., Schink, K.O., Schultz, tacts regulate mitochondrial fission via RAB7 GTP hydrolysis. Nature 554,
S.W., Vietri, M., Nisi, V., Bucci, C., Brech, A., et al. (2015). Repeated ER-endo- 382–386.
some contacts promote endosome translocation and neurite outgrowth. Na- Young, I.T., Zagers, R., van Vliet, L.J., Mullikin, J., Boddeke, F., and Netten, H.
ture 520, 234–238. (1993). Depth-of-focus in microscopy. In Proceedings of the 8th Scandinavian
Richards, B., and Wolf, E. (1959). Electromagnetic diffraction in optical sys- Conference on Image Analysis, B. Braathen, K.A. Høgda, and K. Heia, eds.
tems. II. Structure of the image field in an aplanatic system. Proc. R. Soc. (NOBIM, Norwegian Society for Image Processing and Pattern Recognition),
Lond. A Math. Phys. Sci. 253, 358–379. pp. 493–498.
STAR+METHODS
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dong Li
(lidong@ibp.ac.cn).
Cell culture
Bothe COS-7 and U2OS cells were purchased from American Type Culture Collection (ATCC). COS-7 cells were derived from the
kidney of Cercopithecus aethiops, and U2OS cells were derived from human female moderately differentiated sarcoma of the tibia.
COS-7 cells were grown in DMEM media (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) and 1% penicillin/strepto-
mycin at 37 C and 5% CO2 until 60 to 80% confluency was reached. U2OS cells were grown in MCMM media (GIBCO) supple-
mented with 10% FBS (GIBCO) and 1% penicillin/streptomycin at 37 C and 5% CO2 until 60 to 80% confluency was reached.
For GI-SIM imaging experiments, 35 mm coverslips were pre-coated with 50 mg/ml collagen for 1 hour, and cells were then seeded
onto the coverslips to achieve 70% confluence prior to transfection. Transfections were executed using Lipofectamine 3000 (In-
vitrogen) according to the manufacturer’s protocol. Cells were imaged 16-36 hours post-transfection in a microscope stage top mi-
cro-incubator (OKO Lab) maintained at 37 C and 5% CO2. Where indicated, the cells transfected with HaloTag plasmids were
labeled with JF646 ligand following the published protocol (Grimm et al., 2015), and the cells were imaged immediately afterward.
Drosophila embryo
The transgenic Drosophila line stably expressing a GFP-tagged Actin Binding Domain of fly Moesin (GFP-Moe-ABD, referred to as
sGMCA) was generated by Dan Kiehart lab (Kiehart et al., 2000). Embryos were prepared as previously described (Kiehart et al.,
1994). Briefly, adult flies laid eggs for 3-4 hours at 25 C which were subsequently aged at 16 C for 24 hours. Embryos were dechor-
ionated in 50% bleach for 1.25 min. Dorsal closure staged embryos were lined up on a grape juice agar pad, secured to a coverslip
using embryo glue (Kiehart et al., 1994; Sullivan et al., 2000) and desiccated on the coverslip in a tightly sealed jar containing Drierite
(W. A. Hammond Drierite Company, Ltd.) for 6-18 minutes depending on the length of time between dechorionation and desiccation.
This step helps flatten the embryos against the coverslip when mounted. Note that the desiccation time is less than what is typically
used to prepare embryos for microinjection of DNA constructs for creating transgenic flies. The desiccated embryos typically hatch,
and when rescued to a food source, development progresses normally.
METHOD DETAILS
GI-SIM system
GI-SIM systems at Janelia and the Institute of Biophysics were used for the experiments. Both setups (Figure S7A) represent
improved versions of our previous TIRF-SIM system (Li et al., 2015). Briefly, three laser beams of 488 nm (500 mW, Coherent,
Genesis-MX-SLM), 560 nm (500 mW, MPB Communications, 2RU-VFL-P-500-560), and 642 nm (500 mW, MPB Communications,
2RU-VFL-P-500-642) were collinearly combined and passed through an acousto-optic tunable filter (AOTF; AA Quanta Tech,
AOTFnC-400.650). The beam was then expanded to a diameter of 20 mm, and directed into a phase-only modulator composed
of a polarization beam splitter, an achromatic half-wave plate, and a ferroelectric spatial light modulator (SLM; Forth Dimension Dis-
plays, SXGA-3DM). The incident light was diffracted by the grating pattern displayed on the SLM, and then passed through a home-
made azimuthally patterned achromatic half-wave plate (HWP, Bolder Vision Optik), which consisted of three pairs of segments. The
fast axes of each pair of HWP are properly oriented (Figure S7B), so that the linearly polarized diffracted light from the grating pattern
with a given orientation can be rotated into the desired s-polarized light. Therefore, the contrast of the illumination pattern formed
inside the specimen was maximized. In contrast to the liquid crystal device-based polarization rotator that takes 10 ms to accom-
plish each polarization rotation, the azimuthal HWP requires no settling time, which permitted us to achieve the maximum imaging
speed limited only by the readout time of the sCMOS camera. The diffracted light was spatially filtered by a mask that only allowed the
desired ± 1 diffraction orders to pass through. The ± 1 order light spots were then relayed onto the back focal plane of the high
numerical aperture (NA) objective (Olympus APON 100XHOTIRF 1.7 NA, or UAPON 100XOTIRF 1.49 NA for Drosophila embryo spec-
imen). The distance between the two light spots determined the excitation NA (i.e., the incidence angle onto the interface of the spec-
imen and coverslip), which could be adjusted by changing the period of the pattern displayed on the SLM. The pattern generation
algorithm used for patterned activation nonlinear SIM allowed us fine tune the period of the grating pattern to search the GI NA. After
identifying the GI NA, the fluorescent image generated by the applied excitation pattern of each phase and orientation was collected
by the same objective, separated by a dichroic beam splitter (Chroma, ZT405/488/560/647tpc), and focused by a tube lens onto a
sCMOS camera (Hamamatsu, Orca Flash 4.0 v3). The nine raw images consisting of 3-orientation 3 3-phase for each time point were
reconstructed into a super-resolution (SR) image based on the previous algorithm (Gustafsson et al., 2008). In order to suppress the
noise in the high frequency region, we adopted a Gaussian apodization function with standard deviation, s % 45 nm. Changing the
standard deviation allowed flexible adjustment of the cut-off frequency of the reconstructed optical transfer function (OTF) according
to the practical signal-to-noise ratio (SNR) offered by different specimens. This strategy permitted case-by-case quality control to
minimize the reconstruction artifacts, which usually originate from the high frequency region with low SNR.
Interleaved reconstruction
In the conventional reconstruction algorithm, the raw images of each pattern orientation are only employed once to generate a GI-
SIM image. At the next time point, the GI-SIM image is reconstructed from another set of 3 orientation images (upper in Figure S7C).
However, the high resolution information is independently reconstructed for each pattern orientation, and there is no inherent require-
ment for the acquisition order among the 3 pattern orientations. Therefore, regrouping the data stream with either orientation 2 or 3 as
the starting orientation would generate another two GI-SIM images interleaved between two consecutive conventional time points
(lower in Figure S7C). For continuously acquired datasets (i.e., without intervals between frames, e.g., Video S1, part II), we utilized
the interleaved reconstruction strategy (Ma et al., 2018) to improve the GI-SIM frame rate by 3-fold. The advantage of interleaved
reconstruction is that the new temporal information can be incorporated to update the GI-SIM image as soon as it is available in
the raw data of a single pattern orientation. As demonstrated in Figure 1C, the ultra-rapid constrictions and bulges along ER tubules
can be clearly visualized with interleaved reconstruction.
Using Equation (1) and the vector model of diffraction (Richards and Wolf, 1959), we simulated the OTFs at different path length dif-
ferences (i.e., different amounts of defocus; Figure S7E). As shown, defocus distorts the OTF curve. In particular, when the path
length difference is greater than 66% of the emission wavelength in the immersion medium (dDz > 0.66l/n), a sign reversal occurs
in the OTF (blue and red curves in Figure S7E) (l = the emission wavelength in a vacuum, n = the refractive index of the immersion
medium of the objective lens). Thus, the contrast of some features in the acquired image will be reversed, complicating our under-
standing of the detected modulation pattern in the GI-SIM raw images. To avoid this, the maximally allowable path length difference
for a green fluorescence image with a central emission wavelength of 525 nm is dDz = 0.195 mm. This corresponds to the defocus
amount of Dz = 0.614 mm, so the corresponding DOF is 2∙Dz = 1.228 mm.
However, in this work we have used a more restrictive criterion to define the DOF of GI-SIM: the area under the defocused OTF
curve must be greater than half of the area under the in-focus OTF curve (Figure S7F). This standard ensured that the contrast of
each raw image was sufficient for artifact-free GI-SIM image reconstruction (Figure S7G). Using this restricted criterion, the DOF
of a GI-SIM system equipped with a 1.7 NA objective lens was determined as: DOFGI-SIM = 2∙Dz half area = 0.986 mm z1 mm (Figures
S7F and S7G).
where l = 0.488 mm is the wavelength of excitation light, f = 180 mm is the focal length of the tube lens of the GI-SIM system, and rL =
3.43 mm is the radius of the collimated beam at the tube lens. Therefore, the radius of the focal spot is rF = 8.15 mm, which corre-
sponds to a small DNA = 0.0045. Consequently, the focal spot of excitation light actually crosses the critical NA for TIRF, but
most of the excitation light within the focal spot is at an excitation NA just below the critical NA for TIRF. At this pre-critical excitation
NA, the excitation light is refracted at the interface of coverslip and cell specimen with a refraction angle > 85 , and therefore forms the
grazing incidence illumination.
By plotting d as a function of excitation NA, we determined the irradiation depth z at each NA through Equation (3) (Figure S1K). In
particular we found that the irradiation depth at 1.33 NA was very close to the DOF of our GI-SIM system (i.e., the cyan and orange
dashed circles coincided each other, green boxed image in Figure S1K).
Next, we characterized the total fluorescence intensity as a function of the excitation NA (Figure S1L). Similar to the live-cell mea-
surements, the curve could be fitted by the combination of an exponential function for the TIRF region and a linear function for the
non-TIRF region. The intersection of the two curves represents the critical NA for TIRF, which is 1.333 ± 0.002 for a polystyrene
bead submerged in water. Therefore, 1.33 NA is the GI NA for the polystyrene bead specimen.
In summary, the illumination depth at GI NA is well matched to the objective DOF. The extended illumination depth at GI excites
more fluorescent molecules than TIRF, and all these excited molecules, being in focus, contribute to a high contrast raw image.
In particular, compared to the images acquired at the TIRF NA of 1.55, the signal strength of the GI image was generally improved
by > 10-fold (Figures S1B and S1D). GI illumination therefore provides optimal imaging conditions for live-cell imaging.
which usually presented as fluctuations with amplitudes smaller than the GI-SIM resolution (labeled in red in Figure 2D). The velocity
of each growth or shortening phase was calculated as the slope of the linear fitting of the phase. Consistent with the previous
definition (Gierke et al., 2010), the catastrophe frequency was defined as the number of transitions from growth to shortening divided
by the time the MT spent growing. The rescue frequency was defined as the number of transitions from shortening to growth divided
by the time the MT spent shortening.