Our understanding of the structure-photochemistry relationships of green fluorescent protein (GFP) (Tsien, 1998) has enabled the development of genetic calcium probes based on a circularly permuted GFP (cpGFP) in which the amino and carboxyl portions have been interchanged and reconnected by a short spacer between the original termini (Baird et al., 1999). The resulting new amino and carboxyl termini of the cpGFP have been fused to calmodulin and its target peptide M13, generating a chimeric protein named pericam (Nagai et al., 2001). This new protein was fluorescent, and its spectral properties changed reversibly with Ca2+ concentration, probably due to the interaction between calmodulin and M13, which alters the environment surrounding the chromophore. Three types of pericam have been obtained by mutating several amino acids adjacent to the chromophore. Of these, "flash pericam" becomes fluorescent with increasing Ca2+, whereas "inverse pericam" dims. However, "ratiometric pericam" has an excitation wavelength that changes in a Ca2+-dependent manner, thereby enabling dual-excitation ratiometric Ca2+ imaging. Ratiometric dyes permit quantitative Ca2+ measurements by minimizing the effects of several artifacts that are unrelated to changes in the concentration of free Ca2+ ([Ca2+]), such as uneven loading or partitioning of dye within the cell or varying cell thickness. This article presents an outline of an imaging experiment using HeLa cells expressing ratiometric pericam to measure receptor-stimulated changes in intracellular [Ca2+] ([Ca2+]i) (Protocol 1).
In contrast to cameleons (Miyawaki et al., 1997), which are fluorescence resonance energy transferbased Ca2+ indicators, pericams can be easily targeted into the mitochondria matrix using an upstream targeting sequence encoding subunit IV of cytochrome c oxidase. Ratiometric pericam has been used successfully to monitor changes in [Ca2+] in mitochondria ([Ca2+]m) (Nagai et al., 2001). In most dual-excitation imaging experiments, the excitation wavelength is alternated using a rotating wheel containing two bandpass filters. However, it is also possible to use a highspeed grating monochromator to increase the rate at which the ratio measurement is conducted to approximately 10 Hz. The latter instrumentation enables the measurement in spontaneously contracting cardiac myocytes of beat-to-beat changes in [Ca2+]m, contributing to the demonstration that [Ca2+]m oscillates synchronously with cytosolic [Ca2+] during beating (Robert et al., 2001). This article discusses factors to be considered when using such a monochromator with ratiometric pericam.
Although the aforementioned measurements are typically performed using conventional microscopy, the monitoring of changes in [Ca2+] is often severely limited by the poor spatiotemporal resolution of such wide-field techniques. To obtain a more reliable representation of changes in subcellular [Ca2+], it is necessary to increase the z-axis resolution and the speed of production and collection of the ratios of the excitation peaks. This article provides a detailed protocol describing a modified laser-scanning confocal microscopic (LSCM) system for ratiometric pericam (Protocol 2). In our experiment (Shimozono et al., 2002), fast exchange between two laser beams was achieved using acousto-optic tunable filters (AOTFs). Samples were scanned on each line sequentially by a violet laser diode (408nm) and a diode-pumped solid-state laser (488 nm). In this way, the ratios of the excitation peaks can be obtained at a frequency of up to 200 Hz.
HeLa cells (ATCC # CCL-2.2)
Culture medium: Add fetal bovine serum (Gibco BRL, Life Technologies) to Dulbecco's modified Eagle's medium (DMEM) (Sigma Aldrich) to a final concentration of 10% (v/v)
Hank's balanced salt solution (HBSS): Dissolve CaCl2 (0.14g), KCl (0.40g), KH2PO4 (0.06g), MgCl2·6H2O (0.10g), MgSO2·7H2O (0.10g), NaCl (8.00g), NaHCO3 (0.35g), Na2HPO4 (0.048g), and D-glucose (1.00 g) in 800ml of distilled H2O, adjust the pH to 7.4, and then adjust the volume to 1 liter.
Histamine solution: Dissolve 1.841 mg of histamine dihydrochloride (Sigma Aldrich, H7125) in 1 ml of HBSS to make a 10 mM stock solution.
Ratiometric pericam cDNA
Ratiometric pericam mitochondrial cDNA
35-mm glass-bottom cell culture plates (Matsunamiglass, Osaka, Japan)
400DF15, XF1006, Omega
485DF15, XF1042, Omega
535AF45, XF3084, Omega
505DRLP, XF2010, Omega
505DRLP-XR, XF2031, Omega
500ALP, XF3092, Omega
A. Conventional Microscopy for Time.Lapse [Ca2+]i Imaging
Dual-excitation imaging with ratiometric pericam uses two excitation filters (485DF15 and 400DF15), which are alternated by a filter changer (Lambda 10-2, Sutter Instruments, Novato, CA), a 505DRLP-XR dichroic mirror, and a 535AF45 emission filter. The excitation and emission spectra of ratiometric pericam in the presence and absence of Ca2+ with passbands of the emission filter and two excitation filters are shown in Fig. 1A. Also, the transmittance of the dichroic mirror (505DRLP-XR) is superimposed. The dichroic mirror has an eXtended Reflection region below 505nm; it was designed originally for the measurement of Ca2+ (fura-2) and pH (BCECF). In this situation, the broad reflection of a dichroic mirror is imperative. A more common long-pass dichroic mirror (505DRLP) cannot be used; superimposition of its transmission spectrum (Fig. 1B) makes one notice a complex transmission band at short wavelengths, which prevents reflection of the 400-nm light. The author strongly recommends that researchers make graphs of spectra plotted as percentage transmittance for the interference filters (excitation filters, emission filters, and dichroic mirrors) that are actually used, together with excitation and emission spectra of the relevant fluorescent dyes. The transmittance curves for the filters (normal incidence) and dichroic mirrors (45° incidence) can be measured easily using a conventional spectrophotometer. This measurement also provides a chance for researchers to check the quality of their interference filters, which may deteriorate with time.
B. A Modified LSCM System for Ratiometric Pericam
Although the following procedure assumes familiarity with LSCM, and with the Olympus LSCM in particular, the procedure can be adapted easily to other LSCM types. The microscope and lasers we use are described in Fig. 2. The beam from a diode-pumped solid-state laser (Sapphire 488-20, COHERENT) directly enters an AOTF (AOTF1; AOTE8C, AA Opto- Electronic). The light is relayed through an optical fiber (FV5-FUR, Olympus) to a confocal microscope scan head and passes through a dichroic mirror (DM1; DM420SP, Olympus). A laser diode (NLHV3000E, NICHIA) is mounted on a heat sink with temperature control, which consists of an LD mount and heat sink [F125-4A (Suruga Seiki)], LD current driver [LDX-3525 (ILX Lightwave)], and LD temperature controller [LDT5525 (ILX Lightwave)]. The output of the laser diode is collimated (made parallel) using multiple lenses, and its power is stabilized by a feedbackregulated device. The beam is directed into another AOTF (AOTF2; AOTE4C UV, AA Opto-Electronic). The diffracted light is sent through another fiber-optic line (FV5-FUR-UV, Olympus) to the scan head and is then reflected on DM1. The two AOTFs are controlled electronically, allowing the reciprocal choice of one of the two laser lines. The chosen laser beam is reflected on a second dichroic mirror (DM2; FV5-DM442, Olympus). The scan head is coupled to an inverted microscope (IX70, Olympus) through its right-side port. The objective lens used is a PlanApo 60× N.A. 1.00 WLSM (Olympus). The fluorescent light is descanned, passed through DM2, a pinhole, and an emission filter (BA505-525 (Omega)), and detected by a photomultiplier. Fluorescence signals with excitation wavelengths of 408 and 488nm are distributed into two channels during analog-to-digital conversion. The pair of galvanometer mirrors, the digitized detector output, and the AOTF controller are orchestrated. The excitation and emission spectra of ratiometric pericam in the presence and absence of Ca2+ , together with the passband of the emission filter (BA505-525) and the two laser lines, are shown in Fig. 3.
The procedure for time-lapse [Ca2+]i imaging in HeLa cells is as follows.
The following is a procedure for confocal imaging of [Ca2+]m in HeLa cells using the modified LSCM system.
A. pH and Photochromism as Practical Considerations
Ratiometric pericam is sensitive to changes in pH in both the presence and the absence of Ca2+ (Fig. 6). pHrelated artifacts were not an issue in experiments that used HeLa cells because agonist-induced [Ca2+]c mobilization did not induce any intracellular pH changes detectable by the pH indicator, BCECF (data not shown). However, ratiometric pericam expressed in dissociated hippocampal neurons was perturbed by acidification following depolarization or glutamate stimulation (data not shown).
Pericams are derived from yellow fluorescent protein (YFP). If YFP is excited too strongly, its fluorescence will be reduced. This apparent bleaching is actually photochromism because the fluorescence recovers to some extent spontaneously and can be restored further by UV illumination (Dickson et al., 1997). Intense excitation of ratiometric pericam also causes photochromism, which results in a decrease in the 490/410-nm excitation ratio independent of Ca2+ change. The extent of photochromism is dependent on excitation power, numerical aperture of the objective, and exposure time. Therefore, it is necessary to optimize these factors for each cell sample in order to minimize photochromism while preserving a high signal-to-noise ratio. A good solution is to bin pixels at the cost of spatial resolution. The increased signal-tonoise ratio permits a decrease in intensity of the excitation light with a neutral-density filter and the observation of [Ca2+]i oscillations without significant photochromism of the indicators.
B. Use of a High-Speed Grating Monochromator
Instead of a filter wheel containing 400DF15 and 485DF15 excitation filters, a fast wavelength exchanger based on a grating monochromator (U7773-XX and U7794-16, Hamamatsu Photonics) can be used with a fast-acquisition CCD camera (HiSCA, C6790-81, Hamamatsu Photonics). The spectrum of the excitation light when the wavelength was set at 410 or 490nm is shown in Fig. 7. Because light acquired with the setting at 490nm spills over into the observing optical path, it is preferable to eliminate the unwanted light by putting a short-pass filter like 500SP (Fig. 7, broken line) in front of the microscope.
C. Calcium Transients in Motile Mitochondria
When changes in [Ca2+]m are monitored by alternating the excitation wavelength automatically with wide-field conventional microscopy, the rate of acquisition of the excitation ratio is about 10 Hz, which is identical to the frame rate. Despite this rapid rate of data collection, the [Ca2+]m measurements are often affected adversely by the active motion of mitochondria, especially at warmer temperatures. Using our LSCM technique, we attempted to increase the speed of the alternation of excitation wavelengths so that it was faster than the movement of mitochondria. With this method, the frame rate was 5 Hz and the rate of ratio acquisition was 200 Hz. Although the frame rate did not allow us to fully follow the rapid movement of mitochondria, the fast rate of ratio acquisition minimized the time lag between the two measurements used to calculate the ratiometric signal. We feel that this method of imaging [Ca2+]m effectively corrects for mitochondrial movement in all three possible both laterally and into and out of the optical section. After the application of histamine, spots of [Ca2+]m increase within a single mitochondrion were identifiable (Fig. 8A) and the global increase in [Ca2+]m was found to occur relatively slowly (Fig. 8B).
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