2.1.

Chemicals and Solutions

The fluorescein-phalloidin, unlabeled phalloidin, phosphocreatine, creatine kinase, glucose oxidase, and catalase were from Sigma (St Louis, Missouri). The rhodamine-phalloidin was from Molecular Probes (Eugene, Oregon).

2.2.

Preparation of Myofibrils

Rabbit psoas muscle was first prewashed with cold EDTA-rigor solution ( $50\mathrm{mM}$ KCl, $2\mathrm{mM}$ EDTA, $10\mathrm{mM}$ DTT, and $10\mathrm{mM}$ TRIS-HCl pH 7.6) for $\frac{1}{2}\mathrm{h}$ , followed by Ca-rigor solution ( $50\mathrm{mM}$ KCl, $2\mathrm{mM}$ $\mathrm{Mg}{\mathrm{Cl}}_2$ , $0.1\mathrm{mM}$ $\mathrm{Ca}{\mathrm{Cl}}_2$ , $10\mathrm{mM}$ DTT, and $10\mathrm{mM}$ TRIS-HCl pH 7.6). Myofibrils were made from muscle as previously described.²

2.3.

Labeling and Preparation of Myofibrils

Unless otherwise indicated, myofibrils $(1\mathrm{mg}∕\mathrm{mL})$ on glass coverslips were labeled for $5\min$ with $0.01\mu \mathrm{M}$ fluorescent phalloidin $\left(\mathrm{RP}\right)+9.99\mu \mathrm{M}$ unlabeled phalloidin (UP). When indicated, they were labeled for $5\min$ with $0.1\mu \mathrm{M}$ $\mathrm{RP}+9.9\mu \mathrm{M}$ UP. After labeling, myofibrils were washed by centrifugation on a desktop centrifuge at $3000\mathrm{rpm}$ for $2\min$ followed by resuspension in rigor solution. Myofibrillar suspension $\left(15\mu \mathrm{L}\right)$ was placed on uncoated or coated coverslips then covered with glass coverslip (to avoid drying); and washed with three to four volumes of rigor solution containing phosphocreatine, creatine kinase, glucose oxidase and catalase to remove oxygen.⁴

2.4.

Microscope Slides

Microscope slides were covered with metal by vapor deposition by EMF Corp. (Ithaca, New York). A $52\text{-}\mathrm{nm}$ -thick layer of silver, a $48\text{-}\mathrm{nm}$ layer of gold, or a $20\text{-}\mathrm{nm}$ layer of aluminum was deposited on the slides. A $2\text{-}\mathrm{nm}$ chromium undercoat was used as an adhesive background. A $5\text{-}\mathrm{nm}$ layer of silica was layered on top of the silver, to protect it from oxidation by air.

2.5.

Preparation of Silver Island Films

Slides were incubated with poly-L-lysine solution (Sigma, 0.1%) for $1\mathrm{h}$ and rinsed thoroughly with water. Then $500\mathrm{mg}$ of $\mathrm{Ag}\mathrm{N}{\mathrm{O}}_3$ (99%, Aldrich) was dissolved in $60\mathrm{ml}$ of water and 5% NaOH was added until a brown precipitate developed. The precipitate was collected, redissolved by the addition of 30% $\mathrm{N}{\mathrm{H}}_4\mathrm{O}\mathrm{H}$ , and then cooled in ice for $3\min$ . Next, $15\mathrm{ml}$ of glucose $(48\mathrm{mg}∕\mathrm{ml})$ were added to this solution and the coverslips were inserted into it. The solution was then heated for $2\min$ , followed by cooling at room temperature (RT) for $3\min$ . The slides were removed when the solution turned cloudy.

2.6.

Data Analysis

Data were processed using MathCad 2001i Professional.

2.7.

Atomic Force Microscope (AFM)

Imaging was performed on the AFM Explorer (ThermoMicroscopes/Veeco Instruments Inc.) in contact scanning mode with a Non-Conductive Silicon Nitride Probe (Veeco Instruments Inc.). Images were acquired at a rate of $2\text{to}5\mu \mathrm{m}∕\mathrm{s}$ with a resolution of $300\text{pixels}∕\text{line}$ . Images were then processed with WSxM Version 4.0 software for 3-D structure and analyzed with the Veeco SPMLab Version 6.0.2 software for $Z$ , $X$ , and $Y$ distance quantification.

2.8.

Bulk Fluorescence Measurements

The sample was positioned in the sample compartment of a Varian Eclipse Spectrofluorometer (Varian, Inc.) in the front-face configuration. The excitation was at $475\mathrm{nm}$ , and on the observation we used a $540\text{-}\mathrm{nm}$ -long wave pass filter. Under these conditions, there was no detectable signal from bare glass slide or SIF reference slide.

2.9.

Measuring Fluorescence Lifetimes

Fluorescence lifetimes were measured by the time-domain technique using FluoTime 200 fluorometer (PicoQuant, Inc.). The sample was positioned in front-face configuration inside the fluorometer chamber. The excitation was by a $475\text{-}\mathrm{nm}$ laser-pulsed diode, and the observation was through a monochromator at $575\mathrm{nm}$ with a supporting $550\text{-}\mathrm{nm}$ -long wave pass filter. The FWHM of pulse response function was $68\mathrm{ps}$ (measured by PicoQuant, Inc.). The reconvolution procedure was used to take into account the excitation pulse shape. The time resolution was better than $10\mathrm{ps}$ . Less than 0.5% background was detected from the SIF slide. The intensity decays were analyzed in terms of a multiexponential model using FluoFit software (PicoQuant, Inc.).

2.J.

Microscopic Measurements

The microscope was described earlier.¹⁸ Here we used a modification whereby the myofibril is first placed on a metal-covered microscope slide coated with SIF and then immersed in Ca-rigor solution. A small chamber was made by sealing the sides around the sample with Vaseline and the top with a #1 glass coverslip (Fisher) (Fig. 1 ). The chamber is placed coverslip-down on a movable piezostage (Nano-H100, Mad City Labs, Madison, Wisconsin) that is installed on the stage of an Olympus $\mathrm{I}\times 51$ microscope. The piezostage is controlled by a nanodrive. This provides sufficient resolution to place the region of interest (ROI) in a position conjugate to the confocal aperture.¹⁹ The excitation light from an expanded diode-pumped solid state (DPSS) laser beam (Compass 215M, Coherent, Santa Clara, California) enters the commercial TIRF illuminator (Olympus, Center Valley, Pennsylvania). The expanded laser beam is focused at the back focal plane of the objective and directed by the movable optical fiber adapter to the periphery of the objective [Olympus PlanApo $60\times$ , 1.45 numerical aperture (NA)]. At the interface between glass and solution the beam refracts and propagates toward the sample at an acute incidence angle. Note that this angle is not equal to the TIRF angle. TIRF excitation is impossible with this configuration, because the evanescent wave, which is created at the glass-solution interface, never reaches the muscle. The sample is excited directly by the laser beam, not by evanescent wave. The same $\mathrm{NA}=1.45$ objective is used to collect the fluorescent light. The light is projected onto a tube lens, which focuses it at the conjugate image plane. A confocal aperture or an optical fiber (whose core acts as a confocal aperture) is inserted at this plane. An avalanche photodiode (APD, Perkin-Elmer SPCM-AQR-15-FC) collects light emerging from the aperture.

Fig. 1

Experimental arrangement. Myofibril is placed on microscope slide covered with thin layer of metal and coated with SIF. An experimental chamber is made by covering a sample with a glass coverslip. The sample is illuminated directly by the green light, not necessarily at a total internal reflection fluorescent (TIRF) angle, because the evanescent wave produced at the coverslip/buffer interface does not reach a sample in any case. Fluorescence (orange) is collected by the objective, projected by the tube lens onto a confocal aperture, and detected by the APD.

The conditions of the intensity measurements on different substrates were kept strictly the same. The samples were placed sequentially on the microscope stage without altering the focus or angle of illumination. The nature of the metal substrate did not affect the focus. The angle of illumination was unchanged, since samples were not illuminated by TIRF. The only modification allowed was insertion of neutral density filters in the excitation light path. This was necessary because the APD was becoming saturated when measuring fluorescence on highly enhancing substrates (e.g., $\text{silver}+\mathrm{SIF}$ ). The values reported are those obtained within first few seconds after opening the shutter and admitting laser light.

2.K.

Selecting Aperture Size

The projection of the aperture on the object plane defines the lateral $(X,Y)$ dimension of the detection volume, and is equal to the diameter of the confocal aperture $\left(D\right)$ divided by the magnification of the objective $(M=60)$ . The aperture should be as large as possible to maximize the signal, but it makes no sense to make the projection less than $R=0.25\mu \mathrm{m}$ , the optical resolution of the $\mathrm{NA}=1.45$ objective used here. Therefore, the optimal size of the confocal aperture is $D=MR=15\mu \mathrm{m}$ . Since there are no commercial optical fibers of this size, we used $4\text{-}\mu \mathrm{m}$ fibers.

2.L.

Number of Observed Molecules

The detection volume is $\pi {(D∕2)}^{2}H$ , where $H$ is the height of the volume. Since we did not use TIRF illumination, $H=\sim 100\mathrm{nm}$ (the average thickness of a myofibril, see Fig. 2 ), and the detection volume is $\sim 20\times {10}^{-18}\mathrm{L}$ . Actin concentration in muscle $\left(0.6\mathrm{mM}\right)$ implies that there are $\sim 7200$ actin protomers in this volume. The ratio of fluorescent phalloidin to nonfluorescent phalloidin was fixed at 1:1000, suggesting that the signal was contributed on average by about seven to eight actin molecules.

Fig. 2

(a) AFM image of a glass coverslip covered with silver, (b) glass coverslip covered with silver and coated with SIF, and (c) myofibrils on the glass coverslip covered with silver and coated with SIF. All images are $10\times 10\mu \mathrm{m}$ . The peaks in the southwest corner of (a) are dirt particles. The dirt is present because the slides initially contained aqueous buffer. They were used in experiments, dried, and AFM imaged.

3.1.

AFM Images of Surfaces

Figure 2 shows representative AFM images of [Fig. 2(A)] a silver mirror, [Fig. 2(B)] a silver mirror coated with SIF, and [Fig. 2(C)] myofibrils on silver mirror coated with SIF. All images are $10\times 10\mu \mathrm{m}$ . The peaks in the southwest corner of Fig. 2(A) are dirt particles. The $52\text{-}\mathrm{nm}$ layer of silver was protected by a $5\text{-}\mathrm{nm}$ coat of silica, which was responsible for small “bumps” present in Figs. 2(B) and 2(C). The average diameter of SIF was $217\pm 22\mathrm{nm}$ [ $\text{mean}\pm \text{standard}$ deviation (SD)]. We think that the area of a sarcomere projecting above the surface of the coverslip in Fig. 2(C) is the O-band, because it is offering more resistance to the AFM probe as a consequence of having been strengthened by the attachment of phalloidin. The thickness of myofibrils was calculated from AFM images by measuring the distance from the coverglass to the highest point of elevation of the O-band. In 12 measurements on phalloidin-labeled myofibrils, the average height of a myofibril was $97\pm 4\mathrm{nm}$ . The average width was $1.68\pm 0.44\mu \mathrm{m}$ $(\text{mean}+\mathrm{SD})$ . It is clear that the average height of a myofibril is smaller than the $1∕e$ distance of an evanescent wave, and therefore there is no advantage to TIRF illumination.

3.2.

Increase of Brightness

Figure 3 compares the spectra of myofibrils on glass coverslips coated with SIF with the spectra of myofibrils on silver mirror coated with SIF. The emission spectra were recorded in a Cary Eclipse spectrofluorometer by placing the plane of the microscope slide at a $45\text{-}\mathrm{deg}$ angle with respect to the direction of the exciting $475\text{-}\mathrm{nm}$ beam of light. No differences in the shape of emission spectra were detected, but the intensity of fluorescence of myofibrils on $\text{glass}+\mathrm{SIF}$ was enhanced $\sim 3.6\pm 0.2$ times $(\text{mean}\pm \mathrm{SE})$ by the silver mirror in comparison with the intensity of myofibrils on glass alone. Gold and aluminum enhanced the fluorescence by a factor of 4 (Table 1 , column 2). This effect is caused by the presence of the mirror surface, which enhances intensity by a trivial reflection from the reflecting surface. The reflection enhances both illuminating light fluxes (the sample is illuminated directly, and the fluorescent light is reflected from the surface). SIF alone (no mirror) enhanced⁵ intensity by a factor of 4 to 5. The increases in intensities of myofibrils on metal coated glass in the presence of SIF are listed in Table 1 (column 3). The strongest enhancement in fluorescence signal was observed for the SIF deposited on silver mirror, which is consistent with earlier report on enhanced immunoassays.⁷ Intrigued by the high magnitude of the observed enhancements on the metallic surfaces coated with nanoparticles, we decided to study this effect in more detail, exploring the changes in lifetimes and photostabilities.

Fig. 3

Enhancement of fluorescence by SIF on silver. Myofibrils $(1\mathrm{mg}∕\mathrm{mL})$ labeled with $0.1\mu \mathrm{M}$ Rh-phalloidin were placed on a glass coverslip coated with SIF (green) and on a glass covered with silver and then coated with SIF (red). The spectra were measured at a $45\mathrm{deg}$ angle in a Varian Eclipse spectrofluorometer. The excitation wavelength is $475\mathrm{nm}$ . The vertical scale is in arbitrary units (a.u.). The spectrum of SIF in the absence of muscle is less than $10\mathrm{a.u.}$ at all wavelengths. (Color online only.)

Table 1

Enhancement of fluorescence intensity of phalloidin labeled myofibrils with reference to glass alone (column 1) by the mirrored surface alone (column 2), and the mirror together with SIF (column 3).

3.3.

Decrease of Fluorescent Lifetime

To quantify the effect of SIF, we measured the lifetimes of myofibrils on various metallic surfaces. The results show that the fluorescence lifetime is decreased with a simultaneous increase of the brightness. This suggests that the proximity of fluorophores to metallic surfaces increased the radiative rate because the decrease of lifetime is the distinguishing feature of the increase of radiative rate.²⁰ The observed decrease of the lifetime cannot be solely a result of the increase in the nonradiative rate because the quantum efficiency is also increased, and any increase in nonradiative rate decreases the brightness.

3.3.1.

$\text{GLASS}+\mathrm{SIF}$

Figure 4 shows that SIF causes the fluorescent lifetime to significantly decrease. The decay of fluorescence of myofibrils on glass [Fig. 4(A) and Table 2 ] was best fitted by two exponentials with lifetimes ${\tau }_1=0.60\pm 0.14$ and ${\tau }_2=3.18\pm 0.12\mathrm{ns}$ with the relative contributions to the total amplitude of 41.81 and 58.19%, respectively. The amplitude weighted average lifetime was $2.100\mathrm{ns}$ . Addition of SIF [Fig. 4b] augmented the fast decay of fluorescence of myofibrils (arrow). It was now best fitted by three exponentials with lifetimes ${\tau }_1=2.775\pm 0.082$ , ${\tau }_2=0.544\pm 0.033$ , and ${\tau }_3=0.063\pm 0.0018\mathrm{ns}$ with the relative contributions to the total amplitude of 1.51, 4.28, and 94.20%, respectively. The amplitude weighted average lifetime decreased nearly 17 times to $0.125\mathrm{ns}$ .

Fig. 4

Decrease of lifetime caused by SIF. (a) Lifetime signal from myofibrils on glass (blue) is best fitted (black line) by the two exponentials with lifetimes ${\tau }_1=0.60$ and ${\tau }_2=3.18\mathrm{ns}$ and the relative contributions to the total amplitude of 41.81 and 58.19%. The red signal is the exciting pulse from the diode laser. The bottom inset is the residual fit to all 2134 analyzed data points. (b) Lifetime signal from myofibrils on glass coated with SIF (blue) is best fitted (black line) by three exponentials with lifetimes ${\tau }_1=2.775$ , ${\tau }_2=0.544$ , and ${\tau }_3=0.063\mathrm{ns}$ and with the relative contributions to the total amplitude of 1.51, 4.28, and 94.20%. The arrow points to the fast decay of fluorescence. The red signal is the exciting pulse. The bottom inset is the residual fit to all 2245 data analyzed points. (Color online only.)

Table 2

Effect of SIF on different surfaces on the amplitude-weighted component of the fluorescence lifetime of phalloidin.

Substrate	Lifetime in the Absence of SIF (ns)	Lifetime in the Presence of SIF (ns)	Decrease (fold)
Glass	2.100	0.125	16.8
Ag	2.328	0.068	34.2

3.3.2.

$\text{Silver}+\mathrm{SIF}$

Figure 5(A) shows that the decay of fluorescence of myofibrils on silver mirrors was best fitted by two exponentials with lifetimes ${\tau }_1=0.936\pm 0.064$ and ${\tau }_2=3.380\pm 0.041\mathrm{ns}$ with the relative contributions to the total amplitude of 43.05 and 56.95%, respectively (Table 2). The amplitude-weighted average lifetime was $2.328\mathrm{ns}$ . Figure 5(B) shows that addition of SIF augmented the fast decay of fluorescence of myofibrils on silver mirrors (arrow). It was best fitted by the four exponentials with lifetimes ${\tau }_1=0.029\pm 0.001$ , ${\tau }_2=0.215\pm 0.006$ , ${\tau }_3=1.077\pm 0.017$ , and ${\tau }_4=3.318\pm 0.028\mathrm{ns}$ ; and the relative contributions to the total amplitude of 88.66, 7.81, 2.52, and 1.01%, respectively. The amplitude weighted average lifetime decreased 34 times to $0.068\mathrm{ns}$ .

Fig. 5

Decrease of lifetime caused by SIF on silvered glass. (a) Lifetime signal from myofibrils on silver-covered glass (blue) is best fitted (black line) by the two exponentials with lifetimes ${\tau }_1=0.936\pm 0.064$ and ${\tau }_2=3.380\pm 0.041\mathrm{ns}$ and with the relative contributions to the total amplitude of 43.05 and 56.95. The red signal is the exciting pulse. The bottom inset is the residual fit to all 7831 analyzed data points. (b) Lifetime signal from myofibrils on silver-covered glass coated with SIF (blue) is best fitted (black line) by four exponentials with lifetimes ${\tau }_1=0.029\pm 0.000$ , ${\tau }_2=0.215\pm 0.006$ , ${\tau }_3=1.077\pm 0.017$ , and ${\tau }_4=3.318\pm 0.028\mathrm{ns}$ and with the relative contributions to the total amplitude of 88.66, 7.81, 2.52, and 1.01%. The arrow points to the fast decay of fluorescence. The red signal is the exciting diode laser pulse. The bottom graph is the residual fit to all 7924 data points analyzed. (Color online only.)

3.4.

Decrease of Bleaching in SMD Experiments

The signal from a sarcomere of a myofibril was measured by positioning an O-band by a piezostage over the projection of the confocal aperture on the sample plane as described in Ref. 18.

3.4.2.

$\text{Gold}+\mathrm{SIF}$

The intensity of myofibrils on the gold mirror coated with SIF increased on average 10 times in comparison with uncoated glass (Table 1). Figure 6(A) compares photobleaching of the O-band of myofibrils on gold mirrors (green) with myofibrils on gold mirrors coated with SIF (red). The decay on gold mirrors was best fitted by quickly and slowly decaying exponentials with the average contributions to the total intensity of ${\alpha }_1=32$ and ${\alpha }_2=68\%$ . The decay on gold mirrors coated with SIF was best fitted by quickly and slowly decaying exponentials with the average contributions to the total intensity of ${\alpha }_1=26\%$ and ${\alpha }_2=74\%$ . In six separate experiments, SIF caused the average short half-time of myofibrils on gold mirrors to increase $f_1=1.6$ -fold from $19\pm 2\text{to}30\pm 6\mathrm{s}$ . The long half-time increased $f_2=2.9$ -fold from $89\pm 4\text{to}260\pm 39\mathrm{s}$ . The results are summarized in Table 3. These results show that coating gold mirrors with SIF decreased fast photobleaching on average 2.5-fold ( ${\alpha }_1^{\mathrm{aver}}{f}_1+{\alpha }_2^{\mathrm{aver}}{f}_2$ , where ${\alpha }_1^{\mathrm{aver}}$ and ${\alpha }_2^{\mathrm{aver}}$ are the average change in $\alpha$ on the addition of SIF).

Fig. 6

Comparison of the rate of photobleaching of myofibrillar overlap zone on SIF on metal (red) and on plain metal (green). Myofibrils $(1\mathrm{mg}∕\mathrm{mL})$ were labeled with $0.01\mu \mathrm{M}$ rhodamine-phaloidin $+9.99\mu \mathrm{M}$ unlabeled phalloidin. The overlap zone of a myofibril was viewed by SMD microscopy through a $4\text{-}\mu \mathrm{m}$ confocal aperture. The three-parameter single exponential fits are in black. $\mathrm{NA}=1.45$ objective. CPB denotes counts per $100\text{-}\mathrm{ms}$ bin. Signal on (a) gold surface with SIF signal is attenuated 2.5 times and (b) on silver surface. SIF signal is first attenuated threefold using a two-exponential fit and another 50-fold using a three-parameter single-exponential fit. (Color online only.)