MOLECULAR AND CELLULAR BIOLOGY, Dec. 2011, p. 4994–5010
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 24
Superresolution Fluorescence Imaging of Mitochondrial Nucleoids
Reveals Their Spatial Range, Limits, and
Timothy A. Brown,1* Ariana N. Tkachuk,1Gleb Shtengel,1Benjamin G. Kopek,1
Daniel F. Bogenhagen,2Harald F. Hess,1and David A. Clayton1
Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, Virginia 20147,1and
Pharmacological Sciences, BHS T-8, Room 140, SUNY Stony Brook, Stony Brook, New York 117942
Received 24 May 2011/Returned for modification 3 August 2011/Accepted 9 October 2011
A fundamental objective in molecular biology is to understand how DNA is organized in concert with various
proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA),
which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise
locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence micros-
copy techniques that exceed the previous limits of imaging within the small and highly compartmentalized
mitochondria have been recently developed. We have improved and employed both two- and three-dimensional
applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core
dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals
that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on
average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the
freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are
closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner
membrane invaginations. Determinations revealing high packing density, separation from the matrix, and
tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA
and that remain largely unknown.
Mitochondria are multifunctional cellular organelles that
maintain and express their own DNA (mtDNA). Of the several
thousand different proteins comprising the mitochondria, only
13 are encoded by mtDNA, which also contains genes for
ribosomal and transfer RNAs necessary for mitochondrial
mRNA translation. Transcription initiates from a minimal con-
trol region, resulting in strand-specific polycistronic RNAs.
The 13 mtDNA-encoded proteins are essential subunits of the
mitochondrial complexes responsible for ATP production
through oxidative phosphorylation. The function of these com-
plexes is coupled to a multitude of processes involved in inter-
secting metabolic pathways, reactive oxygen generation, cal-
cium homeostasis, growth and differentiation, and apoptosis.
The importance of these processes is emphasized by the nu-
merous human genetic disorders associated with nuclear and
mtDNA mutations leading to defects in mtDNA inheritance,
maintenance, and expression (63). The small circular mito-
chondrial genome is unique both in its simplicity of organiza-
tion and in that its expression is coordinated with that of
nuclear-encoded proteins in response to cellular demands and
mitochondrial capacity. mtDNA is organized into structures
called nucleoids (34, 59). There are ?300 to 800 nucleoids in
a cell, each containing ?1 to 10 mtDNAs, RNA, and proteins.
Although the basic enzymology of mtDNA replication, expres-
sion, and repair is reasonably well understood, challenges re-
main in determining how these processes are integrated within
a cell. As with other organisms, the packaging of mtDNA and
the remodeling of nucleic acid-protein complexes are thought
to regulate mitochondrial genetic events and their signaling
pathways. Nucleoids are associated with proteins that are in-
volved with mtDNA maintenance and expression, along with
others that have no obvious relationship with mtDNA. The
latter include those involved in protein stability and folding,
mitochondrial import, RNA helicases, amino acid, carbon, and
lipid metabolism and others with inner mitochondrial mem-
brane locations (8, 19). In Saccharomyces cerevisiae, the nucle-
oid is thought to have a dynamic protein composition that
responds to various cellular conditions while enrolling alterna-
tive functions of metabolic proteins (31). Although the yeast
paradigm might generally apply to mammalian mtDNA, the
evolutionary patterns with respect to their mitochondrial ge-
nomes, metabolic needs, and nucleoid compositions are dis-
Mitochondrial transcription factor A (TFAM; also known as
mtTFA) is regarded as the predominant core nucleoid protein.
In mammals, this HMG box protein binds to mtDNA with high
affinity at mtDNA promoters, where it functions as a transcrip-
tion factor. It also binds DNA less specifically while function-
ing as an mtDNA packaging factor and is one of the few
proteins with a clear structural role within the nucleoid. At
maximum capacity, there are potentially ?450 TFAM binding
sites on the circular ?16-kb mammalian mtDNA (27). TFAM
* Corresponding author. Mailing address: Howard Hughes Medical
Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ash-
burn, VA 20147. Phone: (571) 209-4000, ext. 3026. Fax: (571) 209-
4059. E-mail: firstname.lastname@example.org.
?Published ahead of print on 17 October 2011.
† The authors have paid a fee to allow immediate free access to
localizes almost exclusively to nucleoids and is unstable in
mitochondria without mtDNA (32, 33). The high DNA binding
capacity and nucleoid-specific localization are properties of
TFAM that have been useful in this work.
High-resolution imaging of submitochondrial structures has
been achieved by electron microscopy (EM), which is man-
dated by spatial constraints within the organelle. Mammalian
mitochondria are compartmentalized by outer and inner mem-
branes, which form intermembrane spaces (IMS) that are typ-
ically only 20 to 50 nm wide (16). mtDNA was initially imaged
using EM and was described as embodying elongated fibrils or
rods residing within electron-lucent regions of the mitochon-
drial matrix (39). Unfortunately, the appearance of mtDNA
was very much dependent upon the sample processing method,
leaving uncertainty about the native organization of mtDNA.
Iborra et al. later used immunogold EM analysis and reported
that mammalian nucleoids are spheres 65 to 70 nm in diameter
(24). Although that range represents a reasonable approxima-
tion, immunogold EM-labeled structures lack dimensional res-
olution. Nucleoids may have been directly imaged by EM ac-
cording to a more recent report (47), but that study lacked any
experimental evidence that the putative nucleoid structures
contained mtDNA. Fluorescence microcopy has also been use-
ful for producing images that reveal nucleoid location and
frequency and for observing the dynamics of fusion and fission
(6, 19, 54). However, in conventional fluorescence microscopy,
the diffraction of light restricts the image resolution of objects
to those that are more than 200 to 350 nm apart. This limit of
diffraction has been surpassed using both two-dimensional
(2D) (7, 22, 28, 52) and three-dimensional (23, 25, 55, 61)
approaches. Other novel strategies continue to advance the
field of superresolution microscopy at a rapid pace (56).
Two-dimensional photoactivated localization microscopy
and three-dimensional photoactivated localization microscopy
(PALM and iPALM, respectively) are two of the fluorescence
techniques capable of locating objects at resolutions of 20 to 50
nm. This is accomplished through the use of photoactivatable
fluorescent proteins (PA-FPs), which are activated stochasti-
cally and at low frequency rates in order to achieve spatial
separation of their diffraction-limited fluorescent images. The
location of each PA-FP molecule is determined with high pre-
cision by fitting the images to 2D Gaussian profiles (7). In
iPALM, interferometry is added to locate molecular positions
in the z dimension (58). Here we describe how the use of
hyperbolic mirrors facilitates discrimination of multiple inter-
ferometric fringes and extends the measurement range along
the z axis from ?250 nm to ?750 nm. We used this extended-
range iPALM method to visualize mitochondrial nucleoids in
500-nm-thick sections. We also describe how imaging of gold
nanoparticles in both channels allows two-color PALM image
registration and accurate estimation of colocalization errors.
Two-color 2D PALM is applied to determine the relative lo-
cations of mitochondrial nucleoids, matrix proteins, and cristae
with unprecedented spatial resolution.
MATERIALS AND METHODS
Plasmid construction and mtDNA analysis. For inducible gene expression, we
used the GeneSwitch system (Invitrogen). A commercial pGeneV5/His plasmid
was modified by inserting an AscI site into the multicloning sequence between
the EcoRI and BamHI sites. The AscI linker was created by hybridizing oligo-
nucleotides 5?-GATCCACTAGTGGCGCGCCAG-3? and 5?-AATTCTGGCG
CGCCACTAGTG-3? to yield single-stranded overhangs suitable for ligation.
The AscI-bearing plasmid (pGS) was then used to subclone mEos2 and Dronpa
fragments into the NotI and AgeI sites, resulting in the plasmids pGS-mEos2 and
pGS-Dronpa. The mEos2 cloning fragment was amplified by PCR using primers
5?-AACTGCGGCCGCATGAGTGCGATTAAGCCAG-3? and 5?-GCAGACC
GGTTATCGTCTGGCATTGTC-3?. The Dronpa cloning fragment was ampli-
fied by PCR using primers 5?-AACTGCGGCCGCATGGTGAGTGTGATTAA
ACCAG-3? and 5?-GCAGACCGGTTACTTGGCCTGCCTCG-3?. TFAM and
LACTB sequences were ligated upstream and in-frame using the AscI and NotI
sites of pGS-mEos2 and pGS-Dronpa, respectively. The TFAM fragment was
PCR amplified using primers 5?-CATTACGGCGCGCCAGTAATGGCGCTG
TTCCGGG-3? and 5?-TAGATGCGGCCGCAATGCTCAGAGATGTCTCC-
3?. The LACTB fragment was amplified by PCR using primers 5?-CATTACGG
CGCGCCAGTCATGTACCGGCTCCT-3? and 5?-TAGATGCGGCCGCTGG
ACTGAATGGGGACGGC-3?. All constructs were analyzed by sequencing.
TFAM has a T-to-C transition at nucleotide position 643, resulting in a lysine-
to-glutamate substitution at amino acid position 215. The cytomegalovirus
(CMV) promoter-driven CoxVIII1–29-Dronpa and CoxVIII1–29-Eos plasmids were
previously described (7, 61). To determine the average number of mtDNA
molecules per cell, 3T3sw cells were harvested during the late log phase of
growth and counted using a Vi-CELL XR analyzer (Beckman Coulter). Total
DNA was prepared from 100,000 cells by the use of 10 independent samples and
the method of Legros et al. (33). To account for the efficiency of mtDNA
recovery, half of the cell samples were spiked with a known amount of reference
plasmid DNA containing the entire mouse mtDNA genome (p501-1). Whole-cell
DNA preparations were then diluted, and mtDNA was quantified against a
standard curve using the p501-1 plasmid in a quantitative PCR (qPCR) assay as
previously described (9).
Cell culture. The mifepristone-inducible 3T3 Switch (3T3sw) mouse fibroblast
cell line was purchased from Invitrogen. These cells express a transcriptional
activator protein consisting of the yeast GAL4 binding domain, a progesterone
ligand binding domain, and a p65 activation domain from NF-?B. Cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM)–10% NCS–1 mM
sodium pyruvate–2 mM L-alanyl-L-glutamine (GlutaMax)–50 ?g/ml hygromycin
B (Gibco/Invitrogen). The inducible expression plasmids carrying TFAM-mEos2
and LACTB(1–68)-Dronpa were transfected into 3T3sw cells by the use of
Amaxa Nucleofector as previously described (10). Stably transfected cell lines
were maintained under conditions of selection using Zeocin (200 ?g/ml). Gene
expression was induced with mifepristone (150 to 200 pM) for 7 to 8 h. For
two-color PALM, the stable cell lines were transiently transfected with the
appropriate second expression plasmid. The regulated genes were induced with
mifepristone 20 h after transient transfection was performed as outlined above.
Confocal microscopy and analysis. Cells were grown in Lab-Tek II chambers
with no. 1.5 borosilicate coverglass bottoms (Nunc) coated with human fibronec-
tin (Millipore) (15 ?g/ml) overnight at 4°C. For mitochondrial imaging, cells
were stained with 0.050 mM MitoTracker Red CMXRos (Invitrogen) in com-
plete media for 15 min at 37°C, rinsed once with dye-free media, and imaged live.
For mtDNA staining, cells were stained with PicoGreen (Invitrogen) diluted
1:500 in complete media for 30 min at 37°C and then incubated in dye-free media
for 1 h. Colocalization by immunofluorescence was accomplished by fixing cells
with 2% paraformaldehyde in 60 mM piperazine-N,N?-bis(2-ethanesulfonic acid)
(PIPES)–25 mM HEPES–10 mM EGTA–2 mM MgCl2, pH 7.0, for 15 min at
37°C, blocking in 5% goat serum, and incubating with anti-single-stranded DNA
(anti-ssDNA) monoclonal antibody (clone BV16-19 [Millipore]) diluted 1:4,000
in 0.25% Triton X-100–phosphate-buffered saline (PBS) overnight at 4°C. Cells
were then reacted with an AlexaFluor 568 goat anti-mouse IgG secondary anti-
body (Invitrogen) diluted 1:3,000 in 0.25% Triton X-100–PBS for 1 h at room
temperature. Cells were imaged using a Zeiss LSM 510 META microscope
equipped with a 100? 1.4-numerical aperture (NA) Plan-Apochromat objective.
A 15-mW DPSS 561-nm diode laser was used for excitation of MitoTracker Red
and Alexa-Fluor 568. The 488 line of a 30-mW multiline gas argon laser was used
for excitation of TFAM-mEos2, PicoGreen, and LACTB1–68-Dronpa. All images
represent 1,024-by-1,024, 12-bit z-stack acquisitions created using sequential
scanning and 0.8-?m steps. Volocity software (Perkin Elmer) was used to identify
and count nucleoids by the following protocol: objects are found using intensity
levels and clipped to region of interest, and touching objects are separated by 0.2
?m3. Microsoft Excel was used for basic statistical analysis.
Cell fixation and cryosectioning. Cells in T flasks (75 cm2) were washed with
PBS and fixed in monolayers with 10 ml of freshly prepared 4% paraformalde-
hyde–0.2% glutaraldehyde–100 mM sodium phosphate (pH 7.4). Initial fixation
for 15 min at 37°C was followed by an additional hour with fresh fixative at room
temperature. Cells were washed with PBS and incubated with 0.05 M glycine–
VOL. 31, 2011SUPERRESOLUTION IMAGING OF MITOCHONDRIAL NUCLEOIDS4995
PBS for 15 min and then scraped from the flask surface into PBS with 1% bovine
serum albumin (BSA), centrifuged at 2,000 ? g for 5 min, harvested, and
repelleted in a 1.5-ml microcentrifuge tube. For embedding and cryosectioning,
cell pellets were suspended in 75 ?l of warm 10% gelatin. Cooled gelatin frag-
ments were infiltrated overnight at 4°C with 2.3 M sucrose–100 mM sodium
phosphate (pH 7.4). Samples were mounted onto specimen pins, immersed in
liquid nitrogen, and transferred to a precooled cryochamber affixed to a micro-
tome. Interferometric PALM data were also collected from samples embedded
in LR White resin. This modified Tokuyasu method performed using LR White
resin embedding was previously described (10).
Two-dimensional superresolution microscopy. Basic PALM methods were
described previously (57). For typical TFAM-mEos2 data collection, images
were taken at intervals of 50 ms, and 15,000 to 25,000 frames were collected at
a 561-nm laser power of approximately 1,000 W/cm2. For typical Dronpa PA-FP
data collection, images were taken at 100 ms, and 4,000 to 8,000 frames were
collected at a 488-nm laser power of approximately 500 W/cm2. Molecule local-
ization accuracy is represented here as the standard deviation of the position
assignment value (?). In these experiments, the average ? for Eos was 3.0 nm and
for Dronpa was 3.1 nm. To determine whether nucleoid dimensions are altered
by expression of TFAM-mEos2, we compared molecular maps from PALM to
those obtained using antibody-based dSTORM imaging (21). For dSTORM
imaging, nucleoids were labeled with a monoclonal antibody (clone BV16-19) as
described above. A 1:8,000 dilution of an Alexa Fluor 568-labeled goat anti-
mouse IgG antibody (Invitrogen) was used for the secondary labeling. dSTORM
imaging of 150-nm-thick cryosections was done using PBS containing 100 mM
mercaptoethylamine and a 488-nm laser at ?3 kW/cm2. Approximately 6,000
images were obtained, with frames acquired every 20 ms. Half-maximum histo-
gram peak widths determined from two perpendicular axes were used to deter-
mine nucleoid diameters for dSTORM and PALM analyses as shown here (see
Measurement, calibration, and validation of z-coordinate positions in iPALM.
Basic iPALM methods were described previously (58). For typical TFAM-mEos2
data collection, images were collected at 12.5 frames/s with an excitation time of
50 mS, collecting 20,000 to 50,000 frames at a 561-nm laser power of approxi-
mately 2,000 W/cm2. Molecule localization accuracy is reported here as the
standard deviation of the position assignment value (?). In these experiments,
the average accuracy values for the x, y, and z dimensions were 11.9 nm, 10.1 nm,
and 10.5 nm, respectively. The z determinations of the iPALM technique in this
work have been improved by extending the range to ?750 nm from a previous
limit of ?250 nm. Hyperbolic mirror parameters were adjusted in order to
ensure that the ellipticity point-spread functions (PSF) depended on the emitter
z coordinate. Modeling suggested that the mirror curvature radius required to
ensure this dependence of PSF ellipticity on z-coordinate values is on the order
of 105cm. The hyperbolic mirrors impart weak saddle-shaped wavefront distor-
tions to infinity-corrected beams at the back focal planes of the microscope
objectives in the iPALM setup. As a result, the diffraction-limited images of
single fluorescent molecules become elliptical, with the degree and angle of
ellipticity being functions of the z coordinate. This allows discrimination of
adjacent interferometric fringes and extension of the measurement range from
?250 nm to ?750 nm. Astigmatic point-spread functions (PSF) can be approx-
imated by determinations performed using 2D Gaussian profiles with different x
and y widths. The x-y ellipticity of the PSF can be defined as follows:
where ?xand ?yrepresent Gaussian widths along the x and y axes. In a typical
calibration run, we translated the sample with gold nanoparticles over a vertical
range of ?800 nm and recorded the fluorescent images every 8 nm. We then
performed 2D Gaussian fitting and extracted x-y ellipticity values as defined in
equation 1. The z coordinate could then be extracted in the following manner.
During the iPALM calibration, the dependence of x-y ellipticity on the sample z
coordinate (see Fig. 3) is recorded along with the standard iPALM interfero-
metric calibration (58). The polynomial fitting is performed according to the
z ? m1? m2? ε ? m3? ε3
where ε is the x-y ellipticity of PSF as defined in equation 1 and m1, m2, and m3
are the fitting parameters determined from the calibration step. Then, during the
iPALM measurements, we extract two values: zinterf(which is the z coordinate
determined from the recorded interferometric patterns as described in reference
58), and Zε(which is the z coordinate determined using equation 2 from the
recorded value of ε and m1, m2, and m3determined during calibration).
The z coordinate is then calculated using the following formula:
z ? zinterf? zinterf.fringe? Round?
where the constant zinterf.fringerepresents a single interferometric fringe value of
The assignment of the z coordinate was validated using biotinylated Eos
fluorescent protein (FP) molecules bound to the surface of a coverslip. No. 1.5
coverglasses (Werner Instruments) were cleaned and coated with 0.1% poly-L-
lysine for 30 min, rinsed, and dried. Gold nanorod particles (NanoPartz) were
then applied by application of a 0.5% solution for 5 min. After being rinsed, the
glass was coated with biotin (1 mg/ml)–BSA–PBS (Sigma Chemical A8549)
followed by streptavidin–PBS (Sigma Chemical S462) (1 mg/ml solution). Biotin-
conjugated mEos (gift of George Patterson) was then bound to the streptavidin
at 3.2 ?g/ml for 5 min. We then took a wide-field (20 ?m by 20 ?m) image of the
molecules while translating the sample along the z axis in 8.75-nm steps (pausing
for 99 frames at each step). The entire sample excursion along the z axis was
?750 nm (or 3 interferometric fringes). We then extracted first interferometric
and then full z coordinates for each detected FP molecule (total count, ?85,000)
by the use of equation 3 and the procedure described above. Data from biological
samples were obtained using 500-nm-thick sections and both the modified
Tokuyasu method and the LR White resin-embedded samples as described
Alignment of two-color PALM images and evaluation of alignment errors.
Two-color superresolution imaging requires a way to differentiate and align two
fluorescent labels. The colocalization of fluorescent labels in 2 channels is de-
termined by the following equation (44):
? ? ??loc1
where ?loc1and ?loc2represent the localization accuracies in each color channel
and ?regrepresents the accuracy with which positions between 2 channels can be
registered. Assuming the simplest case with no chromatic aberrations, the rela-
tionship between the images collected in the 2 color channels should be revealed
by a similarity transformation involving shift, rotation, and slightly different
Y?? ? M?cos ?
? sin ?
where M represents magnification.
In order to determine the transformation parameters in equation 5, we used
100-nm-diameter gold nanoparticles (Microspheres-Nanospheres, Cold Spring
Harbor, NY), which exhibit fluorescence in both channels. These particles exhibit
surface plasmon resonance-enhanced photoluminescence with wide spectra in
the visible region (14, 62). The spectra of individual nanoparticles deposited on
glass coverslips with excitation at 488 nm and 561 nm were determined using a
SPEX 500 spectrometer and averaged over a range of at least 5 nanoparticles in
each case. In order to evaluate the two-color registration accuracy (?reg), we
deposited gold nanoparticles on a standard coverslip and imaged them in both
channels, collecting 5,000 frames from each channel. We used Semrock RU-568/
FF01-593-40 and RU-488/FF01-520-35 long-pass and band-pass filter pairs (the
same filters used in PALM measurements for fluorescent protein localization) to
reject the excitation signal and limit the emission spectra to relevant wavelength
ranges. We then performed the standard localization procedure for all fiducials
within a field of 25 by 25 ?m in both channels. For each nanoparticle, we then
determined the x and y coordinates in each channel, determining the averages of
the distributions over 5,000 frames. The pairs of coordinates determined for each
nanoparticle in the 2 channels should satisfy equation 5, so we can use the linear
regression procedure to extract the transformation coefficients.
First, it is convenient to use complex variable Z ? X ? jY (where j represents
complex unity), so we can rewrite equation 5 as follows:
Z? ? Mc? Z ? ?
where Mc? Mej?, ? ? ?x? j?y, and Mcrepresents complex magnification. We
can derive a least-square optimization (linear regression) procedure for finding
i ? 1
??* ? Mc
4996 BROWN ET AL.MOL. CELL. BIOL.
i ? 1
The least-square condition requires the derivatives to be zero. Taking the com-
plex conjugates of equations 7 and setting them to 0 gives regression conditions
represented by the following equations:
i ? 1
i ? 1i ? 1
i ? 1
Zi? N ? ?
*? ? ??
i ? 1
One can then solve the system shown above by linear decomposition to get the
values of Mcand ?.
In order to evaluate the 2-channel registration accuracy (?reg), we again
determined the averaged x and y coordinates of each nanoparticle in each
channel after transformation. Then we calculated the differences between the
averaged x and y coordinates in 2 channels for each nanoparticle.
EM analysis of mitochondrial matrix dimensions. To determine the average
dimensions of the mitochondrial matrix, 3T3sw and 3T3sw cells expressing in-
ducible LACTB1–68-Dronpa were harvested and fixed as outlined above. Cell
pellets were washed with 1% BSA–0.1 M phosphate buffer (PB) (pH 7.4) and
resuspended in 0.1 M sodium cacodylate buffer prior to immobilization of pel-
leted cells in 1.2% agar–PB at 44°C. Agar pellet fragments were secondarily fixed
overnight in 2% paraformaldehyde, washed three times with cacodylate buffer,
and postfixed with reduced 1% OsO4for 1 h. Samples were again washed with 0.1
M sodium cacodylate, stained for 1 h in 1% uranyl acetate, dehydrated stepwise
in ethanol, and embedded in Epon. EM images were obtained using an FEU
Tecnai G2 20 Twin transmission EM (TEM) at magnifications of ?5,000 to
?11,500. The average matrix space between cristae and the outer edges was
determined using multiple measurements from 80 different mitochondria and
SigmaScan Pro (Systat). Measurements of distances between various membranes
were taken at 2 to 6 perpendicular locations for each.
Immunogold particle electron microscopy. A stable 3T3sw cell line carrying an
inducible LACTB1–68-Dronpa gene was induced, fixed, and frozen as described
above. Cryosections (60 nm thick) were cut and placed on Pioloform-coated,
carbon-stabilized 200-mesh nickel grids. The sections were washed three times
on drops of PBS (pH 7.4) for 5 min with shaking. The sections were then
incubated in 0.05 M glycine–PBS for 15 min followed by a 20-min incubation in
a blocking solution of 1% BSA, 0.1% Tween 20, 0.1% Triton X-100, and PBS
(pH 7.4). The primary anti-Dronpa-green antibody (MBL, Nagoya, Japan) was
diluted 1:20 in blocking buffer and incubated on sections for 2 h, followed by
three 10-min washes in 0.1? blocking buffer. This was followed with 60 min of
incubation with a goat anti-mouse secondary antibody conjugated to a 10-nm-
diameter gold particle (Ted Pella, Redding, CA), three 10-min washes with 0.1?
blocking buffer, and two 5-min washes with PBS. Sections were fixed with 1%
glutaraldehyde–PBS for 15 min, followed by two 5-min washes in PBS and five
3-min washes in distilled water. Lastly, the cryosections were stained with 0.5%
uranyl acetate–2% methylcellulose for 10 min on ice.
Immunogold labeling was analyzed as described by Rabouille (49). Images of
labeled cells were taken at a magnification of ?29,000 for a field of view of
3.26 ?106/nm2with a Tecnai T20 TEM (FEI) operated at 80 kV. The surface
areas of mitochondria, nucleus, and cytoplasm were determined for each elec-
tron micrograph using the point-hit method and ImageJ software (http://imagej
.nib.gov/ij/) to overlay the micrograph with line spacing at 100 nm, representing
a surface area of 10,000 nm2. For submitochondrial localization, we used a
finer-mesh grid such that each point represented 5,000 nm2. The relative labeling
density for each organelle and compartment was determined by dividing the
number of gold particles located on the organelle and compartment by the
surface area as determined by the point-hit method. Nuclear labeling was used as
the background signal, and specific gold labeling was determined by subtracting
the background labeling from the relative labeling density.
TFAM-mEos2 is incorporated into nucleoids without appar-
ent mitochondrial alteration. To label nucleoids, TFAM
(NM_009360.4) was fused into the mEos2 translational reading
frame (FJ707374.1) encoding PA-FPs with green to red fluo-
rescence (36). To limit effects of overexpression, we used the
GeneSwitch system (Invitrogen), which employs an inducible
synthetic promoter. We have also established mouse 3T3
Switch (3T3sw) fibroblasts that stably express the inducible
TFAM-mEos2 gene in order to limit the copy number effects
inherent in transfection. The cells retain the native TFAM
gene. It is difficult to unequivocally state that heterologous
gene expression is without consequence with respect to unin-
tended effects. However, we have made efforts to determine
whether the TFAM-mEos2 fusion protein significantly alters
the relevant properties of the mitochondria.
Figure 1 displays confocal microscopy images showing that
TFAM-mEos2 accumulates at punctate foci within the mito-
chondria. The pattern of these foci is consistent with nucleoid
images obtained by others (19). Mitotracker red staining
showed that the foci are limited to the mitochondria and that
the reticular morphology of the mitochondria was retained
(Fig. 1B to D). This indicates that TFAM-mEos2 expression is
well tolerated, as fragmentation of mitochondria is often asso-
ciated with distressed mitochondrial function and apoptosis
(29). TFAM-mEos2-labeled nucleoids were counted and com-
pared to the number of nucleoids in control cells stained with
the DNA-binding dye PicoGreen (4). The number of TFAM-
mEos2 foci per cell (mean ? 291) was similar to the nucleoid
count in PicoGreen-stained 3T3sw cells not expressing the
fluorescent fusion protein (mean ? 275 [Table 1]). Anti-DNA
antibody immunostaining showed extensive colocalization with
TFAM-mEos2 (Fig. 1E to H). In addition, induction of
TFAM-mEos2 expression did not alter either the mtDNA
amount determined using qPCR or the mitochondrial mor-
phology, as seen in electron micrographs of EPON-embedded
cells (data not shown).
To determine whether TFAM-mEos2 expression affects nu-
cleoid structure, we compared these nucleoid dimensions to
those seen in the absence of TFAM-mEos2 expression. This
was done using dSTORM (21) imaging of nucleoids labeled
with an antibody to DNA. TFAM-mEos2- and anti-DNA an-
tibody-labeled nucleoids are similar in size (Table 1). Thus, it
does not appear that expression of TFAM-mEos2 protein
leads to abnormal packaging of nucleoids. Under the condi-
tions used, dSTORM imaging suffered from a comparatively
low labeling density. Therefore, the resolution of nucleoid
structure determinations performed using dSTORM is less
than that seen with PALM.
PALM and iPALM image display and dimension measure-
ment. The distribution of TFAM-mEos2 molecules within a
nucleoid obtained using PALM can be displayed in several
ways, as shown in Fig. 2. Final PALM data consist of a distri-
bution of points representing assigned locations of molecules
of a fluorescent protein (7). Panel A in Fig. 2 represents the
simplest molecule distribution using point assignments. PALM
images are most often displayed as a space- and color-filled
distribution that expresses the statistical certainty of the as-
signment of each molecular location. An example of such a
probability map is shown in Fig. 2C. Each of the probability
density values represents the probability of an observed mo-
lecular label occurring in a given area. These values are derived
from the Gaussian profile determined for each molecular event
imaged and are reported as the probability divided by the area
VOL. 31, 2011 SUPERRESOLUTION IMAGING OF MITOCHONDRIAL NUCLEOIDS4997
in square nanometers. For example, a point of light with di-
mensions of 20 by 20 nm would be binned over a grid with
dimensions of 20 by 20 nm, with each grid cell value calculated
at 1/400 per nm2, or 0.0025/nm2, as the maximum probability
value for this event. A broader Gaussian function would yield
a lesser value, reflecting the broader area of each grid space. In
the rendered image, each space is given a color on a gradient
that reflects the probability value for each event. These are
then overlaid on a single image. The utility of this image is that
it supplies further information about the quality of the centroid
point assignments. This probability map display is used in the
remaining figures of this work, and a detailed description of
the rendering algorithm appears in earlier work (7) (see sec-
tion 6 of the online supporting information associated with that
work). In images that are densely labeled, the data represent-
ing the localization probability of a given individual molecule
are lost in the summed projection. However, this positional
information is preserved and used in the histogram analysis.
Figure 2B displays a histogram that plots the frequency of the
same molecules at a given position along a single axis. Impor-
tantly, the width of the frequency histogram at half the maxi-
mum peak value (full-width half-maximum of the histogram
peak height; FWHM) represents a statistically robust and con-
servative measurement of the width of the nucleoid in this
dimension. The use of FWHM measurements applied to
PALM has previously been validated using cytoskeletal fibers
with known dimensions (58).
Extension of iPALM measurement range to 750 nm by the
use of hyperbolic mirrors. The original iPALM method was
able to achieve highly accurate lateral (x and y) localization of
fluorescent particles and a vertical z localization range of ?250
nm. Extension of this vertical z range is based on the principle
that point sources of light correspond to point-spread func-
TABLE 1. Properties of 3T3sw mitochondrial nucleoids,
mitochondria, and relative DNA packing density
Property Value Basis or reference
Mean no. of nucleoids/3T3sw
PicoGreen DNA staining
TFAM-mEos2 (green state)
Mean no. of mtDNA copies/
Mean nucleoid vol (nm3)
Mean matrix space between
Mean matrix diam (nm) (?SD)e
Fig. 4 830,000
ECV304 (human) mt nucleoid
P. polycephalum mt nucleoid
3T3 (mouse) mt nucleoid
3.3 ? 109bp/113 ?m3
4.64 ? 106bp/0.9 ?m3
Nucleoid diam (nm)
2D PALM (TFAM-mEos2)
dSTORM (DNA antibody)
aData were determined by whole-cell confocal microscopy.
bData were determined by qPCR.
cAverage mtDNA genomes/cell divided by average nucleoids/cell.
dData were determined by measurements from EM images using the formula
(mitochondrial length/number of cristae) ? average crista width (21.5 nm); n ?
eData were determined from 60 width measurements taken between inner
boundary membranes; sample range, 115 to 430 nm.
fData were determined using the equation ?pack?
represents the number of base pairs and ? represents the package volume in
cubic nanometers. mt, mitochondrial.
gData were determined using averages of 3 genomes/nucleoid and the equa-
tion shown in footnote f.
, where Nbp
FIG. 1. Genetically expressed TFAM-mEos2 localizes to mtDNA. (A and E) Confocal images of cells displaying the green-state
fluorescence of TFAM-mEos2. (B) Mitochondrial staining with Mitotracker Red. (C) Merged images from panels A and B, with box
outlining the magnified region displayed in panel D. Arrows in panel D indicate yellow nucleoids within mitochondria. (F) Alexa-Fluor 568
immunofluorescence staining with an anti-DNA antibody. (G) Merged images from panels E and F, with box outlining the magnified region
displayed in panel H. Left and right arrows in panel H point to nucleoids with lesser and greater TFAM-mEos2 signals, respectively. Bars,
10 ?m (A and E), 5 ?m (D and H).
4998 BROWN ET AL.MOL. CELL. BIOL.
tions (PSF) that can be made elliptical using hyperbolic mir-
rors. Figure 3A shows that the ellipticity of the resulting x-y
PSF is dependent on the location of the light source in the z
dimension. Thus, the ellipticity can be used to determine the z
coordinate of a particular fluorescent molecule. Figures 3B
and C show that the procedure works well over a range that is
greater than ?750 nm, which is close to the depth of field of
the high-NA objective used in the experiment. There was a
small fraction of molecules for which the interferometric fringe
was determined incorrectly (faint traces 250 nm above and
below the center trace in Fig. 3C). The total fraction of these
ghosts, or the error ratio, was 0.75%, and it did not exceed 5%
for any z-position.
Dimensional analysis of ellipsoid-shaped nucleoids deter-
iPALM of TFAM-mEos2-defined nucleoids reveals that about
65% of nucleoids have roughly ellipsoidal shapes. Examples of
three ellipsoidal nucleoids are shown in Fig. 4, where the
TFAM-mEos2 distributions are displayed in two-dimensional
projections, along with a three-dimensional drawing of each
nucleoid with FWHM measurements of each dimension. The
nucleoid shown in Fig. 4D to F has dimensions that are close
to the average within the ellipsoid subset. The short, middle,
and long nucleoid axis measurements are shown as distribu-
tions in Fig. 5A. Ellipsoidal nucleoid dimensions differ over a
10-fold range from shortest to longest dimension (31 to 318
nm). The shapes of the ellipsoids also differed. However, most
nucleoids have either three unequal dimensions or one dimen-
sion that is larger than the other two. The mean values of the
three major axes are 85 by 108 by 146 nm, as shown in Fig. 5A.
The ellipsoidal subset was also used to estimate nucleoid vol-
ume, and that distribution is shown in Fig. 5B. A histogram
plotting the frequency of binned nucleoid volumes reveals that
the distribution is positively skewed. Individual nucleoid vol-
umes, along with the mean value of 830,000 nm3, are plotted in
Fig. 5C. The standard deviation of the mean encompasses a
25-fold range from about 60,000 to 1,600,000 nm3. Giant nucle-
oids above this range exist but are infrequent. A relative esti-
mation of the number of TFAM-mEos2 molecules per nucle-
oid can be approximated using PALM. There is no correlation
between TFAM-mEos2 molecular counts and nucleoid vol-
ume. It is therefore unlikely that the fluorescent molecules
influenced the size of the nucleoids.
Analysis of nucleoid size, relative spatial limits, and esti-
mated mtDNA density. We were interested in learning whether
nucleoid size measurements represented any spatial con-
straints within the mitochondria. Dimensions of mitochondrial
compartments were obtained from EM images of 3T3sw cells
(Table 1). The average diameter of the mitochondrial matrix
between inner boundary membranes measured 238 (?75) nm,
which is sufficient to accommodate most nucleoids. However,
FIG. 2. PALM data representation and measurement of a single
nucleoid defined by TFAM-mEos2 fluorescence localization. (A) Flu-
orescence localization data displayed as a two-dimensional distribution
of points, each representing the centroid position of the diffraction-
limited image of the fluorescent protein (FP) molecule TFAM-mEos2.
(B) Histogram distribution of the FP molecules shown in panel A in
the x dimension. The relative position of each bin is displayed on the
x axis, with marks every 10 nm. Measurement of the nucleoid x axis
width is obtained from the full-width half-maximum of the histogram
peak height (FWHM), shown here as a red line with asterisks. (C) The
same data displayed as a color-coded probability map of FP molecule
locations. The color intensity scale (bottom left) indicates the FP
molecule probability per square nanometer. The FWHM positions are
also shown in panels A and C with white hash marks. For this mole-
cule, the FWHM value for the x dimension is about 125 nm. (A and C)
Bars, 50 nm.
VOL. 31, 2011 SUPERRESOLUTION IMAGING OF MITOCHONDRIAL NUCLEOIDS4999
the mean available matrix space between cristae measured only
68 (? 24) nm, which is inadequate for most nucleoids. The
relative dimensions of the nucleoid and the crista-limited ma-
trix indicate either an intimate association between cristae and
nucleoids or the existence of specialized, crista-free nucleoid
domains within the matrix.
We have also estimated the packing density of mtDNA in
mouse fibroblasts by the use of cell population averages. Table
1 contains a summary of the data used in these calculations.
Quantitative PCR yielded an average value of 821 mtDNA
genomes per cell. Whole-cell nucleoid counts obtained from
confocal microscopy indicated an average of 275 nucleoids per
cell. Therefore, there are, on average, approximately 3 mtDNA
molecules per nucleoid in 3T3sw cells. Although carcinoma
cells typically yield higher estimates, our determined value is
consistent with a previous estimation of 2.4 genomes per nu-
cleoid in fibroblasts (33). The DNA packing density (?pack) is
determined by dividing the volume of a DNA cylinder of
known length by the volume of space that it occupies (48).
Calculated using this method, the mammalian nucleoid has an
average ?packvalue of 0.063, which is similar to the Physarum
polycephalum mtDNA nucleoid ?packvalue of 0.069 (Table 1)
but less than that calculated from measurements made by
Iborra et al. (24). By comparison, Escherichia coli and mam-
malian nuclear genomes, with ?packvalues of 0.031 and 0.005,
are less compact. This level of mtDNA condensation has im-
plications with respect to mechanisms of packaging and
FIG. 3. Point-spread function ellipticity and interferometric z-coordinate display. (A) Three examples of PSF images of gold nanopar-
ticles at different vertical positions are shown. x-y ellipticity is plotted relative to the z position (shown in red). The black line represents the
polynomial fit corresponding to equation 2. (B) Interferometric coordinates are displayed as black dots relative to collected frame number.
(C) The full z coordinate is displayed relative to frame number as the sample is displaced along the z axis. Sample positions from panels B
and C are shown in red.
5000BROWN ET AL.MOL. CELL. BIOL.
iPALM and PALM imaging analysis of variously shaped
nonellipsoidal nucleoids. Although the majority of nucleoids
were roughly ellipsoidal, as described above, the remaining
35% displayed an assortment of shapes. We can loosely cate-
gorize these nucleoids into three equally represented subsets
based on their predominant features. The first category com-
prises nucleoids with either a crescent shape or one side that is
largely concave. Examples of these are shown in Fig. 6A and D.
The second category includes nucleoids that are amorphous or
branched, as displayed in Fig. 6C. The third category consists
of nucleoids that have either an internal gap or are split, as
shown in Fig. 6B and E. It is important that the heterogeneity
in size and shape also applies to the elliptical subset and that
these categories, while not fully discrete, have descriptive
value. The crescent or concave shapes demonstrate that nucle-
oids are not restricted to a globular form and may indicate
structural constraints imposed by mitochondrial membranes.
The amorphous nucleoids show that the TFAM-mEos2 popu-
lation can be unevenly distributed into discrete core domains
with branches that are less condensed. The images of the split
nucleoids are consistent with previous observations of nucleoid
fusion and fission (19). More subtly gapped nucleoids may
encompass protrusions of cristae. Alternatively, uneven distri-
butions of TFAM-mEos2 could represent core TFAM binding
sites, thus resolving discrete mtDNA molecules within a more
loosely organized nucleoid.
Alignment of two-color PALM images and colocalization
accuracy. In this study, we sought to determine the location of
nucleoids relative to those of other mitochondrial compart-
ments. This required the imaging of two fluorescent proteins
within the same sample. Superresolution imaging requires an
accurate and robust method to determine the relative locations
of different fluorescent proteins within the same field. This can
be done by constructing labels with different excitation spectra
and identical emission spectra (5) or by using fluorescent labels
with different excitation spectra (57). In the latter case, PALM
images in 2 channels with different fluorescent wavelengths are
typically not aligned “as acquired,” and images may be shifted
and tilted due to different filter sets used in acquisition and
slight magnification differences of optics at different wave-
FIG. 4. Ellipsoidal nucleoid projections and their graphical dimensions obtained from iPALM imaging of TFAM-mEos2. Panels A to C, panels
D to F, and panels G to I represent three different nucleoids. In panels A, D, and G, each nucleoid image is projected in x (horizontal) and y
(vertical) dimensions. In panels B, E, and H, the z dimension replaces y on the vertical axis. In panels C, F, and I, each nucleoid is graphically
displayed along with measurements of full width at half-maximum (FWHM) in three dimensions. Bar, 50 nm (the scale bar in panel A applies to
all panels). The fluorescent molecule scale maximum values (representing probability per square nanometer) differ among the panels and range
from 0.019 to 0.034.
VOL. 31, 2011 SUPERRESOLUTION IMAGING OF MITOCHONDRIAL NUCLEOIDS5001
lengths. We have used gold nanoparticles as fiducial markers
for dual-label alignment. To characterize the utility of gold
particles for such alignments, we first measured their photolu-
minescence spectra. As can be seen from Fig. 7A, the emission
spectrum of 100-nm gold nanoparticles is fairly broad and
covers the wavelength range of interest. This allows flexibility
in the choice of the fluorescent proteins used. It should be
pointed out that, when one of the fluorescent labels, such as
Alexa 647, produces emissions that are further toward the red
end of the spectrum, the gold particles cannot be used, since
their emission at 650 nm is weak. However, particles with a
properly chosen size/aspect ratio work well (62). An example
of gold particle alignment is displayed in Fig. 7B. This alignment
results in a low localization error, i.e., the distance between aver-
aged coordinates in the 520-nm and 590-nm channels after align-
ment. The distributions of the x and y localization errors are
shown in Fig. 7C. The standard deviations of the x and y local-
ization errors shown in Fig. 7C are 5.1 nm and 6.7 nm, respec-
tively, and thus we can estimate ?reg? ??x
?reg? ?8.4 nm). For the two-color PALM measurement, we
used enough fiducials to follow the same procedure, which
allowed us both to perform image alignment and to estimate
dual-label registration accuracy (?reg), which is typically be-
low 10 nm. This high degree of accuracy of two-color image
alignment is crucial for interpreting colocalization studies at
high resolution, and we have applied this method to the
colocalization of mitochondrial nucleoids within the organ-
elle. Prior to this work, methods for determining two-color
localization accuracy were lacking.
2(in this case,
FIG. 5. Ellipsoidal nucleoid axial dimension and volume distribution. (A) For each nucleoid (n ? 98), a short-, middle-, and long-axis length
is plotted to display the distribution. Bars representing means (? standard deviations [SD]) are plotted above each data set; the long-axis mean
is 146 (?47) nm, the middle-axis mean is 108 (?30) nm, and the short-axis mean is 85 (?27) nm. (B) A 24-bin histogram displaying the frequency
distribution of calculated nucleoid volumes. (C) The individual nucleoid volumes (n ? 98) are plotted as points along the x axis shared with panel
B. The mean (8.5 ? 105nm3) ? SD (7.77 ? 105nm3) is displayed above the raw distribution.
5002 BROWN ET AL.MOL. CELL. BIOL.
TFAM-mEos2 nucleoid location and mitochondrial matrix-
targeted CoxVIII1–29-Dronpa are largely mutually exclusive.
The N-terminal mitochondrial targeting sequence from
CoxVIII (NM_004074.2) is commonly used to deliver fluores-
cent proteins to the mitochondrial matrix and has also been
applied in PALM (7, 51). We used a previously characterized
CoxVIII1-29-Dronpa expression plasmid (61) to deliver the
PA-FP Dronpato themitochondrial
(AB180726.1) encodes a green photoactivatable fluorescent pro-
tein used as a second sequential marker in PALM that is paired
with the green-to-red-fluorescence PA-FP mEos (18, 57). Figure
8 displays examples of 2D PALM imaging data obtained from
cells coexpressing the nucleoid-specific TFAM-mEos2 and the
matrix-specific CoxVIII1–29-Dronpa. The large majority (84%) of
the cells show that the nucleoid and matrix proteins have discrete
distributions (Fig. 8A to E). Gaps between the matrix and nucle-
oid proteins are not uncommon. The remaining 16% show vari-
ous degrees of overlapping expression of the nucleoid and matrix
proteins (Fig. 8F). This could represent a true mixing of the
proteins. However, it is also possible that the two signals were
have been unable to obtain two-color iPALM data to answer the
question of how frequently these proteins truly colocalize. Re-
gardless, the predominantly discrete localization shows that the
nucleoid most often excludes freely diffusing matrix proteins.
Amino-terminal peptides from LACTB faithfully target
Dronpa to the mitochondrial IMS. Prior data indicate that the
protein encoded by LACTB (NM_030717.1) localizes to the
mitochondrial intermembrane space (IMS) within cristae. To
create a marker for the mitochondrial IMS and cristae, we
fused Dronpa to the nucleotide sequence encoding the N-ter-
minal 68 amino acids of LACTB that were previously identified
as the mitochondrial targeting sequence (45). LACTB1–68-
Dronpa is seen exclusively in the mitochondria, according to
the results of confocal microscopy (Fig. 9A, B, and C). As
shown by the use of PALM at higher resolutions, the majority
of LACTB1–68-Dronpa appears on the inside of the mitochon-
dria (Fig. 9D, E, and F). The outside edge of the mitochondria
is not labeled, indicating that neither the outer membrane nor
the boundary IMS exhibits significant fluorescence. Without
further reference structures, this internal labeling is consistent
with a matrix or crista location.
To further specify the location of LACTB1–68-Dronpa, we
performed immunogold-labeling EM with an antibody against
Dronpa (Fig. 9G). The results of immunogold labeling indi-
cated that nearly the entire specific LACTB1–68-Dronpa signal
localized to mitochondria (86% ? 3.4% standard error of the
mean [SEM] [n ? 2,129]). Cytoplasmic labeling, which pre-
sumably included LACTB1–68-Dronpa being translated at
and/or trafficked into the cytoplasm, represented the remain-
ing 14% of specific labeling. The cytoplasmic signal may also
represent some form of labeling in mitochondria that is not
clearly visible in the low-contrast cryosections. Because we
wanted to use LACTB1–68-Dronpa protein as an IMS-crista
marker, we analyzed its submitochondrial localization. The
primary and secondary antibody-gold complex used for immu-
nogold-labeling EM may span a distance of 20 nm; therefore,
a gold particle that is located 20 nm away from a compartment
may still represent detection of an antigen within that com-
partment. Since mitochondrial cristae have an average cross-
sectional diameter of ?30 nm, we assigned a gold particle to a
compartment only when its center was in that compartment
FIG. 6. iPALM imaging reveals that nucleoids are not restricted to ellipsoidal boundaries. TFAM-mEos2-labeled nonelliptical nucleoids exhibit
concave, split, and amorphous forms. The fluorescent molecule scale maximum values (representing probability per square nanometer) differ
among the panels and range from 0.14 to 0.19. Collectively, these examples represent about 35% of the nucleoid population. Bar, 100 nm (the scale
bar in panel A applies to all panels).
VOL. 31, 2011 SUPERRESOLUTION IMAGING OF MITOCHONDRIAL NUCLEOIDS5003
and used a point-hit method to determine specific labeling
densities by adjusting for compartment surface area values (see
Materials and Methods). Using nuclear labeling as background
and subtracting the associated value to determine specific la-
beling, we found that 77% ? 3.6% (SEM) (n ? 1,245) of gold
particles labeled the mitochondrial IMS. The crista IMS was
labeled more frequently than the boundary IMS (48% and
29% of total specific labeling).
are closely associated. To determine the location of nucleoids
relative to the inner mitochondrial membrane, we took advantage
of the IMS localization of LACTB1–68-Dronpa by the use of
FIG. 7. Alignment of dual-label PALM images and colocalization accuracy. Panel A displays emission spectra of 100-nm-diameter gold
nanoparticles. Data representing the emission filters used for registration in the 520-nm channel (Semrock FF01-520-35; green dashes) and 590-nm
channel (Semrock FF01-593-40; orange dashes) are also shown. (B and C) Two-color PALM image alignment. Localizations of gold nanoparticles
at 520 nm (green dots) and 590 nm (red dots) before (B) and after (C) alignment. Bars, 20 nm. Data points were collected from 5,000 frames and
peak coordinates extracted for each channel. (D) Two-color localization error distributions. Distances plotted are between averaged coordinates
in the 520-nm and 590-nm channels after alignment. Localization differences within the given error range are shown in red for the x axis and in
blue for the y axis. The standard deviation for X (?x) is 5.1 nm and for Y (?y) is 6.7 nm.
5004 BROWN ET AL.MOL. CELL. BIOL.
two-color PALM. Figure 10 shows a close association between
the nucleoid TFAM-mEos2 and the LACTB1–68-Dronpa. Two
types of relative positioning are seen. The first is displayed in
Fig. 10A to C, where the nucleoid is immediately adjacent to
the IMS, with various degrees of overlap at their margins. The
second is displayed in Fig. 10D to F, in which the nucleoid
appears to surround a region of IMS. This close proximity
exceeds that most often seen between the matrix and the nu-
cleoid (Fig. 8). Thus, it appears that nucleoids are often either
adjacent to the boundary of crista IMS or partially or fully
wrapped around a crista-like structure. As with the matrix
(CoxVIII1–29-Dronpa) colocalization described above, some
overlapping nucleoid and IMS signals cannot be completely
resolved in the z dimension of the 150-nm-thick sections. In
such cases, the nucleoid and the membrane may reside in
different z planes. In the x and y dimensions, we have deter-
mined that the dual-label image alignment accuracy is high,
with ? values ranging from 22 to 26 nm. The alignment was
quantified using multiple gold fiducials within each field as
Preliminary iPALM measurements of mitochondrial nucle-
oids revealed that the structures were larger than anticipated.
Nucleoids were frequently truncated at the boundaries of 200-
nm-thick sections. To facilitate data collection and achieve
full-length measurements of nucleoids that are larger than 200
nm, we developed and employed a version of the iPALM with
an expanded vertical z-coordinate range. The original iPALM
method allows both high lateral x and y localization and high
vertical z localization of a fluorescent particle over a vertical
range of ?250 nm. Beyond that range, the high precision can
be maintained with the next interference fringe; however, one
cannot discriminate between adjacent interference fringes.
This results in a position ambiguity of n ? zinterf.fringe, where n
represents the integer number and zinterf.fringe(?250 nm) rep-
resents the interferometric fringe.
The problem that the position ambiguity presents may be
overcome by establishing a property that varies with the z
coordinate at a low rate, which would allow discrimination of
adjacent interferometric fringes. This could be done by ana-
lyzing image moments (3), but that approach does not have a
high enough signal-to-noise ratio to work well with relatively
dim fluorescent proteins. Here we replaced the original turning
22.5° mirrors in iPALM with mildly hyperbolic mirrors that
added a saddle-shaped phase shift across the pupil plane. As a
result, the PSF of images of point sources became elliptical,
and, as proposed by Kao and Verkman (26), this ellipticity
should vary with the axial coordinate. The hyperbolic shapes of
the two turning mirrors were matched such that the phase
differences between the two interfering beams remained con-
stant across the pupil plane; thus, the multiphase interferom-
etry critical to the original iPALM method was preserved. The
ellipticity is dependent on the sample z coordinate and can be
used to extract the vertical location of a molecule (Fig. 3). This
method has much lower accuracy than the iPALM technique
but is not limited to a single interferometric fringe. The pro-
cedure can be used to determine the fringe order, which, in
turn, allows accurate determination of the z coordinate over a
Mitochondrial nucleoid dimensions were previously esti-
mated by using anti-DNA immunogold EM to measure the
diameters of clustered particles (24). Those data indicate that
the nucleoids from a human carcinoma are roughly spherical,
FIG. 8. TFAM-mEos2 nucleoid location relative to the mitochondrial matrix-targeted CoxVIII1–29-Dronpa. TFAM-mEos2 nucleoids are
displayed in red. The mitochondrial matrix protein CoxVIII1–29-Dronpa is shown in green. The fluorescent molecule scale maximum values
(probability per square nanometer) differ per panel and range from 0.005 to 0.01 for Dronpa and 0.01 to 0.02 for mEos2. The nucleoid and matrix
proteins have discrete boundaries in most (84%) images, as displayed in panels A to E. These edges are less discrete in the remaining 16%, as
represented by panel F. Bar, 100 nm (the bar in panel A applies to all panels).
VOL. 31, 2011 SUPERRESOLUTION IMAGING OF MITOCHONDRIAL NUCLEOIDS5005
with a diameter that averages 65 to 70 nm and ranges from 31
to 132 nm. In contrast, our data indicate that most (65%)
nucleoids appear ellipsoidal and have a much broader size
range of 31 to 318 nm. The difference in these two measure-
ments is notably at the upper end of the distributions. It is
possible that there are real differences in the nucleoid sizes
between human carcinoma cells and the mouse fibroblasts
used in this study. However, the larger nucleoid sizes detected
with iPALM are perhaps better explained by the 3-dimensional
capacity of iPALM. Immunogold EM is a surface-limited tech-
nique and is sensitive only to those epitopes that are exposed
on a thin section. It is less likely that longer nucleoids would be
exposed on a thin sectional plane that reveals the entire nu-
cleoid length. In contrast, iPALM is a 3-dimensional tech-
nique, revealing fluorescent protein locations within all dimen-
sions of our 500-nm-thick samples. Therefore, we believe that
the larger and more variously shaped nucleoids revealed by
iPALM better represent their true dimensions. This has led us
to reconsider both the relative external and internal organiza-
tions of nucleoids.
We used the dimensions of the examples in the ellipsoidal
subset to estimate nucleoid volumes and found them to vary
over a large range. Previous DNA fluorescence intensity data
indicate that nucleoid sizes reflect their mtDNA content (6).
Although we have not determined numbers of mtDNAs within
individual nucleoids, the distribution of our volumetric data is
FIG. 9. Genetically expressed LACTB1–68-Dronpa localizes to the mitochondrial intermembrane space. (A to C) Confocal images of a single
cell expressing LACTB1–68-Dronpa (A) and mitochondria stained with Mitotracker Red (B); (C) a merged image representing the two labels.
(A) Bar, 10 ?m. (D to E) Two-color PALM images expressing LACTB1–68-Dronpa (green) and the mitochondrial matrix protein encoded by
CoxVIII1–29-mEos2 (red). (D) PALM image showing multiple mitochondria in a cell cross-section; bar, 2 ?m. Five gold fiducial particles are circled
in white. (E and F) High-resolution two-color PALM images of sectioned mitochondria; bars, 200 nm. The fluorescent molecule scale maximum
values (probability per square nanometer) in panels E and F are about 0.0023 for Dronpa and 0.025 for mEos2. (G) Immunogold EM labeling
of cryosections of cells with anti-Dronpa antibody, showing LACTB1–68-Dronpa localization to the mitochondrial inner membrane space. M,
matrix; C, cristae; BM, boundary membrane. Arrows highlight several gold particles localized to cristae.
5006BROWN ET AL.MOL. CELL. BIOL.
consistent with this conclusion. The pattern of volume per
nucleoid distribution shown in Fig. 5A is remarkably similar to
typical distributions of mtDNAs per nucleoid described by
others (11, 42, 54). This correlation is substantiated by the
conversion of volumes to genomes. The average nucleoid vol-
ume in 3T3sw cells was determined to be 830,000 nm3(Fig.
4A). By counting nucleoids and measuring mtDNA content
per cell, we calculate that these cells have an average of 3
genomes per nucleoid (Table 1). If nucleoid volumes scale
primarily with mtDNA content, then a single-genome nucleoid
would have an approximate volume of 276,650 nm3. This con-
version yields a range of 1 to 6 mtDNAs per nucleoid within
the standard deviation shown in Fig. 5 and is congruent with
multiple prior estimates of numbers of genomes per nucleoid
(34). We hypothesize that nucleoid volumes might also be
affected by changes in the packaging of mtDNA. However, in
this analysis of steady-state populations, genome content ap-
pears to have a dominant role in ellipsoidal nucleoid volume
These data also allowed us to determine the degree to which
mtDNA is packaged. Surprisingly, we found that it is more
condensed than both E. coli and mammalian nuclear DNA.
Previous studies have led to the popular view that mtDNA is
loosely packaged. EM imaging and psoralen cross-linking ex-
periments indicated that only the noncoding control region of
mtDNA is densely bound by protein (2, 13, 40, 46). More
recently, the packaging of mtDNA has been reconsidered.
TFAM clearly has mtDNA maintenance functions separate
from transcription and exhibits the capacity to condense DNA
in vitro (15, 27). However, its contribution to packaging is a
subject of current controversy. One argument in this debate is
that TFAM is abundant enough to saturate the mtDNA and
effectively packages DNA at high protein/DNA ratios (1). The
counterargument is that intracellular TFAM levels are, in fact,
insufficient to cover mtDNA and that full binding is incompat-
ible with transcription (12, 35). Considering the very high
mtDNA ?pack value calculated from our data, the most
straightforward conclusion is that, within the ellipsoidal nucle-
oids, mtDNA is maximally bound by TFAM. This may in fact
be the case, and this condensed structure would exclude the
possibility that the core nucleoid is sparsely bound at the
steady state. However, our data also led to a caveat. The high
packing density of mouse fibroblast mtDNA is similar to that
found in the mitochondrial nucleoid of the slime mold P.
polycephalum. This was unexpected, as there are major differ-
ences in the mtDNA packaging proteins used by these organ-
isms. Mammalian TFAM condenses DNA via two HMG box
domains. In addition to these domains, the P. polycephalum
TFAM homolog (Glom) contains additional lysine-rich and
proline-rich domains that are responsible for maintaining this
highly compact yet functional mtDNA (53). Without these
domains in TFAM, it is unclear how mammalian nucleoids
would achieve the same mtDNA packing density. Further-
more, although yeast Abf2 and mammalian TFAM have the
ability to condense DNA in vitro, mathematical modeling and
volume estimates of numbers of fully bound DNA in vitro
appear to be insufficient to account for this large ?packvalue
(17, 27).More recently, methylation protection analyses con-
current with altered TFAM expression indicate that mtDNA is
unlikely to be fully bound by TFAM (50). If this is the case,
then an alternative mechanism of mtDNA condensation is
required to reconcile these data. It is possible that there are
additional strategies for packaging mtDNA within the nucle-
oid. If other packaging mechanisms are present and their rel-
ative contributions are unknown, then full TFAM binding is
not an obligatory feature. mtDNA condensation resulting from
FIG. 10. TFAM-mEos2 nucleoid location relative to the IMS targeted LACTB1–68-Dronpa. Two-dimensional, dual-label PALM images of cells
expressing inducible TFAM-mEos2 (red) and LACTB1–68-Dronpa (green). The fluorescent molecule scale maximum values (probability per square
nanometer) differ per panel and range from 0.002 to 0.01 for Dronpa and from 0.011 to 0.019 for mEos2. TFAM-labeled nucleoids either were
located adjacent to the cristae-IMS as shown in panels A to C or surrounded the cristae-IMS as shown in panels D to F). Bars, 100 nm.
VOL. 31, 2011SUPERRESOLUTION IMAGING OF MITOCHONDRIAL NUCLEOIDS 5007
the presence of other proteins, macromolecular crowding, or
DNA supercoiling has not been investigated.
The high mtDNA packing density finding is also supported
by two-color PALM data obtained using matrix-targeted Cox-
VIII1–29-Dronpa (Fig. 8). A similar fluorescent fusion protein
(CoxVIII-green fluorescent protein) has been shown to be
freely diffusible within the mitochondrial matrix (43). If the
nucleoid were loosely packed, we should see widespread colo-
calization of matrix Dronpa with TFAM-mEos2. However,
Dronpa protein within the mitochondrial matrix is largely ex-
cluded from the nucleoid. Interestingly, this nucleoid exclusion
was previously seen by Iborra and coworkers in a study using
matrix-targeted yellow fluorescent protein. They also noted
that areas surrounding the nucleoids were devoid of fluores-
cence (24). The latter observation was partially replicated here
and may indicate an additional barrier between the nucleoid
and the matrix. A compressed and isolated structure is also
consistent with recent data indicating that nucleoids are genet-
ically autonomous (20). The high packing density and limited
diffusional access by matrix proteins are also directly relevant
in defining how the nucleoid receives signals and alters its
organization. These are physical impediments for an unmedi-
ated exchange with the matrix that have likely been underap-
preciated. Mechanisms for achieving the exchange in bacteria
and in nuclei involve strategies for remodeling of DNA binding
and packaging proteins. The regulation of mtDNA topology
within the mitochondria is only beginning to be explored (50).
It has recently been shown in studies using Drosophila cells that
the mitochondrial Lon protease can regulate TFAM levels,
which in turn alter the rate of transcription of mtDNA (35).
Thus, it may be that local turnover of TFAM, other nucleoid
proteins, or RNA is a general strategy for altering the local
Nucleoid remodeling proteins and intercompartmental com-
munication might also function through interactions with the
inner membrane. Therefore, we have considered nucleoid po-
sition relative to cristae. There is a large amount of heteroge-
neity among nucleoids with respect to both size and shape.
Among examples in the ellipsoidal subset of nucleoids, there is
a 25-fold range in volumes just within the standard deviation.
It is clear that nucleoid size is loosely restricted. One constraint
affecting nucleoid size could be the physical space available
within the mitochondria. The mitochondrial matrix space is
defined by both the width of the space between the inner
boundary membranes and the frequency and size of the cristae
that partition that width. In two dimensions, 3T3sw mitochon-
dria show an average width of 238 (? 75) nm between the
inner boundary membranes. The mean dimensions of a nucle-
oid are 85 by 108 by 146 nm. Therefore, the mitochondrial
matrix is wide enough to easily accommodate the majority of
nucleoids if the cristae are excluded. However, the spaces
between cristae may be more restrictive for the nucleoid. The
average matrix space between cross-sectioned cristae is only 68
(? 24) nm. Although these are average dimensions, they en-
courage speculation that either the cristae membrane influ-
ences nucleoid structure or the inner membrane is arranged to
accommodate the nucleoid.
We present two additional results that point toward a close
relationship between nucleoids and the inner membrane. First,
analyses of nucleoid shape yielded circumstantial evidence that
the inner membrane might influence nucleoid contour. A total
of 65% of nucleoids are ellipsoidal and have no obvious struc-
tural influence from the inner membrane. The remaining 35%
have concave edges, splits, or branches that are consistent with
membrane interactions. In direct support of this proposition,
LACTB1–68-Dronpa localization indicates a very close rela-
tionship with nucleoids (Fig. 10). EM imaging has shown that
mtDNA (24, 41) and TFAM (19) are located in the mitochon-
drial matrix. Therefore, the 5- to 10-nm-wide inner membrane
lies between the nucleoid and the IMS, although this was not
resolved in our PALM analysis. Essentially all of the nucleoids
seen in two-color PALM were very close to or overlap the
LACTB1–68-Dronpa signal. There are two common relative
positions of the nucleoid and IMS marker (Fig. 10). The IMS
(and therefore the inner membrane) either penetrates the nu-
cleoid boundaries or is tangentially positioned. Those nucle-
oids with tangentially oriented IMS might be located next to
either the boundary membrane or cristae. Those that are more
fully engaged with the IMS are likely to be wrapped around an
inner membrane cristae tubule. Our current imaging results do
not always distinguish between the IMS of the boundary mem-
brane and the IMS of cristae, and either location is plausible.
However, prior localization of LACTB and our current immu-
nolocalization of LACTB1–68-Dronpa reveal the novel pros-
pect that there is an intimate association of the nucleoid with
specialized cristae. Conventional microscopy has suggested
that, in yeast, a subpopulation of nucleoids is associated with a
protein complex that spans both the inner and outer mitochon-
drial membranes (37). As we cannot rule out boundary mem-
brane interactions, it remains possible that mammalian nucle-
oids interact with analogous structures. The primary evidence
for membrane association in mammals came from early EM
imaging, which revealed that partially purified mtDNA con-
tains short regions that are bound to detergent-soluble frag-
ments (2, 38). The current PALM data bear more directly and
fully on this interaction.
Although two-color PALM is able to detect fluorescent pro-
teins residing within the cryosections, it is a two-dimensional
method and lacks the capacity to assign molecular locations in
the z dimension. We were therefore unable to confidently
determine the exact frequency of membrane-associated nucle-
oids. However, we propose an initial conservative estimate that
at least 35% of nucleoids are in contact with the inner mem-
brane, as inferred from that fraction of nucleoids that are not
ellipsoidal. However, an ellipsoidal shape does not necessarily
preclude membrane association or even membrane penetra-
tion. It is worth noting that our nucleoid volume calculations
do not account for space that may be occupied by cristae in
some of the ellipsoidal nucleoids. Therefore, the volume oc-
cupied by the mtDNA in this space may be lower than shown
in Table 1. Thus, the nucleoid density value reported here
might also represent a low estimate.
The list of nucleoid-associated proteins that have been iden-
tified is long and increasing (8, 59). A future challenge is to
spatially and temporally categorize members of this list. Some
nucleoid-associated proteins are located in the inner mem-
brane. Others are located in the matrix, the intermembrane
space, and even the outer membrane. Many of these have
primary functions that appear unrelated to mtDNA and largely
reside outside the nucleoid. Others that have known functions
5008 BROWN ET AL.MOL. CELL. BIOL.
within the nucleoid, such as mtDNA repair enzymes, are prin-
cipally located within the membrane, independently of
mtDNA (60). From a functional standpoint, it is important to
make a distinction between core proteins and those that are
The available data allow us to propose a model for the
organization of mammalian nucleoids that has testable fea-
tures and emphasizes current questions (Fig. 11). The core of
the nucleoid is displayed in Fig. 11 in dark orange. Size appears
to primarily reflect genome content, and shape may indicate
“active” nucleoids. A very high mtDNA packing density com-
monly limits the protein composition within the core to those
that have a direct role in mtDNA packaging. Diffusible matrix
proteins may not freely infiltrate most nucleoids. Other nucle-
oid-associated proteins may gain fuller or more local access to
mtDNA through regulated modifications of nucleoid struc-
tures. This remodeling could be mediated by proteins located
in the matrix or at the inner membrane or both. The nucleoid-
membrane interface remains poorly defined. However, the ex-
change of transiently associated nucleoid proteins could be
mediated by cristae and inner membrane dynamics. The local
availability of the inner membrane proteins and the intermem-
brane space lends additional routes for intercompartmental
communication. Additional analysis of this membrane interac-
tion awaits further development of two-color iPALM or other
high-resolution microscopy approaches.
We thank Yalin Wang for contributions in optimizing and providing
cryosections for PALM, Wei-Ping Li for providing TEM images of
3T3sw cells used for mitochondrial dimension analysis, and Rick Fetter
for LR White resin sample processing. Eric Betzig, Hari Shroff, Jim
Galbraith, and Cathy Galbraith provided invaluable instruction and
advice with PALM.
This work was supported by the Howard Hughes Medical Institute
(T.A.B., A.N.T., G.S., B.G.K., H.F.H., D.A.C.) and grant AGSS237009
from the Ellison Medical Foundation Foundation (D.F.B.).
We disclose that Harald Hess has licensed PALM technology to Carl
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