2.1.

Chemicals and Solutions

Rhodamine-phalloidin (RP) was from Molecular Probes (Eugene, Oregon). Unlabeled phalloidin (UP) was from Sigma (Saint Louis, Missouri). All other chemicals, including phosphocreatine, creatine kinase, glucose oxidase, catalase, and ATP, were from Sigma. Ethylene diamine tetraacetic acid (EDTA)-rigor solution contained $50\text{-}\mathrm{mM}$ $\mathrm{KCl}$ , $2\text{-}\mathrm{mM}$ EDTA, $1\text{-}\mathrm{mM}$ DTT, and $10\text{-}\mathrm{mM}$ $\mathrm{Tris}\text{-}\mathrm{HCl}$ buffer pH 7.5. Ca-rigor solution contained $50\text{-}\mathrm{mM}$ $\mathrm{KCl}$ , $4\text{-}\mathrm{mM}$ ${\mathrm{MgCl}}_2$ , $0.1\text{-}\mathrm{mM}$ ${\mathrm{CaCl}}_2$ , $1\text{-}\mathrm{mM}$ dithiothreitol (DTT), and $10\text{-}\mathrm{mM}$ $\mathrm{Tris}\text{-}\mathrm{HCl}$ buffer pH 7.5. Contracting solution was the same as Ca-rigor, except that it contained in addition $2\text{-}\mathrm{mM}$ ATP.

2.2.

Preparation of Myofibrils

Muscle was first washed with cold EDTA-rigor solution for $1∕2\mathrm{h}$ to avoid shortening when transferred from glycerinating solution to homogenizing solution. It was followed by extensive wash with Ca-rigor solution. Myofibrils were homogenized in Ca-rigor as described before.³⁶

2.3.

Labeling and Sample Preparation

Labeling was carried out in solution. Myofibrils $(1\mathrm{mg}∕\mathrm{mL})$ were labeled for $5\min$ with a mixture containing 0.05 to $100\text{-}\mathrm{nM}$ RP containing always $10\text{-}\mathrm{\mu }\mathrm{M}$ UP in Ca-rigor solution. $25\mathrm{\mu }\mathrm{L}$ of labeled myofibrils were applied to number 1 cover slips $(20\times 60\mathrm{mm})$ (Corning, New York) precleaned with 90% ethanol. A narrow channel was created by applying a thin layer of Vaseline along the edges of the cover slip. To align myofibrils as much as possible, along the long axis of a cover slip, the sample was applied by streaking the pipette along the long axis. The sample was left for 3 min to allow myofibrils to adhere to glass. The long cover slip was covered with a small $(20\times 20\text{-}\mathrm{mm})$ cover slip and washed with at least 5 vol of Ca-rigor solution by applying the solution to one end of the channel and absorbing with number 1 filter paper at the other.

2.4.

Preparation of Nanoparticle Monolayers

All necessary glassware was soaked in a base bath overnight and washed with deionized water. A solution of $18\text{-}\mathrm{mg}∕\mathrm{mL}$ silver nitrate $\left(200\mathrm{mL}\right)$ was heated and stirred in a $250\text{-}\mathrm{mL}$ Erlenmeyer flask at $95°\mathrm{C}$ . $0.5\text{-}\mathrm{mL}$ aliquots of $34\text{-}\mathrm{mM}$ trisodium citrate solution were added drop-wise. The solution was stirred for $20\min$ and warmed to 96 to $98°\mathrm{C}$ . Then five aliquots ( $0.7\mathrm{mL}$ each) of $34\text{-}\mathrm{mM}$ trisodium citrate were added drop-wise to the reaction mixture every 15 to $20\min$ . Stirring was continued for $25\min$ until the milky yellow color remained. Then the mixture was cooled in an ice bath for $15\min$ . The colloids were separated by centrifugation at $3500\mathrm{rpm}$ for $6\min$ . The residues were collected and dissolved in $1\text{-}\mathrm{mM}$ trisodium citrate. For covalent attachment of the nanoparticles to glass cover slips, the cover slips were washed with Alconox soap and rinsed with distilled water. The cover slips were soaked for $2\mathrm{h}$ in $0.1\text{-}\mathrm{M}$ $\mathrm{NaOH}$ to activate the surface, rinsed copiously with distilled water, and soaked overnight at 80 to $90°\mathrm{C}$ in a solution of 5% (v/v) aminopropyltriethoxysilane buffered with $0.1\text{-}\mathrm{M}$ acetic acid (pH 5.5). The coverslips were rinsed with deionized water, air dried, and placed into a petri dish for drop coating with the sol of silver colloids. About $300\mathrm{\mu }\mathrm{L}$ of the colloidal silver sol was drop coated on the desired area of the glass slide/coverslip. The excess of colloidal silver sol was rinsed with deionized water after incubation for two hours at room temperature. The glass coverslips with attached silver nanoparticles were air dried and stored in vials.

2.5.

Atomic Force Microscope Imaging

Imaging was performed on the AFM Explorer ThermoMicroscope (Veeco Instruments, Incorporated, Plainview, New York) in contact scanning mode with a Non-Conductive Silicon Nitride Probe (Veeco Instruments, Incorporated). Images were acquired at a 2- to $5\text{-}\mathrm{\mu }\mathrm{m}∕\mathrm{s}$ scan rate with a resolution of 300 pixels per 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.

2.6.

Measuring Fluorescence Lifetimes

Fluorescence lifetimes were measured by time-domain technique using a FluoTime 200 fluorometer (PicoQuant, Incorporated, Berlin, Germany). The sample was positioned in front-face configuration inside the fluorometer chamber. 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. Full width at half maximum (FWHM) of pulse response function was $68\mathrm{ps}$ (measured by PicoQuant, Incorporated). Time resolution was better than $10\mathrm{ps}$ . Less than 0.5% background was detected from the NML slide. The intensity decays were analyzed in terms of a multiexponential model using FluoFit ver4 software (PicoQuant, Incorporated).

2.7.

Optical Imaging

The instrument was described before,^{37, 38} except that an Olympus IX71 (Olympus, Melville, New York) was used. A Hamamatsu ImagEM CCD camera (Hamamatsu, Bridgewater, New Jersey) was mounted in the right exit port (model IX2 RSPC-2) of the microscope. The left exit port contained a confocal aperture and the avalanche photodiode, as previously described.³⁸ It was used for statistical analysis of the reduction of photobleaching. Excitation light from an expanded diode pumped solid state (DPSS) laser beam (Compass 215M, Coherent, Santa Clara, California) was coupled by the polarization-preserving-fiber to a commercial TIRF attachment (Olympus), which was mounted on the back port of the microscope. The attachment expanded the laser beam, focused it at the back focal plane of the objective, and directed it by the movable optical fiber adapter to the periphery of the objective (Olympus Apo $100\times$ , $1.65\mathrm{NA}$ , or PlanApo $60\times$ , $1.45\mathrm{NA}$ ). Excitation light totally internally reflected at the interface and produced an evanescent wave on the aqueous side of the interface.³⁹ Exciting light was s-polarized (perpendicular to the incidence plane), giving an evanescent field similarly linearly polarized.⁴⁰ Fluorescence was excited with linearly polarized light parallel or perpendicular to the myofibrillar axis. The sample rested on a moveable piezo stage (Nano-H100, Mad City Labs, Madison, Wisconsin) controlled by a nano-drive. The fluorescent light was collected through the same objective, projected onto a tube lens and to a calcite prism (Melles Griot, Carlsbad, California), which split the fluorescent light into two orthogonally polarized components. The light was focused on the photosensitive area of the camera. The insertion of the calcite prism did alter by a few millimeters the position of the conjugate image plane, but in practice it made a negligible difference to the focus because the high magnification objective (with long back focal distance) was used.

The coupling of the DPSS laser to the TIRF module was done through polarization preserving single mode optical fibers. Those fibers have small diameters $(\sim 3\mathrm{\mu }\mathrm{m})$ and are notoriously difficult to couple efficiently with the laser beam. We used a commercial fiber (kineFlex, PointSource, England) mounted into a kinematic adapter (Model KC1, Thor, Newton, New Jersey) to assure high coupling efficiency $(\sim 50\%)$ , i.e., $\sim 25\mathrm{mW}$ of $532\text{-}\mathrm{nm}$ light was launched into a TIRF module.

2.8.

Spatial Sampling

The pixel size of the camera is $16\times 16\mathrm{\mu }\mathrm{m}$ . For the $60\times$ NA 1.45 objective with $$ 532-nm illumination, the Rayleigh resolution limit is $\sim 200\mathrm{nm}$ . According to the Nyquist sampling theorem, the ideal spatial sampling rate should have been $200∕2.3\approx 90\mathrm{nm}$ . However, in our experiments the image was undersampled. The back-projected size of the pixel of the camera is $16\mathrm{\mu }\mathrm{m}∕60=267\mathrm{nm}$ . Therefore, the images are undersampled by a factor of $267∕90\approx 3$ . For the $100\times$ NA 1.65 objective, the back-projected size of the pixel of the camera is $160\mathrm{nm}$ , and the images are undersampled by factor of $\sim 1.8$ . Undersampling allows the light to be concentrated on fewer pixels. Under the present low-light conditions, this creates a signal that has greater amplitude relative to the background noise, and therefore boosts SNR.

2.9.

Choice of Sampling Time

The whole field ( $512\times 512$ pixels) is collected at time intervals $\tau$ . The choice of $\tau$ is crucial in SMD. Decreasing $\tau$ increases time resolution but decreases the number of fluorescent photons detected from each half-sarcomere. Increasing $\tau$ improves SNR but decreases time resolution. We took $\tau =100$ to $400\mathrm{ms}$ as a reasonable compromise, because the characteristic time for ATP hydrolysis by glycerinated skeletal myofibrils is $\sim 0.5\sec$ .⁴¹ The time resolution can be increased by increasing camera gain at the expense of image quality.

2.J.

Image Analysis

HCImage software (Hamamatsu) was used. Rectangular ROI was created corresponding to each O-band in a $512\times 512$ image. The intensity measurement tool was used to measure the mean gray value of all the defined ROIs in all 500 images. ROI was $4\times 4$ pixels (slightly smaller than a half-sarcomere). These data were saved as tabbed text files. The ASCII file was plotted in SigmaPlot (Systat, San Jose, California). The deconvolution of point spread function (PSF) was done to see if weak uniform fluorescence of unstained myofibrils was due to the spread of fluorescence. The analysis showed that it was not. The MATLAB function deconvblind (MathWorks Incorporated, Natick, Massachusetts) was used. It uses a maximum likelihood algorithm to iteratively optimize the PSF when deconvolving the image. The algorithm maximizes the likelihood that the resulting image, when convolved with the resulting PSF, is an instance of the blurred image, assuming Poisson noise statistics. Deconvolution made images less blurred, but the basic observation, that unstained myofibrils were fluorecsent, was unchanged.

3.1.

Number of Observed Molecules

We first measured the volume of muscle sarcomere to estimate the number of observed actin molecules. Figure 1 shows AFM images of unlabeled (top) and labeled (bottom) myofibril. The AFM image reports on the resistance to stress encountered by the atomic probe. The images look different, but it is important to emphasize that the difference in AFM image does not suggest that phalloidin induces changes in the sarcomere structure. Because phalloidin binding makes the ends of thin filaments stiffer, this region of a sarcomere is more difficult for an atomic probe to deform. It is known that the pattern of phalloidin labeling changes with time. Initially only the ends of thin filaments are labeled.³² Redistribution of phalloidin to the I-band takes several hours.⁴² Myofibrils used here were observed 5 to $15\min$ after labeling, so only the ends of thin filaments were labeled.

Fig. 1

AFM image of a native myofibril on glass coverslip and of a myofibril labeled with $0.1\text{-}\mathrm{\mu }\mathrm{M}$ $\mathrm{RP}+9.9\text{-}\mathrm{\mu }\mathrm{M}$ UP. The color scale bar indicates distance from coverslip in nanometers.

The average height of 12 phalloidin-labeled myofibrils was $97\pm 4\mathrm{nm}$ $(\mathrm{mean}+\mathrm{SEM})$ . The typical width and length of a sarcomere are 0.8 and $2.5\mathrm{\mu }\mathrm{m}$ , respectively, so the typical volume of a half-sarcomere is $\sim 0.1\mathrm{\mu }{\mathrm{m}}^3=0.1\times {10}^{-15}\mathrm{L}$ . Since the concentration of actin in muscle is $0.6\mathrm{mM}$ ,¹⁶ this volume contains on average $0.4\times {10}^5$ actin monomers.

For “heavily” labeled muscle (see Fig. 2 ), where 1 in ${10}^2$ molecules are labeled, we have $\sim 400$ labeled actin molecules per half-sarcomere. For “lightly” labeled muscle (see Fig. 3 ), where 1 in ${10}^4$ molecules are labeled, we have on average $\sim 4$ labeled actin molecules per half-sarcomere. For “extremely lightly” labeled muscle (see Fig. 6) where 1 in ${10}^5$ molecules are labeled, we have on average $\sim 0.4$ labeled actin molecules per half-sarcomere.

Fig. 2

The time course of photobleaching of heavily labeled rigor myofibril. Top panel: myofibril at times indicated below each frame. Exciting light polarized vertically, emitted light polarized horizontally. The bar is $5\mathrm{\mu }\mathrm{m}$ . Sarcomere length= $2.6\mathrm{\mu }\mathrm{m}$ . Bottom panel: the time course of photobleaching of the O-band pointed to by the arrow in the top panel (black). The least-square three-parameter exponential fit (yellow) is $y=2469+15291\exp (-6.9\times {10}^{-3}t)$ . The inset is the difference between the signal and the exponential function showing that the exponential is a good fit to the data. Red is the best linear fit to residuals, and green is 95% confidence limit. Myofibril is labeled with $0.1\text{-}\mathrm{\mu }\mathrm{M}$ $\mathrm{RP}+9.9\text{-}\mathrm{\mu }\mathrm{M}$ UP. Myofibrils are on sapphire, viewed by $100\times \mathrm{NA}=1.65$ objective. (Color online only.)

Fig. 3

The time course of photobleaching. Top panel: the whole field. Myofibril enclosed in the box was analyzed. The arrow points to the background ROI. Bottom: intensity of fluorescence of the sarcomere, pointed to by an arrow, was measured every $400\mathrm{ms}$ . The times at which images were taken after opening the shutter are shown at the bottom left. The bar is $10\mathrm{\mu }\mathrm{m}$ . Sarcomere length= $2.1\mathrm{\mu }\mathrm{m}$ . Myofibrils are labeled with $1\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP.

Fig. 6

The time course of photobleaching of an O-band pointed to by an arrow in Fig. 5. Signal change (black) suggests a single step. The least square three-parameter exponential fit (yellow) is $y=35061+33277\exp (-4.42\times {10}^{-3}t)$ . Myofibrils were labeled with $0.1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP. The background has been subtracted.

3.2.

Photobleaching of Heavily Labeled Myofibrils on Glass

If the concentration of the label is high, many actins carry the fluorophores. For heavily labeled myofibrils, we used $100\mathrm{nM}$ RP ( $+9.9\mathrm{\mu }\mathrm{M}$ nonfluorescent label), i.e., 1 in 100 actin monomers carry fluorescent phalloidin. The number of observed actin molecules is $\sim 400$ per half-sarcomere. The top panel of Fig. 2 shows the time course of photobleaching of the myofibrils labeled with $100\mathrm{nM}$ $\mathrm{RP}+9.9\mathrm{\mu }\mathrm{M}$ UP. 500 images were captured every $200\mathrm{ms}$ ; but only frames 1, 50, 100, 200, and 300 are shown. The time course of photobleaching of the O-band pointed to by the arrow in the top panel is plotted in the bottom panel of Fig. 2. The three-parameter exponential (yellow) fits the data closely. The half-time is $\sim 20\sec$ .

It is important to point out that we measure polarized intensity of fluorescence. The sample was always illuminated with light polarized vertically relative to the laboratory frame of reference. The microscope field of view is split into two orthogonally polarized parts by the calcite prism inserted before the camera (see Sec. 2). This causes the camera to see two images simultaneously, obtained with the analyzer oriented horizontally and vertically. The myofibril shown in Fig. 2 was viewed with the analyzer oriented horizontally.

3.3.

Photobleaching of Lightly Labeled Myofibrils on Glass

In contrast to smooth photobleaching of the heavily labeled O-bands, sarcomeres of lightly labeled myofibrils bleached in step-wise fashion. The example is shown in Fig. 3. For myofibrils labeled with $1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP and using the value of $0.4\times {10}^5$ actin monomers per half-sarcomere, we estimate that it contains $\sim 4$ molecules of phalloidin. Figure 3 shows images that were captured every $400\mathrm{ms}$ . The time course of photobleaching of the O-band pointed to by the arrow in the bottom panel of Fig. 3 after subtracting the background is plotted in Fig. 4 . It suggests the presence of two discrete steps. Most of the O-bands in the top panel of Fig. 3 also bleached in step-wise fashion. However, not all of the steps were as clearly defined. We estimate that only $\sim 50\%$ of 480 spots analyzed in 12 separate experiments in which sarcomeres were not uniformly labeled bleached in a clearly defined step-wise fashion. The pattern of photobleaching varied from O-band to O-band. Even the O-bands immediately adjoining each other gave different patterns. For example, the O-band immediately to the right of the one pointed to by an arrow in Fig. 3 suggested that bleaching occurred in four steps. The fact that not all sarcomeres bleached in a step-wise fashion is perhaps due to the fact that some sarcomeres we selected for analysis were not containing fluorophores at all, but were fluorescent due to waveguide effect.

Fig. 4

(a) The time course of photobleaching of the O-band pointed to by the arrow in the bottom panel of Fig. 3, suggesting the presence of discrete steps. Other O-bands in this frame also bleached in a step-wise fashion. Signal change suggests two steps. Note that the vertical scale is the intensity after subtracting background. The inset shows the time course of photobleaching of an O-band labeled with $10\text{-}\mathrm{nM}$ RP. (b) shows the time course of photobleaching of the background pointed to by the arrow in the top panel of Fig. 3. The least square three-parameter exponential fit (yellow) is $y=6447+3356\exp (-6.90\times {10}^{-3}t)$ . (Color online only.)

The time course of the bleaching of the background from the same sized ROI used to collect data from the O-band (pointed to by an arrowhead in the top panel of Fig. 3) is plotted as an inset to Fig. 4. It displays fluorescence equal to $\sim 60\%$ of the signal (recall that the signal is plotted after subtracting the background). However, the inset shows that the bleaching occurs smoothly. The residuals of the least-squares fit to the three-parameter exponential decay function (data not shown) suggested that the exponential is a good fit to the data. This bleaching is most likely due to residual fluorophores remaining stuck to the glass after washing. The rest of the background $(\sim 6000\mathrm{units})$ is due to the autofluorescence of glass. It does not photobleach at all.

3.4.

Photobleaching of Extremely Lightly Labeled Myofibrils on Glass

To image a half-sarcomere containing on average less than one fluorescent actin per half-sarcomere, we used $0.1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP. For an estimated number of actin molecules in a half-sarcomere of $0.4\times {10}^5$ , this gives $\sim 0.4$ molecules of phalloidin actin in a ROI. Figure 5 shows the time course of photobleaching of the myofibrils irrigated with $0.1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP. The time course of photobleaching of the O-band pointed to by the arrow in Fig. 5 is plotted in Fig. 6 . It suggests the presence of a single discrete step.

Fig. 5

The time course of photobleaching of myofibrils labeled with $0.1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP. Intensity of fluorescence of the sarcomere pointed to by an arrow was measured every $100\mathrm{ms}$ . The times at which images were taken after opening the shutter are shown below each frame. Sarcomere length= $2.0\mathrm{\mu }\mathrm{m}$ .

3.5.

Photobleaching of Heavily and Lightly Labeled Myofibrils on the Nanoparticle Monolayer

Figure 7 (top panel) shows the representative examples of images of myofibrils on glass. The images are of good quality, as evidenced by the fact that the Z-bands (pointed to by the arrowheads) and the H-zones (pointed to by the arrows) are well resolved. The bottom panel of Fig. 7 shows representative examples of myofibrils on the NML. The comparison with images on glass shows that image quality remains equally good, as evidenced by the resolution of the Z-bands (arrowheads) and the H-zones (arrows). The intensity is larger in the bottom panels than in the top because colloids increase brightness an average of ten times. This is not reflected in brightness of images because camera gain was adjusted automatically. We did not observe any photobleaching within $100\sec$ . The same was true for lightly labeled myofibrils ( $1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP).

Fig. 7

Representative images of myofibrils on glass (top panels) and on NML (bottom panels). Arrowheads point to the Z-lines and arrows point to the H-zones. Myofibrils are labeled with $0.1\text{-}\mathrm{\mu }\mathrm{M}$ rhodamine phalloidin $+9.9\text{-}\mathrm{\mu }\mathrm{M}$ unlabeled phalloidin. Bar is $10\mathrm{\mu }\mathrm{m}$ . TIRF excitation. The fact that myofibrils are on average 10 times brighter on NML than on glass is not well illustrated in this figure, because the gain of the camera adjusted itself automatically.

3.6.

Photobleaching of Extremely Lightly Labeled Myofibrils on the Nanoparticle Monolayers

Nanoparticle monolayers reduced photobleaching principally through the reduction of fluorescent lifetime (see Discussion Sec. 4). The top panel of Fig. 8 shows the image of a myofibril labeled with $0.1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP on NML. It is important to point out that NML signal from myofibril was enhanced approximately ten times in comparison with signal on glass (see next). The increase of brightness reduced photodamage because it allowed illumination with a weaker laser light. The bottom panel compares signal from a sarcomere containing $\sim 1$ fluorophore (red) with a signal from the background (black). The transient is caused by “blinking” of silver particles on glass. Metallic silver nanoparticles display curious “blinking,” even in the absence of the fluorophores.^{43, 44} Fortunately, blinking is rare. In one half-sarcomere, we have detected on average only three to four events during $100\sec$ .

Fig. 8

The time course of photobleaching of myofibrils labeled with $0.1\text{-}\mathrm{nM}$ $\mathrm{RP}+10\text{-}\mathrm{\mu }\mathrm{M}$ UP. The intensity of fluorescence of the sarcomere pointed to by an arrow in the top panel was measured every $100\mathrm{ms}$ . The horizontal scale is the times at which images were taken after opening the shutter. Note that NML increased the brightness ten fold. The data were taken through 1 OD filter. (Color online only.)

3.7.

Rate of Photobleaching

To obtain an average reduction of photobleaching, we measured signal by avalanche photodiode. We used $\mathrm{NA}=1.45$ , $60\times$ objective and $8\text{-}\mathrm{\mu }\mathrm{m}$ confocal aperture. This makes the depth of the detection volume (equal to the depth of the evanescent wave) $\sim 100\mathrm{nm}$ . The $x\text{-}y$ dimension of the detection volume is equal to the diameter of the confocal aperture divided by the magnification of the objective. With an $8\text{-}\mathrm{\mu }\mathrm{m}$ aperture and $60\times$ objective, it is smaller than the diffraction limit of the objective i.e., the diameter of the volume is $0.25\mathrm{\mu }\mathrm{m}$ ,⁴⁵ giving the detection volume of $18\times {10}^{-18}\mathrm{L}$ . Actin concentration in muscle $\left(0.6\mathrm{mM}\right)$ implies that there are $\sim 6800$ actin protomers in this volume. The ratio of fluorescent phalloidin to nonfluorescent phalloidin was fixed at 1:1000, suggesting that the signal was contributed by approximately seven to eight actin molecules. Figure 9a shows that the NML enhanced intensity of fluorescence ten fold in comparison with glass. Figure 9b compares the rate of photobleaching of the myofibrillar overlap zone on glass (green) and NML (red). NML signal from myofibril was enhanced ten times in comparison with signal on glass, so signal on NML was divided by this factor to make intensities at time 0 the same. The half-time of decay on glass and on large colloids was increased approximately four times, but in 12 experiments this time increased on average from $14\pm 4$ to $69\pm 25\sec$ (approximately four to five times).

Fig. 9

(a) Comparison of spectra of myofibrils $(1\mathrm{mg}∕\mathrm{mL})$ labeled with $0.1\text{-}\mathrm{\mu }\mathrm{M}$ fluorescein-phalloidin on a glass coverslip coated with monolayers containing colloids (circles). The spectra were measured at a $45\mathrm{deg}$ angle in a Varian Eclipse spectrofluorometer. Excitation wavelength was $475\mathrm{nm}$ . The spectrum of glass alone is shown as triangles. The spectrum of a colloid monolayer in the absence of muscle was comparable to glass alone. (b) Comparison of the rate of photobleaching of the myofibrillar overlap zone on glass and NML. The overlap zone of a myofibril was viewed through a $133\text{-}\mathrm{nm}$ back-projected confocal aperture. Gray—myofibrils on glass. Decay of fluorescence is best fitted by three-parameter exponential fit $y=84.5+369\exp (-3.8\times {10}^{-3}t)$ (black curve). Black—myofibrils on NML. Decay of fluorescence is best fitted by three-parameter exponential fit $y=255+221\exp (-9.9\times {10}^{-4}t)$ (black curve). Intensity is measured in counts-per- $100\mathrm{ms}$ bin. Colloid enhanced the signal $\sim 10$ times, so the signal on MNL was divided by this factor to make intensities at time 0 the same. Myofibrils $(1\mathrm{mg}∕\mathrm{mL})$ were labeled with $0.01\text{-}\mathrm{\mu }\mathrm{M}$ rhodamine-phalloidin $+9.99\text{-}\mathrm{\mu }\mathrm{M}$ unlabeled phalloidin. TIRF illumination, sarcomere viewed through an $8\text{-}\mathrm{\mu }\mathrm{m}$ aperture. $\mathrm{NA}=1.45$ objective.

3.8.

Decrease of Fluorescent Lifetime

Figure 10 shows that the enhancement of intensity and decrease of photobleaching is indeed due to decrease of lifetime. NML augmented the fast decay of fluorescein-labeled myofibrils (arrow). The fluorescence decay (black) after a laser flash (gray) is best fitted (black) by the two exponentials with lifetimes ${\tau }_1=0.92$ and ${\tau }_2=4.02\mathrm{ns}$ , with the relative contributions to the total amplitude of 48.5 and 51.5%, respectively. The average amplitude weighted lifetime was $2.52\mathrm{ns}$ . The arrows point to the fast decay of fluorescence. NML significantly decreased the lifetime signal. It is now best fitted (white) by three exponentials with lifetimes ${\tau }_1=1.181$ , ${\tau }_2=3.421$ , and ${\tau }_3=0.066\mathrm{ns}$ with the relative contributions to the total amplitude of 2.0, 1.8, and 96.2%, respectively. The average amplitude weighted lifetime was $0.150\mathrm{ns}$ . The arrows point to the fast decay of fluorescence. The average decrease of lifetime was 17 times, but it is worth pointing out that, whereas there was no ultrafast component of decay of myofibrils on glass, such a component constituted 96% of a signal on NML.

Fig. 10

Decrease of fluorescent lifetime by NML. (a) Lifetime signal from myofibrils on glass (black). The signal is best fitted (white line) by the two exponentials with lifetimes ${\tau }_1=0.92$ and ${\tau }_2=4.02\mathrm{ns}$ , with the relative contributions to the total amplitude of 48.5 and 51.5%. The average amplitude weighted lifetime was $2.52\mathrm{ns}$ . The arrow points to the fast decay of fluorescence. The gray signal is the exciting pulse from the diode laser. The bottom inset is the residual fit to all 9567 data points. (b) Lifetime signal from myofibrils on glass coated with a SIF monolayer (black) is best fitted (white line) by three exponentials with lifetimes ${\tau }_1=1.181$ , ${\tau }_2=3.421$ , and ${\tau }_3=0.066\mathrm{ns}$ , with the relative contributions to the total amplitude of 2.0, 1.8, and 96.2%, respectively. The average amplitude weighted lifetime was $0.150\mathrm{ns}$ . The arrow points to the fast decay of fluorescence. The gray signal is the exciting pulse. The bottom inset is the residual fit to all 5308 data points. $1\text{-}\mathrm{mg}∕\mathrm{mL}$ myofibrils were labeled with $0.1\text{-}\mathrm{\mu }\mathrm{M}$ rhodamine-phalloidin.