Construction of DiMPS

We constructed an optical system to obtain two images having distinct pupil functions that were arbitrarily modulated (Fig. 1A, B, and see materials and methods). The DiMPS has two optic relays. The first relay is for splitting the image into two polarized images and for modulating the pupil function of each. An image passes through a spatial mask positioned at the imaging plane to reduce the field of view by half (S in Fig. 1 A and B). The optical pathway is split into two by a polarizing beam splitter before the Fourier plane (BS1). Reflective LCOS (Liquid Crystal On Silicon) micro mirror arrays (LCMs) are set on two of the Fourier planes of lens L1. Same mask pattern was used for both of LCMs to generate two images with opposite polarization, except for siDiMPS where two images were subtracted to generate one resolution enhanced image (see below). The polarization state of each pixel can be controlled by using a video graphics array signal from a computer at a refresh rate of 60 Hz. The light whose polarization has been modulated by the LCMs is transmitted through or reflected by the polarizing beam splitter. The system composed of the LCMs and the beam splitter can modulate only the amplitude but not the phase. A phase spatial light modulator based on LCOS technology is also available for similar equipment, however, it is not suitable for fluorescent multi-color imaging because of its strong wavelength dependency. The applicability for multi-color imaging is necessary for the recent biology. Because we here had focused on applicability and user-friendliness in vast cases of biological assay, the amplitude modulation, which has the low wavelength dependency such as one for a video projector, is preferred. Its lower price is another reason that amplitude modulation is preferable for practical use. The second relay is for optimizing the in-focus positions of the two images [15]. Moving either L3 or L4 along the optical axis changes the optical length between the two lenses, resulting in a difference in the in-focus positions. Finally, the images at two pathways are combined into one image, or projected side by side onto a charge-coupled device (CCD) camera by a second polarizing beam splitter (BS2). When the specimen had an anisotropic polarization property such as a birefringent sample, a quartz depolarizer was set at the back of the objective to negate any effect of the polarization.

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Figure 1. Basic of the DiMPS.

(A) Photograph of the DiMPS. (B) Schematic drawing of the DiMPS. S, spatial mask; BS, polarizing beam splitter; LCM, liquid crystal micro mirror array; P, prism mirror; L, lens; FP, Fourier plane; CP, conjugate plane. See methods for the optics construction. (C) Typical modulations of the pupil function. The pupil function is the polarization pattern in the LCM. Black part shows where the light is blocked. The PSF is represented by the fluorescence distribution of a φ100 nm bead with an emission peak at 515 nm (Invitrogen). Red lines show the one-dimensional fluorescence intensity profile of the PSF along the transverse and longitudinal axes. Gray lines are the one-dimensional fluorescence intensity profile of a normal PSF. Scale bars, 1 µm.

https://doi.org/10.1371/journal.pone.0044028.g001

To examine the effect of pupil function modulation using the DiMPS, we observed a fluorescence image of a fluorescent bead of 100 nm in diameter as a PSF (Fig. 1C and Fig. S1). The longitudinal (Z) section was obtained by sequential scanning at 50 nm interval. The typical shape of the pupil function is a full circle. In our case, the diameter of the pupil on the surface on an LCM is approximately 1.5 mm with a 150× objective. Decreasing the diameter of the pupil function blurred the fluorescence image of the bead in both the longitudinal (optical) and transverse (horizontal) axes (Fig. 1C, center). Meanwhile, masking of the center of the pupil function shrunk the image (Fig. 1C, right) down to 180 nm at minimum (Fig. S1, top-left). We can apply arbitrary pupil patterns on the LCMs and the fluorescence images are modulated in correspondence to these pupil functions (Fig. S1, lower). Thus, though masking of the pupil function decreases the signal intensity from a specimen, the image can be easily modulated.

Fourier High-pass Filtering and Extension of Focal Depth with the DiMPS

The spatial resolution of an optical microscope is significantly constrained by optical diffraction limits [16]. To overcome this limitation, a number of techniques have been developed that are called superresolution [11]. The DiMPS achieves superresolution-like effect by masking the center of the pupil function (superresolving-masked) [13], though the effect is smaller than recent superresolution techniques. The spatial resolution of a microscope is usually defined as the full width at half maximum (FWHM) of the PSF profile. When using a ring with an inner diameter of 624 µm and an outer diameter of 1456 µm as a pupil function, the FWHM is improved to 190 nm from 230 nm by the high-pass filtering effect at the Fourier plane (Fig. 1C, right). Note that, there is a trade-off between the improvement of FWHM and the intensity, which is directly related to the signal to noise (S/N) ratio of the image, for example, the intensity is decreased 10-fold to obtain 180 nm spatial resolution (Fig. S2). The LCM used here, whose contrast ratio is 1000∶1, cannot absolutely block 100% of the light. The reflection efficiency of the beam splitter used was not also 100% (>90% from 500 nm to 650 nm wavelength) while the transmission was almost 100%. Therefore, when the area of the mask become too large, the light that was not blocked become dominant elements than one that was transmitted and the improvement of FWHM is diminished (Fig. S2, right). To confirm the effect of the superresolving-mask on cell imaging, we observed actin bundles labeled with Alexa488-phalloidin in a fixed cell which had enough intensity to compensate for the S/N ratio decrease (Fig. 2A and B). The actin bundles were more clearly observed by the high-pass filtering effect with the superresolving-mask. The fluorescence intensity profile of the images shows the obvious improvement of the spatial resolution with the superresolving-mask (Fig. 2C). Thus, we can easily obtain a resolution enhanced image by using DiMPS without the use of other equipment.

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Figure 2. Fluorescent imaging with the DiMPS.

(A) Fluorescent images of actin bundles labeled with Alexa488 phalloidin in a HeLa cell. Left, non-masked image. Right, high-pass filtered image with a superresolving-mask. Scale bars, 2 µm. (B) Enlarged images of the yellow rectangles in A. Scale bars, 1 µm. (C) One-dimensional fluorescence intensity profile of the yellow lines in B. Blue, non-masked. Red, superresolving-masked. (D) Extended PSF. Left panels, the pupil functions used. Right panels, the PSFs. The PSF is the fluorescence distribution of a φ100 nm fluorescent bead (right panels). Scale bars, 1 µm. (E) Two typical fluorescent images of actin bundles in overlapping cells. Upper and lower panels are non-masked images and ones obtained with the extended PSF, respectively. Yellow arrowheads indicate the points that are not visible with the normal PSF but are with the extended one. Scale bars, 2 µm. (F) Observation of GFP-fused EB1 in a living KPL4 cell. Upper and lower panels are non-masked images and ones obtained with the extended PSF, respectively. Left panels, schematic drawing showing how EB1 is illuminated. Right panels, snapshots of the EB1 observation. The movies are available as supplementary material (movies S1 and S2). Scale bars, 2 µm.

https://doi.org/10.1371/journal.pone.0044028.g002

The DiMPS also enables one to achieve an extended PSF on the longitudinal axis, which increases the focal depth [17] (Fig. 2D). The focal depth of the extended PSF in our case was roughly 3 µm, while that under the normal condition is about 1 µm. When observing the place where cells overlapped, the DiMPS enable us to observe both cells using the extended PSF (Fig. 2E). This ability of DiMPS to give an extended PSF is applicable to live cell imaging. To confirm the effect of the extended PSF on live cell imaging, we performed live cell imaging of GFP-labeled EB1, which is one of the microtubule-binding proteins targeting the plus ends of polymerizing microtubules [18]–[20] (Fig. 2F, and movies S1 and S2). While the fluorescent image of EB1 usually forms a comet shape [19], [20], the length of the comet is estimated to be slightly shorter because of the thin focal depth on conventional fluorescent microscopy (Fig. 2F, upper). In contrast, the DiMPS with an extended PSF visualized more EB1-comets, and each comet was longer than that observed with a conventional microscope (Fig. 2F, lower). Thus, by using the DiMPS, microscope user can obtain various effects on the image in real time and on site just by changing the pupil function.

Subtractive Imaging with the DiMPS (siDiMPS) for Resolution Enhancement

We have shown the effect of modulation of the pupil function so far. Here, we demonstrate an application involving image computation with two images acquired by distinctly modulating the pupil functions; i.e., a resolution enhancement method with which the spatial resolution can be further enhanced by subtracting the apodizing-masked image from the superresolving-masked image, similar to the subtraction performed for a confocal pair with different pinhole sizes [21]. The only requirement for better resolution is to identify two images with distinct PSF profiles such that the subtracted PSF is as narrow as possible. Because of various aberrations in the optics including the objective, it is difficult to precisely pre-calculate the PSF shape. Moreover, the effect of contrast ratio of the LCMs, which, is appeared when the reflective area is small (Fig. S2), also complicates the pre-calculation. Therefore, we sought the optimal pupil function by actual measurements since the DiMPS can easily produce a huge number of the mask pattern. By testing more than 200 pairs of images, we identified the best combination of pupil functions to achieve the narrowest PSF that is defined by the fluorescence distribution of a fluorescent bead of 100 nm in diameter (Fig. 3, A–F). Although outer rim of the Fourier plane contains high spatial information of the image, we noticed that PSF elongates in the longitudinal direction when pupil function of large diameter ring was used (Fig. S1, top left). This is probably due to the large effect of the aberration in this area, and worsens the resolution along the longitudinal axis. Thus, the best mask we used consists of the mask that blocks both inner and outer area of the Fourier plane. The best resolution was achieved with two ring-shaped masks; one with an inner diameter of 208 µm and an outer diameter of 832 µm (Fig. 3, B and D, blue), and the other with an inner diameter of 624 µm and outer diameter of 1248 µm (Fig. 3 C and D, red), the FWHM of the subtracted PSF improved from 230 nm to 150 nm along the transverse axis, and from 940 nm to 530 nm along the longitudinal (optical) axis (Fig. 3 E). The in-focus positions of the two pathways were unmatched, most likely due to spherical aberrations (Fig. 3 D, right panel). By moving lens L4 in Fig. 1, we can adjust the in-focus positions of the two images, which are defined as where the contrast is highest, independently [15]. This adjustment was made so that the subtracted PSF was narrower (Fig. 3 F). In the present study, we achieved a spatial resolution of 110 nm along the transverse axis and 460 nm along the longitudinal one, respectively. We named this method siDiMPS (subtractive imaging of DiMPS). An obtained image consists of a superresolving-masked image (upper) and an apodizing-masked one (lower) in 512×512 pixels (22×22 µm²) (Fig. S3 A). The non-weighted subtracted image of the two images was displayed on site in real time (Fig. S3 B). When observing an image of actin bundles labeled with Alexa488-phalloidin in fixed mouse fibroblasts cells, the siDiMPS not only enabled us to obtain a resolution-enhanced image but also decreased the background fluorescence since the resolution along both the transverse and longitudinal axes was improve (Fig. 3 G and H). Additionally applying the deconvolution process [22], we could obtain the clearer image (Fig. 3 G, bottom), and it was possible to individually discriminate two actin bundles within 250 nm that is close to the resolution limit of the conventional microscope (Fig. 3 H, red).

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Figure 3. Resolution enhancement with the siDiMPS.

(A, B, C) Pupil functions and fluorescence distributions of a φ100 nm bead with a non-masked (A), apodizing-masked (B), and superresolving-masked PSF (C). Upper panels, pupil functions; lower panels, PSF images in the longitudinal-transverse plane. (D) One-dimensional fluorescence intensity profiles of the non-masked (black), apodizing-masked (blue), and superresolving-masked PSF (red). Values indicate spatial resolution determined at FWHM. (E, F) Fluorescence distributions determined by subtracting an apodizing-masked PSF from a superresolving-masked one without (E), and with optimizing symmetry of PSF by adjusting lens L4 position along the light path (F). Left panels, PSF images in the longitudinal-transverse plane; middle and right panels, one-dimensional fluorescence intensity profile of each PSF along the transverse axis (middle) and longitudinal axis (right), respectively. Values indicate spatial resolution determined at FWHM. Red and gray lines are one dimensional fluorescence profiles of the non-masked (E) and extended PSF (F), respectively. All scale bars, 1 µm. (G) siDiMPS image of Alexa488-phalloidin stained actin bundles in a fixed cell. Top, non-masked. Middle, siDiMPS. Bottom, siDiMPS+deconvolution. Inserts, magnifications of the areas in yellow rectangles, respectively. Scale bars, 2 µm. The images were obtained with a 100 ms exposure time. (H) One-dimensional fluorescence intensity profiles of the yellow broken lines in G (black, conventional; green, siDiMPS; red, siDiMPS+deconvolution).

https://doi.org/10.1371/journal.pone.0044028.g003

GFP Live Observation Using siDiMPS

To further demonstrate the effect of siDiMPS, we observed the localization of E-cadherin fused with GFP in a living cell. E-Cadherin is a calcium-dependent cell adhesion molecule that plays important roles in cell morphology and architecture in a wide variety of cells, and its loss promotes tumor progression [23], [24]. A cell that overexpresses E-cadherin-GFP was seeded onto a glass surface coated with purified E-cadherin to mimic cell-cell adhesion [25]. SiDiMPS enabled us to observe the fiber-like structure of E-cadherin more clearly than under a conventional microscope (Fig. 4, A and B, and movie S3). Furthermore, the background fluorescence, which was most likely caused by E-cadherin-GFP in the cytosol, was dramatically reduced due to the subtraction of low-spatial frequency image. The fiber-like structure resembled a string of beads composed of small E-cadherin. This bead-like localization was more clearly observed in the siDiMPS image (Fig. 4, A and B, insertions, and C). A histogram of the distances between localizations showed peaks at 251, 457 and 617 nm, suggesting that cadherin clusters were arranged every ∼200 nm (Fig. 4 D), which is in good agreement with that observed with other nanoscopic methods [26], [27]. This distance (∼200 nm) is similar to the sizes of the compartmentalization structures or actin meshwork along the cytoplasmic surface of the plasma membrane [28]–[30]. The siDiMPS enabled us to obtain the resolution-enhanced image where E-cadherin clusters are arranged along actin fibers underneath the membrane in a living cell.

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Figure 4. Biological applications of siDiMPS using green fluorescent protein.

(A,B) Fluorescence images of localized E-cadherin-GFP in a living cell obtained by conventional microscopy (A) and siDiMPS (B). Scale bars, 1 µm. The exposure time for the camera was 30.28 ms. The images shown are the averages of 100 acquired images (total frame rate, 0.3 Hz). Inserts, magnifications of the areas in orange rectangles, respectively. Scale bars, 200 nm. (C) One-dimensional fluorescence intensity profiles inside the yellow rectangle in A (blue, conventional) and B (red, siDiMPS). Arrowheads indicate the periodic peaks of fluorescence intensity. (D) Histogram of the distance between two peaks within the periodic localization of E-cadherin-GFP. Arrowheads and values represent the peaks in the histogram. (E, F) Fluorescence images of autophagosomes labeled with GFP-LC3 in a living cell acquired by conventional microscopy (E) and siDiMPS (F). Scale bars, 1 µm. Inserts, the intensity profiles of the one-dimensional fluorescence intensity profile (yellow line) in each panel. (G) siDiMPS time lapse images of a small organelle labeled with GFP-LC3. Scale bar, 400 nm. The exposure time for the camera was 30.28 ms. Images E, F and G are averages of 10 acquired images (total frame rate, 3 Hz). (H, I) Three dimensional images of autophagosome acquired by conventional microscopy (H) and siDiMPS (I). Left and middle panels are XY-image and XZ-image, respectively. Right panels are one-dimensional fluorescence intensity profile along longitudinal axis (Z) at the center of the images. Scale bars, 1 µm. The exposure time for the camera was 100 ms.

https://doi.org/10.1371/journal.pone.0044028.g004

E-Cadherin observations were limited to the juxtamembrane region underneath the glass surface. To further demonstrate the potential of siDiMPS for intracellular imaging, we observed autophagosomes containing GFP-tagged microtubule associated protein light chain 3 (LC3) [31]–[33]. Autophagy is a catabolic process comprising degradation of cytoplasmic constituents via the lysosomal machinery and is induced by starvation [31], [33]. At 30 minutes after starvation in a mouse embryonic fibroblast (MEF) cell, many organelles labeled with GFP-LC3 were observed intracellularly under a fluorescence microscope (Fig. 4 E). SiDiMPS emphasized the periphery of organelles that were most likely autophagosomes (Fig. 4 F and movie S4). Time-lapse imaging showed that a small, 300 nm diameter, organelle was moving and changing in shape in a manner similar to an amoeba (Fig. 4 G and movie S5). Though most GFP-LC3 labeled organelles over 1000 nm had a round shape, which is also indicative of autophagosomes, amoeba-like organelles were occasionally observed among them (movie S6). To confirm the resolution enhancement along the longitudinal axis, we compared the three dimensional image of the autophagosome obtained by Z-scan at 50 nm interval, and compared images taken by conventional microscope and siDiMPS (Fig. 4, H and I). The sectioning effect along the longitudinal axis was clearly confirmed by the siDiMPS image. Furthermore, siDiMPS can obtain movies with the resolution enhanced. The images taken at 300 ms gave us to observe unidirectional movement of the small autophagosomes (movies S7). Thus, siDiMPS can be used to simultaneously observe structural changes and movements of small organelles in living cells in real time, which would be advantageous for interpreting the observed phenomenon.

Resolution Enhancement at High Frame Rate Using siDiMPS

Frame rate of the imaging system with DiMPS is defined by the frame rate of the camera. However, increasing the camera frame rate results in reduction of the S/N ratio because of the lack of photons the camera receives. The subtraction process in siDiMPS further reduces the S/N ratio [21]. We tested the effect of siDiMPS by increasing the camera frame rate with a quantum dot (QD), which is a semi-conductor fluorescent particle exhibiting intense and stable fluorescence [34]. Though the S/N ratio was obviously decreased by the siDiMPS process, FWHM improved to 100∼140 nm (122±17 nm) from 205 nm with a high-speed CCD camera at a frame rate of 2 ms (1.93 ms exposure time) when the individual QDs were fixed on a glass surface (Fig. 5, A and B).

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Figure 5. Individual quantum dots observation with 2 ms temporal resolutions with the siDiMPS.

(A) Four typical fluorescence images of QDs captured by a high speed CCD camera with a conventional microscope (upper) and the DiMPS (lower). One pixel, 64.5 nm. (B) Ten PSFs of fluorescence QD images acquired with the DiMPS with 2 ms temporal resolution. The gray line is the fitted Gaussian curve, which indicates the spatial resolution (122 nm). (C, D) Fluorescent images of E-cadherin-QD on the cell membrane acquired by conventional microscopy (C) and DiMPS (D). Scale bars, 1 µm. Inserts are magnifications of the red rectangles. Scale bars, 200 nm. Dotted cyan circles indicate individual QDs. (E) Time courses of the dissociation of the E-cadherin complex (upper) and kymograph of the cyan broken line showing dissociation (lower left) and association (lower right). Numbers in the panels indicate elapsed time (ms). Position scale bars, 200 nm; time scale bar, 100 ms. (F) Time course of the cross-sections (cyan broken line) for the upper panel in E. Red and blue lines are fitting results using double Gaussian functions and two single Gaussian functions used to make the fitted double Gaussian, respectively.

https://doi.org/10.1371/journal.pone.0044028.g005

We then observed the diffusive movements of individual E-cadherin labeled with QD on a cell membrane at 120 nm spatial and 2 ms temporal resolutions (Fig. 5, C, D, movie S8 and S9). More QDs could be independently identified in DiMPS than in conventional microscopy (Fig. 5, C, D, inserts and Fig. S4). Additionally, we could detect the association-dissociation cycle of an E-cadherin complex (Fig. 5, E and movie S10). By examining the time course of the distance between two QDs, which was obtained by fitting two Gaussian functions to one-dimensional cross sections, we estimated the dissociation velocity to be 8.7 nm/ms (Fig. 5, F). It is difficult to detect this rapid and small association-dissociation cycle with current superresolution methods because their temporal resolution is too low [11]. This observation shows the capability of siDiMPS, which allows 120 nm spatial resolution at 2 ms temporal resolution.

On Site Fourier-filtering in Transmission Observation

With the DiMPS, the mask pattern of the pupil function can be changed on the computer during the image acquisition, leading to the realization of on site optical Fourier filtering with arbitrary pupil functions. We can also apply the DiMPS to transmission imaging. We obtained modulated images of a living cell and the power spectrum of Fourier-transformed images with various pupil function to investigate the effect of Fourier-filtration by pupil function modulation on bright field transmission microscopy (Fig. 6 A and Fig. S5). With decreasing the diameter of transmission area of the pupil function, the obtained image loses the high spatial frequency information. On the other hand, on masking of the center of the pupil function, the low spatial frequency information was lost, resulting in enhanced resolution, which can be seen in observation of thin filopodia.

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Figure 6. Transmission observation with DiMPS.

(A) Effect of Fourier-filter with DiMPS. Left panels, the pupil functions used. White indicates transmission and black indicates blocking. Middle panels, images of a KPL4 cell on transmitted light irradiation. Right panels, two-dimensional power spectra of the middle images. Yellow lines are the intensity profile of the center of the 2D power spectrum, that is, the power spectrum along X direction. Scale bar, 2 µm. (B) Comparison of normal (left upper), pseudo-relief (right), and high-pass filtered (left lower) images. Inserts, the pupil functions used. (C) One dimensional cross-section of the broken lines in B. Green, normal. Blue, high-pass. Red, pseudo-relief. Scale bars, 2 µm.

https://doi.org/10.1371/journal.pone.0044028.g006

The DiMPS also allows imaging of phase gradient specimens similarly to in the case of Hoffman modulation contrast (HMC), with which one can obtain pseudo-relief images of specimens [35]. On HMC microscopy, a diaphragm with an off-axis slit is placed on the front pupil plane of the illumination condenser lens, and a Hoffman modulator, which has three regions of different neutral densities (100%, 15% and 1% light transmission), is set on the Fourier plane of the objective. With this configuration, one can obtain images composed of light scattered and refracted by the phase difference and thus realize imaging of phase samples with high contrast. The DiMPS cannot modulate the incident light, but is capable of yielding different neutral densities of the pupil function on the detection pathway. When the numerical aperture (NA) of the objective is larger than that of the condenser lens, DiMPS gives an image effect similar to MHC by masking half of the Fourier plane (Fig. 6, B). Unlike a commercial HMC microscope, the DiMPS can arbitrarily change the degree of the phase gradient by changing the pupil function without changing the modulator-slit plate (Fig. S6). While the high-pass filter with the DiMPS enhanced the thin filopodia and the edge of a cell and improved the contrast, pseudo-relief imaging enabled us to observe the cell as a shadowed image; especially the small vesicles could be clearly observed (Fig. 6, B and C, and movie S11,S12,S13). Since the DiMPS can modulate two distinct images simultaneously, the microscope user can simultaneously observe the two effects of the pupil function modulations on an image by projecting these images side by side onto a camera. Thus, the DiMPS can be applicable to the imaging of phase samples, such as cells, by transmission imaging without need of modifying the incident light.

Microscopy

Our microscope setup consisted of an epi-fluorescence microscope (IX-71; Olympus Co., Japan), an objective lens (150X PlanApo, 1.45 NA; Olympus Co., Japan), a relay optics box for dual-view imaging (GA03; G-Angstrom, Japan), and an electron multiplier type CCD camera (EM-CCD, iXon DV887 or DU897; Andor Technology plc., UK). In the relay optics box, the image from the microscope was relayed two times with two 4f-optical systems (Fig. 1). Reflective liquid crystal micro mirror arrays (LCM; DILA-SX073-S; JVC KENWOOD Corporation, Japan) have specifications of 1408×1058 resolution with a 10.4 µm pixel pitch. The contrast ratio of the LCM is 1000∶1. The LCMs were controlled by an Analog/Digital transfer board (D-ILA Evaluation Board, DEB-D4; Victor, Japan). The focal length of the lenses in the DiMPS were 75 mm for L1, 100 mm for L2, 70 mm for L3 and L4, and 130 mm for L5 (see Fig. 1). The total magnification of our setup was ∼370. A quartz depolarizer (DEQ-2S; Sigma Koki, Japan) was set at the back of the objective to negate any effect caused by the anisotropic polarization property of fluorescent probes. The position of the objective can be precisely controlled by a piezo actuator (P-721.CDQ and E-665.CR; Physik Instrumente, Germany). For confocal and multi-color imaging, a scanning confocal unit (CSU-X1; Yokogawa Electric Corporation, Japan) was placed at the image plane of the microscope. The DiMPS was set on the image plane of the scanning confocal unit.

The optical components were purchased from Sigma Koki Co., LTD (Japan), and the holders and stages from Suruga Seiki Co., LTD (Japan). Images obtained with the CCD camera were composed of 14-bit data per pixel. The images were imported into analysis software programmed in Visual C++ (Microsoft, USA) and the OpenCV library (IBM, USA).

Biological Sample and Observation Conditions

For fluorescence imaging, an ∼30×30 µm² area was illuminated with a xenon lamp with an excitation filter of 460∼495 nm. An actin bundle of a mouse fibroblast (L cell, gift from Prof. M. Takeichi) [45] fixed on a glass bottom dish was labeled with Alexa-488 phalloidin. For real time imaging, we used a breast cancer cell, a KPL cell, kindly provided from Prof. J. Kurebayashi [46], that overexpresses EB1 fused with GFP. The exposure time for the camera (iXon DV887) was 200 ms for actin observation and 1.0 s for EB1 observation.

For confocal and multi-color imaging, actin bundles and microtubules were labeled with rhodamine-phalloidin and Alexa488-anti-tubulin in a HeLa cell (purchased from Riken Cell Bank; RCB0007). An ∼30×30 µm² area was simultaneously illuminated with blue (488 nm) and green (532 nm) lasers. The exposure time for the camera (iXon DV887) was 300 ms.

We used two types of cells for transmission imaging. One was a KPL4 cell, as a normal thick specimen. The other is myoblast cell line C2C12 (purchased from Riken Cell Bank; RCB0987), which is a useful model cell line for studying the differentiation of non-muscle cells into skeletal muscle cells [47], which was used as a thin specimen in our study. The light irradiation was performed with light of 460∼480 nm wavelength though a band-pass filter in a halogen lamp. The exposure time for the camera (iXon DV887) was 100 ms.

siDiMPS Observation

We used an E-cadherin system for juxtamembrane observation. To fuse E-cadherin with GFP, we inserted mouse E-cadherin cDNA into a pCA-sal-EGFP-IRES-neo vector in which a neomycin resistance gene was driven by the internal ribosome entry sites (IRES) [48]. pCA-E-cadherin-EGFP-IRES-neo plasmids were transfected into mouse fibroblasts (L cells) by using an Amaxa electroporation system (Amaxa) and selected with 400 µg/ml G418 (EEG-L cells, gift from Prof. M. Takeichi) [45]. Just before observation of E-cadherin-EGFP localization, EEG-L cells were re-plated onto E-cadherin coated glass and cultured for 8–10 hours at 37°C in L-15 medium (Invitrogen). The E-cadherin coated glass was obtained as follows; goat anti-Human IgG-Fc (BETHYL) was nonspecifically bound to the surface of a glass bottom dish (Matsunami), followed by the loading of mouse E-cadherin-Fc (R&D Systems) into the dish. After incubation for 1 hour at 37°C, the glass surface was washed out with PBS and then blocked with 1% BSA containing 5 mM Ca²⁺ for 1 hour at 37°C. Microscopic observations were carried out at 25°C. The exposure time for the camera (iXon DV887) was 30.28 ms. To construct a final image, 100 images were averaged (total frame rate, 0.3 Hz).

For intracellular observation, we choose an autophagy system. The plasmid used for microtubule associated protein light chain 3 (LC3)-fused GFP (GFP-LC3) was kindly provided by Prof. T. Yoshimori (Department of Genetics Graduate School of Medicine, Osaka University) and Dr. S. Yamaoka (Tokyo Medical and Dental University). Mouse embryonic fibroblasts expressing GFP-LC3 were prepared as described previously [49]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. For starvation, DMEM was replaced with phenol red-free Hank’s balanced salt solution, with incubation for 30 min at 37°C. Microscopic observations were carried out at 25°C. The exposure time for the camera (iXon DV887) was 30.28 ms.

Single Particle Tracking of E-cadherin

Before single particle tracking of E-cadherin, cell surface E-cadherins were labeled with anti-E-cadherin mAb for 1 hour at 37°C and then QD goat F(ab’) 2 anti-rat IgG conjugate (Invitrogen) for another hour at 37°C. The final concentration of QD was 200 pM in 2 ml medium. The peak wavelength of the QD fluorescent spectrum was 605 nm. Microscope observations were carried out at 25°C. Exposure time of the camera (iXon DV897) was 1.942 ms (frame rate, 2 ms).

The two dimensional position of an individual QD could be calculated by fitting its fluorescent image with a two dimensional Gaussian function as follows,where I₀ is the peak intensity, (x₀, y₀) is the position of the QD and C is the background intensity. When I₀ was calculated with a fitting three times larger than the standard deviation of the background intensity, the computer could record the position (x₀, y₀) of the fluorescent spot.