High-speed, multichannel fluorescence confocal imaging can be achieved by encoding the spatial location information into the frequency domain. Fluorescence confocal microscopy is an important tool for studying live biological cells, and it can reject out-of-focus fluorescence so that an image with a high signal-to-noise ratio can be obtained over the scattering biomedical media. To understand cellular systems, it is necessary to observe fast biological processes — such as cardiac myocyte contraction — in real time. Multichannel methods can increase imaging speed; however, although several have been developed, they are difficult to use directly with fluorescence confocal microscopy. For example, wavelength division multiplexing cannot be employed because the wavelength of the fluorescence emission is determined by the fluorescent label, which is independent of the wavelength of the incident excitation beam.1,2 Another major challenge comes from the weak fluorescence emission signal. To effectively detect a weak signal at fast speeds, a highly sensitive photomultiplier tube usually is employed. In general, a photomultiplier tube is a single-pixel detector. However, many multiplexing techniques — such as those using microlens and pinhole arrays — require a highly sensitive imaging detector (that is, an array of detectors).3 Although photomultiplier tube arrays recently have been used as the detection modules in commercial systems, they usually have a limited number of pixels (e.g., 32), and, furthermore, these modules are very expensive. On the other hand, the sensitivity of the CCD-based detector is limited by the imaging speed. Commonly available CCD imaging detectors used for multichannel confocal microscopy operate at about 30 fps, which is not fast enough to monitor many of the dynamics that occur in cells. Our group at Pennsylvania State University is studying a long-standing problem in cardiac research: How to obtain the transient 3-D distribution of calcium ions in a cardiac myocyte during excitation-contraction. This type of study requires high spatial (~100 nm) and temporal (~1 ms) resolutions. Frequency division multiplexing To overcome the limitations of existing fluorescence confocal microscopy, we recently developed a frequency division multiplexed fluorescence confocal microscope.4 The device provides high spatial resolution as well as temporal resolution up to the nanosecond range, limited only by the lifetime of the fluorophores and the response time of the photomultiplier tube. This technology can be applied to detection modules based on either single or arrayed photomultiplier tubes, significantly increasing the number of multiplexing channels for both. This enables high-resolution fluorescence imaging in real time. To illustrate the working principle of a frequency division multiplexed fluorescence confocal microscope, consider a two-channel version of the device in which a 488-nm incident beam — emitted from an argon-ion laser from Coherent Inc. of Santa Clara, Calif. — is divided into two by a beamsplitter (Figure 1). Figure 1. This illustration shows a two-channel frequency division multiplexed fluorescence confocal microscope. (BS: beamsplitter; M: mirror; PMT: photomultiplier tube). Images reprinted with permission of Biophysical Journal. The intensities of the two beams are individually modulated at different carrier frequencies — ω;1 = 2 π f1 and ω2 = 2 π f2, respectively — and then recombined by means of another beamsplitter. After passing through a third beamsplitter, the two modulated beams are further coupled into the back aperture of a 60x, 1.4-NA objective lens from Nikon Corp. at slightly different angles. The modulated Gaussian beams are focused at two locations on the sample by the objective, forming two spots. The distance between the two spots is adjustable by tuning the relative angle between two incident beams, which can be realized by adjusting the reflection angles of the mirrors. The fluorescence emission from the sample at the two focusing spots is collected by the same objective lens and reflected to the detection system by a beamsplitter. To view the sample and the location of the focusing spots, a fourth beamsplitter is added in the detection portion of the system. The reflected light from this beamsplitter enters a CCD imaging system, and the light that it transmits passes though a bandpass filter that blocks the excitation laser beams and transmits only the fluorescence light. The transmitted fluorescence light beams from the two focusing spots are then focused into two single-mode optical fibers by two objective lenses, with each fiber corresponding to one spot. The single-mode fibers are used as pinholes to filter out the out-of-focus light and to achieve confocal imaging5,6 and, conveniently, to couple the light beams into a single photomultiplier tube. The intensity detected by the photomultiplier tube is the summation of these two modulated intensities. The output from the photomultiplier tube is connected to a data acquisition board, which converts the analog electric signal into a digital signal. The data is sent to a microcomputer that processes it by taking the Fourier transform of the detected signal. In the frequency domain, there is no overlap between the two signals. Thus, one can easily classify both signals in the frequency domain. The worst case scenario for crosstalk noise among various frequency channels (that is, the highest amount of crosstalk) happens when the two laser spots are focused onto the same location. In this case, the signal separation can be realized only from the difference carrier frequency. The ideal situation (no crosstalk noise) occurs when the lowest carrier frequency and the separation between adjacent frequencies are twice the signal frequency (i.e., Nyquist sampling theorem). However, because other noise (e.g., from the detector) exists, there will be crosstalk among laser channels. Without losing the generality, if one assumes that the noise is Gaussian, then the bandwidth of the noise spectrum can be estimated by observing the detected signal over a finite time interval.7 Then, one can select the frequency and the frequency difference between adjacent channels at least twice this estimated spectral bandwidth, which will ensure low cross-talk noise. Limit on number of channels The maximum number of the frequency division multiplexed channels is limited by the response time of the fluorescence emission and the photomultiplier tube detector and by the dynamic range of the photodetector. The temporal resolution of this method also is determined by the response time of the fluorescence emission and the photodetector, which is on the order of 10 ns. Because this resolution is usually adequate to analyze the dynamic behavior of living cells, the response-time-limited number of channels can be estimated by dividing the signal’s temporal bandwidth (1 ms) with temporal resolution (2 x 10 ns = 20 ns), which is as high as 5 x 104. On the other hand, the dynamic-range-limited number of channels may be estimated by assuming that the useful dynamic range of the photodetector is 30 dB (that is, 1000 in the linear scale, a realistic number) and that the required dynamic range from each frequency channel is 10 dB (10 in the linear scale). In this case, the dynamic-range-limited number of channels is 100 (1000/10). Thus, the maximum number of frequency division multiplexed channels is mainly determined by the dynamic range of the photodetector. The total number of channels can be increased further by employing a photomultipler tube array. A 32-channel array is commercially available, and combining our frequency division multiplexing technique with it can make the total number of channels as large as 100 x 32 = 3200. This is good enough for many real-time confocal imaging applications. The spatial resolution of this frequency division multiplexed fluorescence confocal microscope is the same as conventional fluorescence confocal microscopes. To verify the feasibility of our technique, we set up a two-channel experimental demonstration system (Figure 2). A 488-nm argon-ion laser was used as the excitation source. The channels were modulated at 350 and 400 Hz, respectively, by two optical choppers that are conventionally used for lock-in amplifiers. A 60x, 1.4-NA objective from Nikon was used as the focusing lens, which focused the two modulated beams onto the sample. Figure 2. Researchers used the two-channel frequency division multiplexed fluorescence confocal microscope shown here to study intracellular calcium-ion concentrations. A living rat cardiac myocyte that had an average dimension of ~100 μm long x 20 μm in diameter was used as the sample. The fluorescent calcium-ion indicator fluo-4 AM ester was loaded in freshly isolated adult rat myocytes (1.8 μM, 30 min at 37 °C), which then emitted green light (520 to 540 nm) when they were illuminated by the 488-nm blue light (Figure 3). The fluorescence from the two focusing spots was collected by the same objective lens. A bandpass filter (520 to 540 nm) blocked the excitation laser beam. The transmitted fluorescence emissions were coupled into two single-mode fibers by two objectives. A Hamamatsu photomultiplier tube with a response time of ~10 ns was used to detect the fluorescence emissions. Figure 3. A living rat cardiac myocyte has two spots focused on it. Real-world test As an example of real-world applications of this high-speed, multichannel fluorescent confocal microscopy technology, the experimental system was used to study the simultaneous changes in calcium-ion concentration in a living cardiac myocyte. It is well known that calcium ions occupy a central role in cardiac excitation-contraction coupling.8-10 Recent high-resolution imaging suggests that, even though only a small number of calcium ions enter the cell, the local calcium concentration in this “cleft” between the calcium channel and the ryanodine receptor is likely to be substantially higher than that measured in the myoplasm. Other indirect evidence also suggests the existence of a “submembranous” domain in which the concentrations of calcium and sodium ions are significantly different from those in bulk myoplasm — which has major ramifications for understanding the mechanisms of excitation-contraction coupling in the cardiac myocyte. Despite the importance of answering the question of whether there is a change in local concentrations of calcium and sodium ions in the submembranous domain during the excitation-contraction coupling, very few studies have provided direct measurements because of the limited temporal resolution of conventional fluorescence confocal microscopes. Using frequency division multiplexed fluorescence confocal imaging technology, we have tracked simultaneous changes in calcium concentrations in the submembranous domain and in the bulk cytosol in a living cardiac myocyte during an action potential. Freshly isolated adult rat myocytes were loaded with fluo-4. The confocal laser excitation beams were directed separately onto the cell membrane region as well as into the bulk cytosol. To ensure that one of the modulated laser excitation beams was localized at or near the cell membrane, while the other was in the bulk cytosol, we “doubly” labeled the myocyte with a fluorescent membrane potential indicator (di-4-ANEPPS), which distributes to the charged plasma membrane (surface membranes and transverse tubules) with little to no signal in the cytosol. Because of the overlap between the emission spectrum of di-4-ANEPPS and the fluo-4, a bandpass filter (520 to 540 nm) and a long-pass filter (550 nm) were applied separately to discriminate between the two emitted fluorescence signals. Because fluo-4 is excited by a single wavelength, its fluorescence intensity is proportional to that of the excitation light, the optical light path, the fluorescent probe concentration as well as the free-calcium concentration. To ensure that the intensity of fluo-4 reflected the free-calcium concentration in the region interrogated, the intensities of the excitation beams were at two focusing spots and were further balanced by adjusting one of the beams until both had equal intensity. Second, all of the optical light paths were fixed. Finally, the uniformity of the concentration of fluo-4 was realized by following the standard procedure (that is, loading the fluo-4 at 37 °C at least 15 min before the start of the experiment). To minimize the motion artifact of the myocyte when applying the stimulating electric field, we used cytochalasin D to immobilize the cell while preserving transients in cytosolic calcium concentrations. We stimulated the myocyte to contract using field electrodes at 1 Hz, continuously sampling the fluo-4 signals for 5 to 10 s. The detected data was processed according to the described procedure. Figure 4 shows the measured temporal variation of both the cytosolic calcium concentration around the membrane region and the calcium concentration in the bulk cytosol of the same myocyte. Figure 4. The intracellular calcium-ion concentration exhibits beating curves. The solid curve shows the fluorescence emission from the bulk region, and the dotted curve shows the fluorescence emission from the membrane region. From this experimental result, we draw the following conclusions: First, the calcium-ion concentration changes during the cell contraction. Second, the rate of change is the same as the excitation rate (i.e., 1 Hz; the beating behavior). Third, the calcium concentration at the submembranous region was approximately five to six times higher than that measured at the bulk cytosol region, which was consistent with predictions.8,9,10 Meet the author Stuart (Shizhuo) Yin is a professor in the electrical engineering department at Pennsylvania State University in University Park; e-mail: sxy105@psu.edu. References 1. J. Tearney et al (1998). Spectrally encoded confocal microscopy. OPT LETT, pp. 8214-8221. 2. Z. Yaqoob and N. Riza (2002). Free-space wavelength-multiplexed optical scanner demonstration. APPL OPT, pp. 5568-5573. 3. K. Fujita et al (2000). Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays. OPT COMM, pp. 7-12. 4. F. Wu et al (2006). Frequency division multiplexed multi-channel high speed fluorescence confocal microscope, BIOPHYS J, pp. 2290-2296. 5. M. Gu et al (1991). Image formation in a fiber-optical confocal scanning microscope, J Opt Soc Am A, Vol. 8. 6. S. Kimura (1991). Confocal scanning optical microscope using single-mode fiber for signal detection, APPL OPT, pp. 2143-2150. 7. J. Proakis et al (1992). Advanced Digital Signal Processing, Chapter 8: Power Spectrum Estimation, Macmillan Publishing Co., New York, p. 473. 8. D. Bers (2002). Cardiac excitation-contraction coupling, NATURE pp. 198-205. 9. J. Cheung et al (2004). Exercise training improves cardiac function post-infarction: Special emphasis on recent controversies on Na+/Ca2+ exchanger. EXERC SPORT SCI REV, pp. 83-89. 10. D. Scriven et al (2000). Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes. BIOPHYS J, pp. 2682-2691.