**3. Technologies of Single-molecule Imaging in Living Cells**

### **3.1. Microscope and imaging equipment**

SMI uses optical microscopy, and since most of the biological molecules are translucent for visible light, some kind of labeling is required. For detection of single molecules, labeling with fluorophore is effective, because it provides high-contrast imaging, which is essential both for detection of small signals from a single molecule and for detection of specific spe‐ cies of molecules in crowded conditions of living cells. Labeling by fluorescent proteins has expanded the application of SMI in living cells.

Circular illumination is better in TIR-microscopy than the illumination from a fixed single direction that is usually used in commercial TIR system, especially for observations of bio‐ logical samples having anisotropic structure [19]. It also reduces effects of spatially inhomo‐ geneous illumination pattern often caused by the strong coherence of laser beams. There are several methods to construct circular illumination path using only static optical elements or a rotatory moving mirror with a fixed angle, however, using a pair of Galvanometer, the in‐ cident angle of the illumination beam to the specimen is easily adjusted to the best position

Single-Molecule Imaging Measurements of Protein-Protein Interactions in Living Cells

http://dx.doi.org/10.5772/52386

437

**Figure 2.** Optical path of a total internal reflection fluorescence microscope for single-molecule imaging.The illumina‐ tion laser beams are introduced into an inverted fluorescence microscope from the epi-illumination path. Between the microscope and lasers, a two-demensional beam scanning system is inserted to achieve pseudo-circular illumination (see text for details). The violet (405-nm) laser is used for photoconversion of fluorophores. BE: beam expander, DM: dichroic mirror, FS: field stop, GM: Galvanometer scanner mirror, L: lens, λ/4: quarter wave plate, M: mirror, ND: neu‐

Both the chemical fluorophores and fluorescent proteins are applicable as the probes of SMI in living cells. To obtain good contrast against autofluorescence of cells and optics, fluoro‐ phores with relatively longer wavelength emissions are generally better. As the chemical fluorophores, tetrametylrhodamine (TMR), Cy3, Cy5, Alexa 488, and Alexa 594 are used fre‐ quently. Among the fluorescent proteins, as far as we know, eGFP is the best for SMI be‐ cause of relatively good photostability. As the red fluorescent protein in SMI, tag RFP is applicable. Photoconvertible proteins, Eos and mKikGR, are good in photoactivation locali‐ zation microscopy (PALM), which is an application of SMI [20]. Protein tags, like HaLo and SNAP, which can be conjugated covalently with chemical fluorophores after expression in living cells, are useful, because chemical fluorophores are generally more photostable than the fluorescent proteins, and colors of fluorescence emission can be changed according to the purpose of the experiment. When an especially strong and stable (long observation time) signal is required, Q-dot or other fluorescent beads will be used. In such cases, steric hin‐

drance and multivalency should be controlled carefully.

tral density filter, S: shutter.

**3.2. Fluorophores**

for each specimen by changing the amplitude of vibration of the scanner mirrors.

Fluorescence signal from single fluorophores is small but enough to be imaged individually when recent high-sensitivity video cameras, like EM-CCD or CCD equipped with a multichannel plate image intensifier are used. Standard temporal resolution of SMI in living cells is several tens of ms, and in some cases, under strong illumination, ms sampling has been achieved gathering hundreds of photons from a single fluorophore per single video frame.

For detection of small signals from single molecules, background rejection is essential. Total internal reflection [1] and oblique illumination [18] are useful technologies of wide-field flu‐ orescence microscopy to realize effective background rejection and can be used for SMI in living cells (Figure 1) [19].

**Figure 1.** Single-molecule imaging in living cells. Schemes of total internal reflection (A) and oblique illumination (B) microscope and a single-molecule image of tetramethylrhodamine-labeled EGF on the surface of living HeLa cells under an oblique illumination microscope (C) are shown. Bar in C: 10 μm. Detection of singlemolecules can be examined by single-step photobleaching (D).

Figure 2 shows the optical setup of our TIR microscope for SMI, which was home-built based on a commercial inverted fluorescence microscope. Solid-state continuous wave lasers in different emission wavelengths are used for illumination according to the species of fluo‐ rophore. Between the lasers and the microscope, a two-dimensional beam scanning system is constructed. This system is composed by a pair of diagonallypositioned Galvanometerscanning mirrors and a telescope that inserted between the two scanning mirrors. The two scanning mirrors are moved sinusoidally with a π phase difference in a frequency higher than the frame rate of imaging (30 Hz is the typical frame rate). Therefore, the specimen is illuminated from every direction during the acquisition of single frames. Thus, the system achieves pseudo-circular illumination.

Circular illumination is better in TIR-microscopy than the illumination from a fixed single direction that is usually used in commercial TIR system, especially for observations of bio‐ logical samples having anisotropic structure [19]. It also reduces effects of spatially inhomo‐ geneous illumination pattern often caused by the strong coherence of laser beams. There are several methods to construct circular illumination path using only static optical elements or a rotatory moving mirror with a fixed angle, however, using a pair of Galvanometer, the in‐ cident angle of the illumination beam to the specimen is easily adjusted to the best position for each specimen by changing the amplitude of vibration of the scanner mirrors.

**Figure 2.** Optical path of a total internal reflection fluorescence microscope for single-molecule imaging.The illumina‐ tion laser beams are introduced into an inverted fluorescence microscope from the epi-illumination path. Between the microscope and lasers, a two-demensional beam scanning system is inserted to achieve pseudo-circular illumination (see text for details). The violet (405-nm) laser is used for photoconversion of fluorophores. BE: beam expander, DM: dichroic mirror, FS: field stop, GM: Galvanometer scanner mirror, L: lens, λ/4: quarter wave plate, M: mirror, ND: neu‐ tral density filter, S: shutter.

#### **3.2. Fluorophores**

**3. Technologies of Single-molecule Imaging in Living Cells**

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

SMI uses optical microscopy, and since most of the biological molecules are translucent for visible light, some kind of labeling is required. For detection of single molecules, labeling with fluorophore is effective, because it provides high-contrast imaging, which is essential both for detection of small signals from a single molecule and for detection of specific spe‐ cies of molecules in crowded conditions of living cells. Labeling by fluorescent proteins has

Fluorescence signal from single fluorophores is small but enough to be imaged individually when recent high-sensitivity video cameras, like EM-CCD or CCD equipped with a multichannel plate image intensifier are used. Standard temporal resolution of SMI in living cells is several tens of ms, and in some cases, under strong illumination, ms sampling has been achieved gathering hundreds of photons from a single fluorophore per single video frame.

For detection of small signals from single molecules, background rejection is essential. Total internal reflection [1] and oblique illumination [18] are useful technologies of wide-field flu‐ orescence microscopy to realize effective background rejection and can be used for SMI in

**Figure 1.** Single-molecule imaging in living cells. Schemes of total internal reflection (A) and oblique illumination (B) microscope and a single-molecule image of tetramethylrhodamine-labeled EGF on the surface of living HeLa cells under an oblique illumination microscope (C) are shown. Bar in C: 10 μm. Detection of singlemolecules can

Figure 2 shows the optical setup of our TIR microscope for SMI, which was home-built based on a commercial inverted fluorescence microscope. Solid-state continuous wave lasers in different emission wavelengths are used for illumination according to the species of fluo‐ rophore. Between the lasers and the microscope, a two-dimensional beam scanning system is constructed. This system is composed by a pair of diagonallypositioned Galvanometerscanning mirrors and a telescope that inserted between the two scanning mirrors. The two scanning mirrors are moved sinusoidally with a π phase difference in a frequency higher than the frame rate of imaging (30 Hz is the typical frame rate). Therefore, the specimen is illuminated from every direction during the acquisition of single frames. Thus, the system

**3.1. Microscope and imaging equipment**

Applications

436

expanded the application of SMI in living cells.

living cells (Figure 1) [19].

be examined by single-step photobleaching (D).

achieves pseudo-circular illumination.

Both the chemical fluorophores and fluorescent proteins are applicable as the probes of SMI in living cells. To obtain good contrast against autofluorescence of cells and optics, fluoro‐ phores with relatively longer wavelength emissions are generally better. As the chemical fluorophores, tetrametylrhodamine (TMR), Cy3, Cy5, Alexa 488, and Alexa 594 are used fre‐ quently. Among the fluorescent proteins, as far as we know, eGFP is the best for SMI be‐ cause of relatively good photostability. As the red fluorescent protein in SMI, tag RFP is applicable. Photoconvertible proteins, Eos and mKikGR, are good in photoactivation locali‐ zation microscopy (PALM), which is an application of SMI [20]. Protein tags, like HaLo and SNAP, which can be conjugated covalently with chemical fluorophores after expression in living cells, are useful, because chemical fluorophores are generally more photostable than the fluorescent proteins, and colors of fluorescence emission can be changed according to the purpose of the experiment. When an especially strong and stable (long observation time) signal is required, Q-dot or other fluorescent beads will be used. In such cases, steric hin‐ drance and multivalency should be controlled carefully.

### **3.3. Sample preparation**

For SMI, cells are cultured on a coverslip and set on the microscope. Since contamination of small dust on the coverslip prevents SMI, the coverslip must be washed thoroughly before transfer of cells onto it [21]. Usage of glass coverslips (not conventional plastic cell culture dishes) that has the high refractive index was necessary to achieve total internal reflection; however, some cell culture dishes or chambers made of plastic with the refractive index sim‐ ilar to that of glass (1.52) can be purchased recently. One day or more before the observa‐ tion, the culture medium should be replaced to one that does not contain phenol red to reduce background fluorescence. The culture medium used during observation should also not contain phenol red.

bution of single molecules should be Gaussian, because the photon emission from a fluoro‐ phore is a Poisson process; however, when the intensities are small, the distribution

Single-Molecule Imaging Measurements of Protein-Protein Interactions in Living Cells

http://dx.doi.org/10.5772/52386

439

Photobleach of the fluorophore seems to be the most serious problem in SMI. This brings a trade-off between S/N of the single-molecule measurments and the observation length of each single molecule. By increasing the illumination power, the signals from single mole‐ cules increase to improve S/N, which in turn improves the temporal resolution and the accu‐ racy of position determination; however, at the same time, the observation length of each single molecule must be decreased due to increased photobleacing rates. In typical condi‐ tions, the emission photon numbers from a single chemical fluorophore, including TMR and Cy3, before photobleach is less than 1 million, and those from fluorescent proteins are sever‐ al times smaller. Since SMI in typical conditions requires thousands of photon emissions from a fluorophore per frame (due to limited numerical aperture of the objective and tran‐ mittance of the optics, <10% of which reach to the camera), only hundreds of frames can be acquired for each single molecule. If a video rate movie is taken, single molecules could be

Signal intensity (photon flux) of single fluorophores is limited, because the fluorescence emission cycle requires a finite time. The fluorescence lifetime, which is the rate-limiting pa‐ rameter under strong enough illumination, of typical fluorophores used in SMI is about 1 ns, meaning that the maximum photon flux is about 109 s-1. However, strong illuminations that cause such high-rate emission induce higher-order excitation that could be the reason of undesired photochemical reactions. Practical photon emission rate is no more than about 106 s-1. This means that because thousands of photons are required to acquire a snapshot of SMI, temporal resolution of SMI is difficult to be improved to more than 1 ms. Accuracy of posi‐ tion detemination depends on the signal intensity. When more than 10,000 photons are ob‐ tained on the camera for a single frame, the centroid of a single-molecule image can be determined with 1 nm of accuracy [22]. Such high accuracy cannot be obtained with a tem‐

The special resolution of the optical microscopy is worse than 200 nm. This limits the densi‐ ties or the concentrations of the molecule to be observed, because in dense conditions, im‐ ages of molecules overlap to inhibit single-molecule detection. The practical limits of the molecular density and concentration are about 10 μm-2 and 10 nM, respectively. Concentra‐ tions of most cell signaling proteins are thought to be within these limits, but those of struc‐

becomes binominal or sometimes looks as a log-normal distribution.

**3.5. Technical limitations specific for SMI**

observed only for about 10 s in average.

poral resolution better than subseconds.

tural proteins could exceed these limits.

When proteins tagged with fluorescent proteins, like GFP, are expressed and observed in cells, conditions for the transfection of cDNAs should be carefully controlled to avoid over‐ expression that prevents SMI (see section 3.5). Similarly, when HaLo or SNAP tag is used, staining should be carried out with a much lower concentration of fluorescent regents than that recommended by the manufactures.

### **3.4. Image processing**

The signal-to-noise ratio (S/N) in SMI is usually not good due to small signals and, especial‐ ly in cells, due to rather large background autofluorescence and scattering. Temporal aver‐ aging over successive video frames improves S/N under the sacrifice of temporal resolution. Spatial filtering of the images is also used to improve image quality. But, one must be care‐ ful to use any temporal and spatial filtering, because they sometimes do not preserve the lin‐ earity of signal intensity. Background subtraction is usually carried out before quantification of single-molecule signals. In cells, because background signals are highly inhomogeneous, the background levels should be determined locally.

After the appropriate pretreatments, the position and signal intensity are determined for in‐ dividual single-molecule images. For this purpose, fitting with a two-dimensional Gaussian distribution is usually used. Fitting functions can include background signals instead of the pretreatment of background subtraction. We usually use a Gaussian distribution on an in‐ clined background plane as the fitting function [21]. Positions of the molecule can be deter‐ mined as the centroid of the distribution with sub-pixel spatial resolution. Signal intensity can be calculated by integration of the distribution function. Accuracy of these parameters depends on the measurement system and should be determined statistically from the re‐ peated measurements of the same single molecules.

There are several criteria to judge whether single molecules are really detected or not [4]. Single-step photobleach is the most convenient and used criterion (Figure 1D). To distin‐ guish photobleach from disappearance by the movements of molecules, like release into the solution, illumination intensity should be changed. Photobleaching rate, but not the rate of most of other phenomena, depends on the illumination intensity. Because the size of fluoro‐ phores is much smaller than the spatial resolution of the optical microscope, the profile of single-molecule images must be the point spread function of the optics. The intensity distri‐ bution of single molecules should be Gaussian, because the photon emission from a fluoro‐ phore is a Poisson process; however, when the intensities are small, the distribution becomes binominal or sometimes looks as a log-normal distribution.

#### **3.5. Technical limitations specific for SMI**

**3.3. Sample preparation**

Applications

438

not contain phenol red.

**3.4. Image processing**

that recommended by the manufactures.

the background levels should be determined locally.

peated measurements of the same single molecules.

For SMI, cells are cultured on a coverslip and set on the microscope. Since contamination of small dust on the coverslip prevents SMI, the coverslip must be washed thoroughly before transfer of cells onto it [21]. Usage of glass coverslips (not conventional plastic cell culture dishes) that has the high refractive index was necessary to achieve total internal reflection; however, some cell culture dishes or chambers made of plastic with the refractive index sim‐ ilar to that of glass (1.52) can be purchased recently. One day or more before the observa‐ tion, the culture medium should be replaced to one that does not contain phenol red to reduce background fluorescence. The culture medium used during observation should also

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

When proteins tagged with fluorescent proteins, like GFP, are expressed and observed in cells, conditions for the transfection of cDNAs should be carefully controlled to avoid over‐ expression that prevents SMI (see section 3.5). Similarly, when HaLo or SNAP tag is used, staining should be carried out with a much lower concentration of fluorescent regents than

The signal-to-noise ratio (S/N) in SMI is usually not good due to small signals and, especial‐ ly in cells, due to rather large background autofluorescence and scattering. Temporal aver‐ aging over successive video frames improves S/N under the sacrifice of temporal resolution. Spatial filtering of the images is also used to improve image quality. But, one must be care‐ ful to use any temporal and spatial filtering, because they sometimes do not preserve the lin‐ earity of signal intensity. Background subtraction is usually carried out before quantification of single-molecule signals. In cells, because background signals are highly inhomogeneous,

After the appropriate pretreatments, the position and signal intensity are determined for in‐ dividual single-molecule images. For this purpose, fitting with a two-dimensional Gaussian distribution is usually used. Fitting functions can include background signals instead of the pretreatment of background subtraction. We usually use a Gaussian distribution on an in‐ clined background plane as the fitting function [21]. Positions of the molecule can be deter‐ mined as the centroid of the distribution with sub-pixel spatial resolution. Signal intensity can be calculated by integration of the distribution function. Accuracy of these parameters depends on the measurement system and should be determined statistically from the re‐

There are several criteria to judge whether single molecules are really detected or not [4]. Single-step photobleach is the most convenient and used criterion (Figure 1D). To distin‐ guish photobleach from disappearance by the movements of molecules, like release into the solution, illumination intensity should be changed. Photobleaching rate, but not the rate of most of other phenomena, depends on the illumination intensity. Because the size of fluoro‐ phores is much smaller than the spatial resolution of the optical microscope, the profile of single-molecule images must be the point spread function of the optics. The intensity distri‐ Photobleach of the fluorophore seems to be the most serious problem in SMI. This brings a trade-off between S/N of the single-molecule measurments and the observation length of each single molecule. By increasing the illumination power, the signals from single mole‐ cules increase to improve S/N, which in turn improves the temporal resolution and the accu‐ racy of position determination; however, at the same time, the observation length of each single molecule must be decreased due to increased photobleacing rates. In typical condi‐ tions, the emission photon numbers from a single chemical fluorophore, including TMR and Cy3, before photobleach is less than 1 million, and those from fluorescent proteins are sever‐ al times smaller. Since SMI in typical conditions requires thousands of photon emissions from a fluorophore per frame (due to limited numerical aperture of the objective and tran‐ mittance of the optics, <10% of which reach to the camera), only hundreds of frames can be acquired for each single molecule. If a video rate movie is taken, single molecules could be observed only for about 10 s in average.

Signal intensity (photon flux) of single fluorophores is limited, because the fluorescence emission cycle requires a finite time. The fluorescence lifetime, which is the rate-limiting pa‐ rameter under strong enough illumination, of typical fluorophores used in SMI is about 1 ns, meaning that the maximum photon flux is about 109 s-1. However, strong illuminations that cause such high-rate emission induce higher-order excitation that could be the reason of undesired photochemical reactions. Practical photon emission rate is no more than about 106 s-1. This means that because thousands of photons are required to acquire a snapshot of SMI, temporal resolution of SMI is difficult to be improved to more than 1 ms. Accuracy of posi‐ tion detemination depends on the signal intensity. When more than 10,000 photons are ob‐ tained on the camera for a single frame, the centroid of a single-molecule image can be determined with 1 nm of accuracy [22]. Such high accuracy cannot be obtained with a tem‐ poral resolution better than subseconds.

The special resolution of the optical microscopy is worse than 200 nm. This limits the densi‐ ties or the concentrations of the molecule to be observed, because in dense conditions, im‐ ages of molecules overlap to inhibit single-molecule detection. The practical limits of the molecular density and concentration are about 10 μm-2 and 10 nM, respectively. Concentra‐ tions of most cell signaling proteins are thought to be within these limits, but those of struc‐ tural proteins could exceed these limits.
