Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens.
ABSTRACT The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or approximately 100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy.
- SourceAvailable from: Martin Oheim[Show abstract] [Hide abstract]
ABSTRACT: Most structured illumination microscopes use a physical or syn-thetic grating that is projected into the sample plane to generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics. In this study, we designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination pat-terns of arbitrary orientation and period, limited only by the illumination aperture (NA = 1.45), the response time of a fast, piezo-driven tip-tilt mirror (10 ms) and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz. The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrate the performance of our instrument by imaging mitochondrial dynamics in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope provides a versatile, compact and cost-effective arrangement for super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles - important questions in both cell biology and neurophysiology.Optics Express 11/2013; 21(22):26162-73. · 3.53 Impact Factor
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ABSTRACT: We present a versatile scheme for two-dimensional (2D) resolution enhancement in standing wave fluorescence microscopy (SWFM). This SWFM scheme consists of an interferometer, where both beams are focused at the back focal plane of the objective lens. Their position is controlled by a pair of acousto-optic deflectors (AODs). This results in two collimated beams that interfere in the focal plane, creating a lateral periodic excitation pattern with controlled fringe spacing and orientation. The phase of the standing wave (SW) pattern is controlled by the phase delay between two RF sinusoidal signals driving the AODs. An enlarged fluorescence image formed using the same objective lens is captured by a cooled CCD camera. Data collection involves acquiring images with excitation pattern of three equi-polar orientations (0°, 60° and 120°) and three different phases (0°, 120°, 240°) for each orientation. The SWFM image is algebraically reconstructed from these 9 acquired images. The SWFM image has enhanced 2D lateral resolution of about 100 nm with nearly isotropic effective point-spread function (PSF). As a result of the acousto-optic scanning, the total acquisition time can be as short as 100 mus and is only further limited by the fluorescence intensity, as well as sensitivity and speed of the CCD camera. Utilizing acousto-optic laser scanning for advanced SWFM provides the exceptional precision and speed necessary for real-time imaging of subresolution processes in living biological systems.Proceedings of SPIE - The International Society for Optical Engineering 02/2009; · 0.20 Impact Factor
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ABSTRACT: Because of the small sizes of most viruses (typically 5–150 nm), standard optical microscopes, which have an optical diffraction limit of 200 nm, are not generally suitable for their direct observation. Electron microscopes usually require specimens to be placed under vacuum conditions, thus making them unsuitable for imaging live biological specimens in liquid environments. Indirect optical imaging of viruses has been made possible by the use of fluorescence optical microscopy that relies on the stimulated emission of light from the fluorescing specimens when they are excited with light of a specific wavelength, a process known as labeling or self-fluorescent emissions from certain organic materials. In this paper, we describe direct white-light optical imaging of 75-nm adenoviruses by submerged microsphere optical nanoscopy (SMON) without the use of fluorescent labeling or staining. The mechanism involved in the imaging is presented. Theoretical calculations of the imaging planes and the magnification factors have been verified by experimental results, with good agreement between theory and experiment.Light: Science & Applications 09/2013; 2(9). · 8.48 Impact Factor
Two-Dimensional Standing Wave Total Internal Reflection Fluorescence
Microscopy: Superresolution Imaging of Single Molecular
and Biological Specimens
Euiheon Chung,* Daekeun Kim,yYan Cui,§Yang-Hyo Kim,yand Peter T. C. Soyz
*Harvard-Massachusetts Institutes of Technology, Division of Health Sciences and Technology, Departments ofyMechanical Engineering,
andzBiological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and§Department of Physics,
Tianjin Polytechnic University, People’s Republic of China
biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a
total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at
an interface between a high and a low index medium. The ,100 nm penetration depth of evanescence field ensures a thin
excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by
use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to
generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal
drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization
and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission
wavelength or ;100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-
directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single
semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high
signal/background ratio from TIRF microscopy.
The development of high resolution, high speed imaging techniques allows the study of dynamical processes in
Historical review of TIRF microscopy and
In the modern era of biological research, fluorescence
microscopy is a powerful tool to visualize specific biomol-
ecules to which fluorescent dyes can be selectively bound.
Total internal reflection fluorescence (TIRF) microscopy
processes near the basal plasma membrane of adherent cells
by illuminating a very thin region on the order of 100 nm
(1,2). The evanescent wave intensity decays exponentially
from the interface, and this near-field excitation volume
allows intrinsic optical sectioning to less than one-fifth of the
excitation wavelength while keeping wide-field (WF) imag-
ing capability. Although conventional WF fluorescence exci-
of TIRF removes the out-of-focus noise and reduces photo-
bleaching of fluorophores outside the focal plane and is thus
ideal for single-molecule imaging (3,4). On the other hand,
TIRF does not allow deeper imaging into the interior of cells,
and the lateral resolution of TIRF is identical to that of
standard WF imaging.
The quest to image structural and functional biological
information using far-field microscopy at high resolution has
been hindered by the diffraction limit of light. The Abbe
diffraction limit originates from the wave nature of light and
depends on the wavelength and the numerical aperture (NA)
of an objective lens. When an object that is substantially
smaller than the diffraction limit is imaged by a microscope,
its image would be significantly broadened compared to the
original object. The intensity distribution of this effective
point object is defined as the point spread function (PSF).
The commonly accepted Rayleigh criterion defines the res-
olution as the distance between two point objects when one
PSF falls on the first zero point of the other PSF, which can
be barely distinguished in incoherent imaging. Also Sparrow
suggested a resolution criterion when the gradient of the
summed profile is zero or no dip at the midpoint (5). These
resolution definitions are roughly equal to the full width at
half-maximum (FWHM) of the PSF. With typical visible
light emission and oil-immersion objectives, optical resolu-
tion is over 200 nm.
The challenge of achieving superresolution in optical
microscopy beyond the diffraction limit has been overcome
in practice only during the last couple of decades. In
particular, extending lateral or transverse resolution has
been exemplified with several techniques such as i) stimu-
lated emission depletion (STED) microscopy, ii) saturated
structured illumination microscopy (SSIM), iii) solid im-
mersion lens (SIL), iv) structured illumination micros-
copy (SIM), and v) harmonic excitation light microscopy
STED has achieved the highest far-field optical resolution
of ,30 nm using nonlinear photon-induced saturation
Submitted September 27, 2006, and accepted for publication April 13, 2007.
Address reprint requests to Peter T. C. So, E-mail: firstname.lastname@example.org.
Editor: Enrico Gratton.
? 2007 by the Biophysical Society
Biophysical JournalVolume 93September 20071747–17571747
depletion of the excited state in the outer regions of the ex-
citation PSF. However, this technique suffers from relatively
slow speed due to the point scanning nature (6–8). SSIM is
almost an inverse version of STED, using WF mode pro-
viding comparable superresolution to STED. Photobleaching
in SSIM is particularly a challenge under saturating light
intensities (9). SIL microscopy takes advantage of high re-
fractive index material replacing immersion oil, utilizing the
evanescence field of SIL for near-field imaging, which also
requires point scanning (10). In contrast to the point scanning
methods, SIM or HELM use WF camera detection, allowing
faster image acquisition by encoding either the diffraction
grating illumination structure or the standing wave (SW)
illumination. This contains high frequency patterned illumi-
nation onto specimen, providing up to a factor of 2 lateral
resolution enhancement (11,12–14). To decode the high fre-
quency information, only several images need to be taken
with phase shift, and general lateral resolution enhancement
requires rotation of this pattern.
Resolution enhancement by use of evanescent
Even higher lateral resolution in WF mode can be achieved
by a combination of SW illumination and TIRF microscopy
(15,16). Evanescent SW keeps the SW spacing narrower due
to a higher refractive index of the substrate resulting in
enhanced resolution. The SW total internal reflection fluo-
rescence (SW-TIRF) microscope has been recently imple-
Since the SW modulation is inherently sinusoidal, to re-
imum of three image acquisitions at different phases of the SW
are required. Since phase shift is in principle fast, SW-TIRF
does not necessarily increase the total image acquisition
time compared to conventional WF imaging.
The lateral resolution mostly depends on the evanescent
SW fringe period, which is proportional to the excitation
wavelength in the substrate. As with other high resolution
techniques based on interference, there exists a side-band
artifact. This artifact can be easily removed by linear decon-
volution if the emission PSF is narrow enough to keep the
side-band lower than 30% (18). Thus this effectively en-
hances the resolution much more than twofold, as demon-
strated in this article.
The advances in SW-TIRF microscopy achieved in this
work include: 1), the first (to our knowledge) superresolution
WF imaging of single molecular and biological specimens
with 2D SW-TIRF images; 2), a numerical analysis of SW-
TIRF microscopy PSF to explore the effect of more than
two SW directions; and 3), a demonstration that a feedback
control stabilization of the evanescent SW phase ensures that
this technique is sufficiently robust for routine biomedical
Point-spread function engineering with
For one WF image with structured excitation modulation
ontothe specimen,the fluorescence imagecan bedescribedas
IðxÞ ¼ ½OðxÞEðxÞ?5PðxÞ;
and the final SW-TIRF PSF enhanced in the x-direction is
I9ðxÞ ¼ OðxÞ5½EðxÞPðxÞ?:
where the reconstructed image I9ðxÞ is the convolution of the
fluorophore concentration distribution in the object OðxÞ
with EðxÞPðxÞ, which is the multiplication of the conven-
tional PSF PðxÞ and the structured excitation intensity EðxÞ.
This final image with narrowed PSF is obtained by the
weighted sum of several intermediate images. The excitation
EðxÞ, which carries the high spatial frequency component
of structured illumination, has been transferred into the PSF
to generate an effective PSF, EðxÞPðxÞ. The detailed mathe-
matical formulation of one-directional SW-TIRF (1D SW-
TIRF) image reconstruction can be found in So et al. (15).
The excitation intensityprofile from evanescent SW above
the surface of a high refractive index substrate can be ap-
proximately described as
EðxÞ ¼ 11acosð4pnsinux=lÞ;
where a is the contrast of the SW, n is the refractive index of
substrate, u is the incident angle of the excitation beam at the
interface, and l is the vacuum excitation wavelength. This
formulation gives lateral resolution enhancement in the
direction of the SW. The extension of this 1D SW-TIRF
theory to uniform lateral resolution enhancement can be
achieved by superimposing the finite number of rotational
SW-TIRF images (17).
SW-TIRF image reconstruction
To generate a 1D SW-TIRF image, three WF images are
required to be taken for each phase of the interfering SW
excitation changed by 120? while the specimen remains
the same. The phase difference of 120? is chosen to provide
the highest signal/noise ratio (15). The superposition of the
appropriately weighted images gives a high resolution image
in the SW direction.
For two-dimensional SW-TIRF, finite equipolar angles are
chosen and the corresponding 1D SW-TIRF is superposed,
requiring a total of 3N WF images (N ¼ number of equipolar
SW-TIRF) for demonstration. The numerical simulation in
the following section shows that the anisotropy of effective
PSF decreases to ,10% for two orthogonal directions. In
addition, the images are recorded with a camera with square
pixels; this choice simplifies the image analysis.
1748 Chung et al.
Biophysical Journal 93(5) 1747–1757
enhancement can be obtained by the superposition of the
enhanced images in several directions. To determine how
PSF in a practical sense, numerical simulation has been per-
formed using a MATLAB program (MathWorks, Natick,
MA) and described in Fig. 1. For N number of directional
scan, uniform angular separation was used and only the pro-
lies between these two. Fig. 1 A shows the shape of lateral
PSFs with corresponding contour plots for N ¼ 0, 1, 2, 3, and
4. For N ¼ 0, the PSF is the same as conventional TIRF or
wide-field (WF) PSF. For N ¼ 1, the thinnest PSF FWHM is
achieved in the direction of SW propagation, resulting in
;230% narrower PSF in the SW direction whereas the PSF
conventional TIRF (or N ¼ 0 case). For N ¼ 2, the difference
of PSF FWHM in the thinnest and the thickest directions is
that the two directional PSF profiles virtually converge as N
increases. Though N ¼ 3 or 4 would be better in terms of the
isotropic shape of PSF, the 2D SW-TIRF will be used for the
experimental demonstration since using more directions will
lengthen the total exposure time and compromise fast speed
imaging. Future implementations of SW-TIRF will address
thislimitation.ThesimulatedPSFFWHM ofTIRF is199nm
with the actual experimental parameter values. In actual
measurement, the best measured PSF FWHM was ;260 nm,
which corresponds to that of NA 1.1 from numerical simu-
lation. A similar result was observed in the literature (13,19)
code for 2D SW-TIRF numerical simulation will be made
available through the Supplementary Material online.
MATERIALS AND METHODS
The design of the TIRF microscope with SW excitation is depicted in Fig. 2.
There are two ways to generate the TIRF system. One is prism launched and
the other is objectivelaunched. Prismless or objective-launched TIRF allows
easy access to the specimen from the other side, which is particularly
suitable for biological specimens in cell culture dishes, and thus is chosen in
our setup (2). The excitation light from a laser (532 nm, Verdi-10, Coherent,
Santa Clara, CA) is delivered to the setup via a single mode fiber through a
fiber coupler (PAF-X-11-532-PC fiber coupler, OFR, Caldwell, NJ).
Isolation of the laser source from the optical setup is necessary to minimize
the effect of mechanical vibration of laser cooling. The fiber-delivered beam
comes out of a fiber collimator and enters through a 50:50 beam splitter. One
beam is reflected from a retroreflecting mirror attached with PZT (piezo-
actuated transducer) to vary the optical path length for controlling the SW
interference fringe phase. The other beam passes through a combination of a
half-wave plate and a linear polarizer to match the intensity of two final
PSF decreases as N increases (N: number of scan directions). (B) Comparison of the PSF profile in the thinnest and the thickest directions. All the other cross
sectional profiles lie between these two profiles for corresponding N. Simulation condition: NA ¼ 1.45, a ¼ 1.0, lexc¼ 532 nm, lemi¼ 560 nm, n ¼ 1.52,
uincidence¼ 67? (or fringe period ¼ 190 nm).
(A) Effective PSF simulation of 2D SW-TIRF for directions N ¼ 0, 1, 2, 3, and 4. N ¼ 0 corresponds to the conventional TIRF. Anisotropy of
SW-TIRF Imaging of Biological Specimens1749
Biophysical Journal 93(5) 1747–1757
polarization-preserving optical fiber (Oz Optics, Ottawa, Canada), and the
end fiber tips are housed in mirror mounts placed on XYZ-translators.
Precise orientation and position control of the fiber tips are required with
5 degrees of freedom (X, Y, Z, yaw, and pitch) to place the corresponding
excitation foci at the back aperture of the objective.
The separation of two fiber tips with simple translation determines the
final incidence angle onto a coverslip. The divergent beams emitted from
these fiber tips are collimated by a collimation lens (f ¼ 200 mm) and
the s-polarization state, a linear polarizer is put in between the collimation
lensandthe tubelens (not showninthe diagram).The excitationbeamsenter
a modified epiillumination light path of an inverted microscope (Olympus
IX-71, Tokyo, Japan) through its side port and are reflected by a dichroic
mirror (z532dc, Chroma, Rockingham, VT) and focused at the back aperture
of a high NA objective lens (Olympus, Plan Apo 603, NA 1.45). The
collimated beams out of the objective enter the specimen/coverslip interface
at a supercritical angle with beam diameter of several hundred micrometers.
The incident angle is set at 67? 6 0.5?, which is above the critical angle of
62? assuming the index of specimen as 1.38 and that of coverslip as 1.52. By
simply translating the excitation fiber launcher position, the setup can
systematically vary the incidence angle over a range of 41.2?–72.5?, which
makes it easy to switch imaging modes between SW fluorescence micros-
copy to SW-TIRF. The fluorescence emission is collected by the same ob-
jective and transmitted through the dichroic mirror along the emission path.
An additional barrier filter (HQ545LP, Chroma) is used to further
attenuatethe scatteredexcitationlight.The emissionoutof the bottomport is
expanded 16 times by relay optics and is focused onto a 12-bit intensified
charge-coupled device (iCCD) camera (Pentamax, Princeton Instrument
(now Roper Scientific), Trenton, NJ) with a field of view of 12 3 12 mm2.
The residual excitation beams leaked through the back of the dichroic mirror
exit through a hole bored at the back wall of the dichroic mirror cube. The
residue excitation beams were expanded to form an interference pattern on a
complementary metal oxide semiconductor (CMOS) sensor for SW phase
With the current design, conventional TIRF images can be simply
obtained by blocking one beam andconventionalWF images can be taken by
locating one beam at the center of the optical axis while blocking the other
beam. This will be used for the comparison of different imaging modes.
Calibration of evanescent standing wave fringe period
As mentioned in the above section, the direct imaging measurement of the
evanescent SW period is not trivial, due to its subdiffraction limited size.
Instead, a thin uniform layer of fluorescent sample was prepared and SW
emission was imaged by gradually increasing the incident angles from a
subcritical angle, where SW can be imaged, to a supercritical angle where
the SW cannot be imaged due to the resolution limit. This actual mea-
surement of imaging SW fringes was matched with theoretical calculation
from geometry within 2.5% and could be extrapolated to the setup angle of
67? 6 0.5?. We further need to calibrate the pixel size of the CCD camera.
The pixel resolution was determined to be 23.9 nm by imaging a Ronchi
ruling with known spacing (Edmund Optics, Barrington, NJ).
System stability and feedback control of the standing
The SW-TIRF system is basically an interferometer. Due to the thermal
expansion and mechanical instability of the excitation beam path, the phase
of the SW can drift over time (;100?/min) and degrade the quality of final
reconstructed image. Thus it is required to use closed loop feedback control
of the SW phase (Fig. 3). Since the evanescent SW spacing (;190 nm) on
the coverslip is below the resolution of the imaging system (;260 nm), an
indirect way of phase estimation was devised. This is done by generating
another alternative interference pattern onto a CMOS camera (S9227,
Hamamatsu, Bridgewater, NJ) with ;103 magnification relay optics at the
back of the dichroic mirror mount. From the output of the CMOS array, an
algorithm is implemented to calculate the fringe period and estimate the SW
phase in real time. Assuming the Nyquist limit is satisfied and the alternating
current component of the normalized fringe pattern is described by
cosð2px1fÞ, where x is the position along the CMOS array, the fringe
phase,f, can be obtained from f ¼ tan?1ðsinf=cosfÞ after forming and
solving the matrix equation below.
In our setup,the CMOS array has 512 pixelswithsize of 6.4 mm. Here M,
which is approximately equal to six pixels, denotes the number of pixels
corresponding to one period of fringes, which can be estimated by Fourier
transform and taking the lowest but closest integer of the pixel values. ? piis
launched SW-TIRF microscope. Note
that the diagram is very simplified (see
1750Chung et al.
Biophysical Journal 93(5) 1747–1757
the average intensity of the ith pixel in a period. Since 50 fringes can be
recorded by the CMOS array, 50 values were averaged to provide a single
average pixel value.
The SW phase drift was removed by a feedback control system utilizing
an embedded microprocessor (SBC0486, Micro/sys, Montrose, CA) driving
the PZT-driven retroreflector. The feedback signal is provided by the
estimated phase of the interference pattern on the CMOS. A typical pro-
portional, integrative, and derivative control scheme was used and the gains
were tuned using the Zigler-Nichols method (20). The drift of the phase
could be minimized by covering most of the beam path and attaching optical
fibers on the rigid part. The fluctuation was reduced by isolating any
mechanically moving sources from the optical table except a cooling fan
inside the iCCD.
Data acquisition and image reconstruction
The raw images from the iCCD camera were stored using a desktop
computer with Windows 98, running the WinView (Roper Scientific,
Trenton, NJ) program. The typical exposure time for each raw image was
;0.1– 0.2 s with total excitation power entering into the objective ,10 mW.
To minimize the photobleaching of fluorophore, a mechanical shutter was
usedto restrict the illumination during the image acquisition. Nonfluorescent
immersion oil (Olympus, n ¼ 1.516 at 23?C) was used. Each image was
subtracted by the background image of a sample without fluorophore under
the same condition. The image acquisition was synchronized with the real-
time feedback control of the SW fringe phase by the embedded micropro-
cessor using custom-written software in the C programming language
(Turbo C11 version 3.0). Postimage reconstruction and digital image
analysis was performed on an IBM (Armonk, NY) Thinkpad laptop
computer (T40) using MATLAB software. The electronic postprocessing
takes a few seconds, and potentially real-time reconstruction would be
feasible with the development of computing technology.
To generate a 2D SW-TIRF image, the specimen was mechanically
rotated by 90? between the recordings of two 1D SW-TIRF image sets that
were subsequently combined. The registration of two-directional enhanced
images was performed by using cross correlation of two images to find the
relative shift of images before superposition. However, care needs to be
taken since this cross correlation may not work well when the overall
intensity distribution of each image is not the same. The mechanical sample
rotation may be avoided in the future by the use of multiple directional
Fluorescent polystyrene spheres and
semiconductor nanocrystals (quantum dots)
Fluorescent polystyrene microspheres (F-8792, Molecular Probes, Eugene,
OR), with a nominal diameter of 44 nm and peak emission at 560 nm, were
sonicatedfor severalminutesand were loaded onto a cover slide followedby
covering with a standard coverslip (22 3 22 mm2, No. 1.5 thickness). The
coverslip was sealed with nail polish to prevent the evaporation of water.
Initially most beads were moving due to Brownian motion. After overnight,
most beads are attached on the coverslip and ready for imaging. The number
concentration of microspheres was adjusted to get ;10–30 beads in the
microscope field of view (12 3 12 mm2).
The sample preparation of quantum dots was similar to that of
polystyrene microspheres except using quantum dots (Qdot565 streptavidin
conjugated, Invitrogen, Carlsbad, CA). It was recognized that the quantum
dots are prone to lose their signal if stored in diluted solution. In addition,
their blinking nature was adverse to the SW-TIRF technique based on the
quantitative signal of intermediate images.
microcontroller monitors the output phase from the CMOS detector and this estimated phase is used to control input in real time (;70 Hz). (B) Typical fringe
pattern on CMOS image sensor. The invisible evanescent SW on the sample can be indirectly monitored in this alternative way. (C) SW fringe phase
measurement with a feedback loop open (left) and closed (right). Reference angle: 120? and measurement duration: 0.2–45 s. The standard deviation of the
fluctuation is ,3?.
SW phase control with closed loop feedback controller. (A) Schematic diagram of the SW phase closed loop control unit. The embedded
SW-TIRF Imaging of Biological Specimens1751
Biophysical Journal 93(5) 1747–1757
Cell culture and fluorescent labeling of the
Fibroblasts were grown in standard 100 mm 3 20 mm cell culture dishes
(Corning, VWR, West Chester, PA) in Dulbecco’s modified Eagle’s
medium (Cellgro, Mediatech, Herndon, VA) supplemented with 10% fetal
bovine serum (Invitrogen, Carlsbad, CA) and penicillin-streptomycin (100
units of penicillin per ml media, and 100 mg streptomycin per ml media;
Invitrogen). Cells were cultured at 37?C in 5% CO2. At 24 h before the
labeling experiments, fibroblasts were plated on 35-mm glass-bottom cell
culture dishes (MatTek, Ashland, MA) coated with collagen I (1 mg/cm2;
Cohesion Tech, Palo Alto, CA).
Onthe dayof the experiments,the cell confluency had reached;60%.At
room temperature, cells were then fixed with 3.7% formaldehyde in
phosphate buffered saline (PBS, Mediatech) for 10 min, washed twice with
PBS, and extracted with 0.1% Triton X-100 in PBS for 5 min. To reduce
nonspecific background staining, fixed cells were then incubated in PBS
containing 1% bovine serum albumin (Polysciences, Warrington, PA) for 20
min. For F-actin labeling, cells were then incubated with 165-nM
AlexaFluor 532 phalloidin (Molecular Probes) for 20 min and washed three
times with PBS.
Imaging fluorescent polystyrene spheres
A comparison of conventional TIRF PSF and 1D SW-TIRF
PSF images is shown in Fig. 4. The sample contains fluo-
rescent polystyrene microspheres in water (see Materials and
Methods). The intensity profiles in the SW direction show
that SW-TIRF PSF has narrowed by 239% over TIRF in
terms of FWHM. However, SW-TIRF has two prominent
side lobes with amplitudes slightly ,30% of the main peak.
This observationis consistent with theoretical prediction, Eq.
2, if the NA of the microscope objective is ;1.1. Although
the theoretical NA of the objective is supposed to be 1.45, the
lower NA value is consistent with conventional TIRF mea-
surement where PSF with FWHM of 260 nm is measured.
This performance characteristic of the objective is consistent
within-house measurementbyOlympus(Eiji Yokoi,Olympus
America, Inc., personal communication, 2005) and will be
further investigated in the Discussion. Nonetheless, since the
relative height of side lobes compared to the main peak is
,30%, linear deconvolution canquicklyeliminate side lobes
as shown in Fig. 4 c (18). The PSF FWHM of the main peak
remains unchanged after linear deconvolution. No filtering to
the raw images before image reconstruction was applied.
Imaging semiconductor nanocrystals
Quantum dots are gaining popularity for biological imaging.
Their long-term photostability is far superior to organic
fluorescent dye molecules. They also possess broad excita-
tion and narrow emission spectrums, allowing simultaneous
labeling of different structures (21). The imaging of single
molecular quantum dots allows us to demonstrate the sen-
sitivity of this technique.
Quantum dots are also ideal objects for PSF measurements
due to their small size of 10–25 nm, which is far below the
diffraction limit. Theresultisdisplayed inFig.5withvertical
intensity profiles of several regions of interests (ROI). The
total input power into the objective was ;4.6 mW. The
results of 1D SW-TIRF imaging showed lateral resolution
enhancement of more than 2.5 times in ROI I and II.
Interestingly, some spots did not show enhanced resolu-
tion, which is likely due to the blinking of the quantum dot.
Although the mechanism for quantum dot blinking is not
fluorescent bead. The evanescent standing excitation is in the vertical direction. The corresponding vertical intensity profile is (a), (b), and (c). These vertical
profiles show a narrower PSF FWHM for SW-TIRF in comparison with that of conventional TIRF. (Experimental condition is the same as that of numerical
simulation: NA ¼ 1.45, lexc¼ 532 nm, lemi¼ 560 nm, n ¼ 1.52, and uincidence¼ 67? 6 0.5? except the contrast was typically measured ;0.9.)
Effective PSF measurement of (A) conventional TIRF, (B) 1D SW-TIRF, and (C) 1D SW-TIRF with linear deconvolution with a 0.04-mm
1752 Chung et al.
Biophysical Journal 93(5) 1747–1757
fully understood, several different ways of blinking sup-
pression were documented (22). We have tried a number of
these approaches without great success except for the use of
betamercaptoethanol; however, betamercaptoethanol is toxic
to biological specimen and hence has minimal long-term
utility. Recently, we observed significantly reduced blinking
behavior in quantum dot products from a different source
(Evident Technology, Troy, NY). The manufacturing pro-
cess of quantum dots seems to play an important role in their
blinking behavior. Finally, we also found that quantum dots
mounted on microscope slides tend to lose their signals and
degrade in a few days. Unless there are reliable methods to
suppress the blinking of quantum dots and to maintain their
photostability in specimens, they may be less than ideal
probes for SW-TIRF microscopy.
Imaging actin cytoskeleton: 1D SW-TIRF
Imaging of biological specimen is demonstrated in Fig. 6.
Since the SW propagation is in the vertical direction, 1D
SW-TIRF gives resolution enhancement only in the vertical
along the vertical direction, the exposure time is 1.0 s, and other experimental conditions are the same as in Fig. 4.
The comparison of conventional TIRF, 1D SW-TIRF, and SW-TIRD images of semiconductor quantum dots. The evanescent SW direction is
SW-TIRF Imaging of Biological Specimens1753
Biophysical Journal 93(5) 1747–1757
direction. WF image A (a) has a higher background noise
level, whereas conventional TIRF image B (b) shows signif-
icantly lower background noise compared to WF due to the
shallow evanescent excitation of ;73 nm. However, the lat-
eral resolution remains about the same with WF and TIRF.
Images in B (c) and B (d) show the SW-TIRF image and that
with linear deconvolution, respectively. The presence of
horizontal stripes in B (c) is due to the side lobes of the
original 1D SW-TIRF image and is reasonably suppressed
after linear deconvolution in B (d). The SW-TIRF images
reveal finer detail of the cellular actin cytoskeleton, resulting
in 235% enhancement in terms of lateral resolution com-
pared to the WF image, which demonstrates the high reso-
lution capability of this system in the application of imaging
cellular actin cytoskeleton.
Imaging actin cytoskeleton: 2D SW-TIRF
The F-actin cytoskeleton structure of a single fibroblast cell
was imaged by 2D SW-TIRF in Fig. 7 with the resolution
enhancement in both directions. Here, the superposition of
two perpendicular directional 1D SW-TIRF was used to
generate one 2D SW-TIRF image. Fig. 7, A (a) and (b), show
that TIRF decreases the out-of-focus noise to improve the
signal/background ratio compared to conventional WF imag-
ing at the cost of observing only near the surface region. In
inset 1of Fig. 7 B, SW-TIRF can reveal the190 nmseparated
In Fig. 7 C, second row, FWHM of actin fiber is narrowed
SW-TIRF can distinguish actinfibers separated by 190 nm. It
is recognized that not all the actin fibers in one image are in
focus due to the slight changes of actin fiber height from the
or TIRF. A major difficulty of imaging biological specimen
continuous excitation, whereas the actin phalloidin needs
extra care to minimize the total illumination time before fad-
ing away. Since the intermediate images were recorded se-
some photobleaching is unavoidable. To compensate for
photobleaching, the two 1D SW-TIRF image set along two
withlineardeconvolution(SW-TIRD).B(a–d)are enlargedinsetsof corresponding imagefrom A(a–d). B(a1–d1) arethe vertical profilesof dashedline in the
corresponding image B (a–d). The evanescent standing excitation is in the vertical direction.
F-actin cytoskeleton in mouse fibroblast cells (NIH 3T3) imaged with (a) WF, (b) conventional TIRF, (c) 1D SW-TIRF, and (d) 1D SW-TIRF
1754Chung et al.
Biophysical Journal 93(5) 1747–1757
with linear deconvolution (SW-TIRD). Subfigure A compares the four different imaging modes and subfigures B and C are the enlarged insets 1 and 2 marked
in subfigure A. In subfigure C, the second and third rows are the vertical profiles of (1) and (2) in subfigure C (a).
F-actin cytoskeleton in mouse fibroblast cells (NIH 3T3) imaged with (a) WF, (b) conventional TIRF, (c) 2D SW-TIRF, and (d) 2D SW-TIRF
SW-TIRF Imaging of Biological Specimens1755
Biophysical Journal 93(5) 1747–1757
orthogonal directions are superposed after normalization
based on the maximum intensity level of each image. How-
ever, the photobleaching of the three sequential images along
one direction was not compensated since the degree of pho-
tobleaching was not significant. For samples that may be
photobleaching faster, further compensation can be applied
by assuming a simple linear photobleaching rate model (23).
TIRF microscopy and a lateral version of SW microscopy
were combined. This new technique has been demonstrated
with fixed biological specimen. The comparison between
WF, TIRF, and SW-TIRF shows that SW-TIRF can reveal
the fine structural details of actin cytoskeleton with a high
signal/background ratio close to the basal plasma membrane
of cells. Other complementary imaging modalities such as
atomic force microscopy could be combined with TIRF
(24,25) to provide high resolution imaging on the apical
surface. We have further implemented SW-TIRF in objec-
tive-launched geometry instead of using a prism-launched
method to facilitate the imaging of cellular specimens. One
drawback of objective-launched TIRF is the presence of
stray illumination light arising from the scattering inside
the objective contributing nonevanescent fluorescence back-
From the theory of diffraction-limited imaging provided
by the Airy function, the ideal PSF FWHM is calculated to
be 199 nm with an NA of 1.45 and an emission wavelength
of 560 nm. In contrast, the measured PSF FWHM was 260
nm under the same condition with a 44-nm bead. Since in
SW-TIRF the height of side lobes depends on the original
PSF, a discussion regarding the origin of this nonideal PSF
might be useful. First, the high NA microscope objective
may not have an ideal modulation transfer function from
manufacturing due to strong attenuation of marginal rays
(19). Second, the emission from the fluorescent beads is not
actually monochromatic at 560 nm but peaked at 560 nm
with a long spectral tail into the longer wavelength region.
Third, the effect of bead size adds some broadening to the
final PSF FWHM. However, numerical simulation by
convoluting the finite bead model with the Airy function
gives the broadening of PSF FWHM of ,5% with a 44-nm-
size bead. Fourth, there exists cross talk between CCD pixels
due to the coupling fibers which connect the microchannel
plate in the image intensifier to the CCD chip. The pixelation
error is increased by a factor of 1.5 due to the fiber coupling
ratio, resulting in the effective pixel size of 22.6 mm instead
of the physical pixel size of 15 mm (26). In fact, iCCD is not
our first detector choice for two reasons but is used since it is
available in our laboratory. Our image field of view is
significantly limited by the relatively small CCD chip (512 3
512 pixels) used in this camera. Our imaging speed (;11
fps) is limited by the slow readout rate of this device.
A larger format, electron bombardment CCD, with a fast
readout rate may be a better choice. Other potential reasons
could be the misalignment of the optical setup and the
aberration of relay optics. The broadening of PSF FWHM by
20% was also reported in other systems (13).
In principle, the SW-TIRF resolution is not inherently
limited by the emission PSF. But it is mostly limited by how
small structured excitation could be generated. If a nonlinear
excitation is incorporated, even further resolution could be
achieved (9,15). Without incorporating any nonlinear optical
technique, the PSF FWHM could have reached down to ;90
nm with the setting here. However, the Gaussian laser beam
needs a small margin to generate a uniform SW overlapping
field onto the field of view. A further resolution of 69 nm
could be achieved if the currently available NA 1.65 ob-
jective with 457-nm excitation is used.
We are currently developing a real-time SW-TIRF micro-
scope for live cell imaging with multicolor capability. Semi-
conductor quantum dots might be suitable chromophores for
long-term live cell imaging because of their excellent pho-
tostability and low photobleaching (21). Most importantly,
single excitation wavelength, eliminating the instrumental
and computational complications of imaging using different
colors and periods of standing evanescence waves. However,
quantum dots exhibit characteristic blinking behavior. Blink-
ing could interfere with SW-TIRF microscopy where precise
intensity information is needed for image processing. The
problem might be overcome by biochemical suppression
methods (22). This development could allow the study of
dynamic processes of live cells such as cellular attachment or
and with single molecular sensitivity.
Further resolution improvement may require nonlinear
modalities such as STED or two-photon microscopy with the
additional advantage of side-lobe suppression. However, an
even higher signal/noise ratio may be obtained by utilizing
surface plasmon resonance-coupled emission (27). The
resolutionofanyimaging system depends onthesignal/noise
ratio, which is ultimately limited by the number of collected
photons. For fluorescence methods, this imposes an intrinsic
limit to any approaches to improve resolution by decreasing
the size of PSF due to the relatively increased noise in low
light fluorescence imaging (26). On the other hand, the
nonfluorescent or scattering version of SW-TIR with novel
metal nanoparticles may open up broader applications
(28,29). Since the photon scattering process is instantaneous
the signal photon production rate of metal nanoparticles in
biological systems is only limited by heating processes and
canbe orders ofmagnitudehigher than fluorescence.Further,
there is no concern about photobleaching for metal nano-
particles. These noble metal nanoparticles with very high
scattering power may serve as fluorescent analogs in biology
for SW-TIRF imaging although the imaging process is co-
herent rather than incoherent, as in fluorescence imaging.
1756Chung et al.
Biophysical Journal 93(5) 1747–1757
TIRF microscopy has provided a way to peek into the
dynamics of subcellular structures in the near membrane
region of cells, and a high lateral resolution version of TIRF
has been described in this article. Utilizing the subdiffraction
limit evanescent SW excitation, the lateral resolution has
been improved to reveal finer details of biological specimen
which otherwise would be hidden in a blur of out-of-focus
fluorescence. The first, to our knowledge, two-dimensional
superresolution SW-TIRF imaging with cellular actin cyto-
skeleton is demonstrated by achieving more than 230%
lateral resolution improvement. A 1D SW-TIRF is proved to
be sufficient for substantial improvement of lateral resolution
in the SW direction by imaging beads, achieving a lateral res-
olution of 1096 10 nmbeyond the classical diffraction limit.
In addition, we have shown this system has enough sensi-
tivity to image single quantum dot molecules with superres-
technique that has significantly faster imaging speed over the
point-by-point scanning approaches. SW-TIRF microscopy
To view all of the supplemental files associated with this
article, visit www.biophysj.org.
The authors thank Ms. Maxine Jonas of Massachusetts Institute of
Technology for assisting in the preparation of biological specimens and
Wai Teng Tang of the National University of Singapore for technical
This research was funded by the National Science Foundation (research
grant MCB-9604382) and the National Institutes of Health (P01HL64858).
1. Axelrod, D. 1981. Cell-substrate contacts illuminated by total internal-
reflection fluorescence. J. Cell Biol. 89:141–145.
2. Axelrod, D. 2001. Total internal reflection fluorescence microscopy in
cell biology. Traffic. 2:764–774.
3. Webb, S. E. D., S. R. Needham, S. K. Roberts, and M. L. Martin-
Fernandez. 2006. Multidimensional single-molecule imaging in live
cells using total-internal-reflection fluorescence microscopy. Opt. Lett.
4. Tokunaga, M., K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida.
1997. Single molecule imaging of fluorophores and enzymatic
reactions achieved by objective-type total internal reflection fluores-
cence microscopy. Biochem. Biophys. Res. Commun. 235:47–53.
5. Goodman, J. W. 1996. Introduction to Fourier Optics. McGraw-Hill,
6. Westphal, V., and S. W. Hell. 2005. Nanoscale resolution in the focal
plane of an optical microscope. Phys. Rev. Lett. 94:143903.
7. Westphal, V., L. Kastrup, and S. W. Hell. 2003. Lateral resolution of
28 nm (lambda/25) in far-field fluorescence microscopy. Appl. Phys.
B-Lasers Opt. 77:377–380.
8. Hell, S. W., and J. Wichmann. 1994. Breaking the diffraction
resolution limit by stimulated-emission- depletion fluorescence mi-
croscopy. Opt. Lett. 19:780–782.
9. Gustafsson, M. G. L. 2005. Nonlinear structured-illumination micros-
copy: wide-field fluorescence imaging with theoretically unlimited
resolution. Proc. Natl. Acad. Sci. USA. 102:13081–13086.
10. Mansfield, S. M., and G. S. Kino. 1990. Solid immersion microscope.
Appl. Phys. Lett. 57:2615–2616.
11. Gustafsson, M. G. L. 2000. Surpassing the lateral resolution limit by a
factor of two using structured illumination microscopy. J. Microsc.
12. Fedosseev, R., Y. Belyaev, J. Frohn, and A. Stemmer. 2005. Structured
light illumination for extended resolution in fluorescence microscopy.
Opt. Lasers Eng. 43:403–414.
13. Frohn, J. T., H. F. Knapp, and A. Stemmer. 2000. True optical
resolution beyond the Rayleigh limit achieved by standing wave
illumination. Proc. Natl. Acad. Sci. USA. 97:7232–7236.
14. Gliko, O., G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau.
2006. Standing wave total internal reflection fluorescence microscopy
to measure the size of nanostructures in living cells. J. Biomed. Opt.
15. So, P. T. C., H. S. Kwon, and C. Y. Dong. 2001. Resolution
enhancement in standing-wave total internal reflection microscopy: a
point-spread-function engineering approach. J. Opt. Soc. Am. A Opt.
Image Sci. Vis. 18:2833–2845.
16. Cragg, G. E., and P. T. C. So. 2000. Lateral resolution enhancement
with standing evanescent waves. Opt. Lett. 25:46–48.
17. Chung, E., D. K. Kim, and P. T. C. So. 2006. Extended resolution
wide-field optical imaging: objective-launched standing-wave total
internal reflection fluorescence microscopy. Opt. Lett. 31:945–947.
18. Hanninen, P. E., S. W. Hell, J. Salo, E. Soini, and C. Cremer. 1995.
2-photon excitation 4pi confocal microscope—enhanced axial resolution
microscope for biological-research. Appl. Phys. Lett. 66:1698–1700.
19. Neil, M. A. A., R. Juskaitis, and T. Wilson. 1997. Method of obtaining
optical sectioning by using structured light in a conventional micro-
scope. Opt. Lett. 22:1905–1907.
20. Palm, W. J. 2000. Modeling, analysis, and control of dynamic systems.
Wiley, New York.
21. Michalet, X., F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J.
Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. 2005.
Quantum dots for live cells, in vivo imaging, and diagnostics. Science.
22. Hohng, S., and T. Ha. 2004. Near-complete suppression of quantum
dot blinking in ambient conditions. J. Am. Chem. Soc. 126:1324–1325.
23. Yu, W., P. T. C. So, T. French, and E. Gratton. 1996. Fluorescence
generalized polarization of cell membranes: a two-photon scanning
microscopy approach. Biophys. J. 70:626–636.
24. Mathur, A. B., G. A. Truskey, and W. M. Reichert. 2000. Atomic force
and total internal reflection fluorescence microscopy for the study of
force transmission in endothelial cells. Biophys. J. 78:1725–1735.
25. Sarkar, A., R. B. Robertson, and J. M. Fernandez. 2004. Simultaneous
atomic force microscope and fluorescence measurements of protein
unfolding using a calibrated evanescent wave. Proc. Natl. Acad. Sci.
26. Stelzer, E. H. K. 1998. Contrast, resolution, pixelation, dynamic
range and signal-to-noise ratio: fundamental limits to resolution in
fluorescence light microscopy. J. Microsc. (Oxford). 189:15–24.
27. Kostov, Y., D. S. Smith, L. Tolosa, G. Rao, I. Gryczynski, Z.
Gryczynski, J. Malicka, and J. R. Lakowicz. 2005. Directional surface
plasmon-coupled emission from a 3 nm green fluorescent protein
monolayer. Biotechnol. Prog. 21:1731–1735.
28. Yguerabide, J., and E. E. Yguerabide. 1998. Light-scattering submi-
croscopic particles as highly fluorescent analogs and their use as tracer
labels in clinical and biological applications: I. Theory. Anal. Biochem.
29. Yguerabide, J., and E. E. Yguerabide. 1998. Light-scattering submi-
croscopic particles as highly fluorescent analogs and their use as tracer
labels in clinical and biological applications: II. Experimental charac-
terization. Anal. Biochem. 262:157–176.
SW-TIRF Imaging of Biological Specimens1757
Biophysical Journal 93(5) 1747–1757