Spinning Disk Confocal Microscopy of Calcium Signalling in Blood Vessel Walls

Abstract

Spinning disk confocal laser microscopy systems can be used for observing fast events occurring in a small volume when they include a sensitive electron-multiplying CCD camera. Such a confocal system was recently used to capture the first pictures of intracellular calcium signalling within the projections of endothelial cells to the adjacent smooth muscle cells in the blood vessel wall. Detection of these calcium signals required high spatial and temporal resolution. A newly developed calcium ion (Ca²⁺) biosensor was also used. This exclusively expressed in the endothelium and fluoresced when Ca²⁺ concentrations increased during signalling. This work gives insights into blood vessel disease because Ca²⁺ signalling is critical for blood flow and pressure regulation.

INTRODUCTION

With conventional confocal laser scanning microscopy (CLSM), there is a trade-off between image resolution and speed. The laser beam is scanned point by point in a raster pattern and signal is detected sequentially from each point. If the array consists of a 512×512 pixel array and each point is illuminated for 1 microsecond, then each scan will take about 262 milliseconds. The signal from each point must be acquired in that 1 µs and there is time ‘skew’ of 0.26 second between the first and last points in the scan. To compensate for the brief illumination of each pixel, an intense laser beam is required, and if the specimen is dynamic the time skew can lead to errors in observation.

Spinning disk confocal laser microscopy overcomes this problem by exploiting the multiplex principle. This was originally proven by Felgett in spectroscopy and shows that using parallel detection delivers enhanced sensitivity [1]. A recent publication by Wang [2] provides a quantitative comparison of point and disk scanning systems for imaging live-cell specimens.

In this article we describe the use of spinning disk confocal laser microscopy (SDCLM) to study calcium ion (Ca²⁺) signalling in the projections of mammalian vascular endotheliai cells and the adjacent smooth muscle. The SDCLM system could image novel Ca²⁺ release events (Ca²⁺ pulsars), which had a mean duration of 270 ms over a mean area of 14 µm². This is the first study to directly observe these Ca²⁺ events.

SPINNING DISK MICROSCOPY

A further limitation in conventional CSLM is the use of photomultiplier tubes (PMTs) whose quantum efficiency (QE, the probability of converting a photon to an electron) is rather low – typically 15–30%. In contrast the SDCLM technique uses a camera as detector that can have a very high QE; e.g. an iXon+ 897 EMCCD has a peak QE of more than 90%, making it a near-perfect detector.

Figure 1 shows how the Yokogawa dual spinning disk confocal laser scanner operates. Unlike a conventional laser-scanning microscope, where a narrow laser beam sequentially scans the sample, in SDCLM an expanded beam illuminates an array of microlenses arranged on a (collector) disk. Each microlens has an associated pinhole laterally co-aligned on a second (pinhole) disk and axially positioned at the focal plane of the microlenses. The disks are fixed to a common shaft that is driven by an electric motor.

Figure 1.

Schematic showing the principle and optical component in a spinning disk confocal microscope. The collector disk contains a pattern of microlenses, each of which concentrates its fraction of an expanded laser beam into a matching element on pinhole disk, as shown. The microlens and pinhole patterns are aligned and the disk are fixed to an electric motor shaft where they are separated by a distance equal to the focal length of the microlenses. When the pinhole disk is located in a primary image plane of the microscope, spinning the disks causes an array of focused laser beams to be scanned across the specimen. The fraction of the in-focus fluorescence emission light which returns along the illumination path is preferentially passed to the pinhole array and reflected into the camera port by the dichroic mirror located between the two disks. This geometry delivers low-background confocal images which can be detected and amplified in the ultrasensitive EMCCD camera.

When the disks spin and the scanner is coupled to a microscope with the pinhole disk located in its primary image plane, an array of focused laser beams scan across the specimen. The pinholes (and microlenses) are arranged in a pattern [3], which scans a field of view defined by the array aperture size and the microscope objective magnification. The scanning laser beams excite fluorescent labels in the specimen. Fluorescence emission will be most intense where this array is focused – the focal plane. Some fraction of this light will return along the excitation path where it will be preferentially selected by the same ‘confocal’ pinholes.

A dichroic mirror, which reflects emission wavelengths, is located between the two disks. This separates the laser emission from any excitation light reflected or scattered from the microscope optics. The geometry of the emission path results in a confocal fluorescence signal with extremely low background noise. When the detector is a highly-sensitive, back-illuminated electron multiplying CCD camera (EMCCD) the instrument can deliver results with high speed and unequalled signal-to-noise (SNR).

MATERIALS AND METHODS

Animal Procedures

To study signalling between endothelial and smooth muscle cells, a mouse was engineered to express a Ca²⁺ biosensor (GCaMP2) only in the endothelium [4]. This innovative, tissue-specific, gene-encoded Ca²⁺ biosensor facilitates Ca²⁺ measurements because the tissue does not require prolonged loading with dye and the fluorescence signal from the endothelium was not confused with fluorescence from other cell types [5].

Endothelial Calcium Ion Imaging

The Revolution confocal system (Andor Technology) used for Ca²⁺ imaging featured a spinning disk unit (CSU-22, Yokogawa) and an Andor iXon DV-887-BV back-illuminated electron-multiplying CCD camera (16 µm² pixel size, peak quantum efficiency = 95% at 550 nm) on an upright Nikon Eclipse E600FN microscope using a 60× water-dipping objective (NA 1.0) (Figure 2). The camera’s EM gain was 3500 and the camera was cooled to −70°C.

Figure 2.

The Andor revolution spinning disk confocal imaging system in the author’s laboratory. A Nikon fixed stage upright microscope provides the optical platform for the spinning disk scanner and the EMCCD camera. The laser combiner, control hardware and workstation are to the left of the microscope.

Revolution TL acquisition software (Andor Technology) was used to acquire images of 430×512 pixels, which corresponds to a 115×137 µm field of view. Images were acquired at 15 or 30 frames per second with exposure times of 64 ms and 32 ms, respectively. The field of view corresponded to around 25 to 30 partial and whole cells and 13 active cells per field.

Endothelial cells expressing the Ca²⁺ biosensor GCaMP2 were illuminated at 488 nm with 20% of the available power of a 5 mW solid-state laser. Emitted fluorescence above 510 nm was collected. Fractional fluorescence (F/F₀), a measure of relative change in the fluorescence signal, was calculated to normalise for spatial variations in the specimen. The fluorescence of the region of interest (ROI) in the collected image was divided by an average fluorescence calculated from 50 images without activity in the same ROI. Calculations were made using custom-designed software [6–8].

Ca²⁺ levels were measured over the entire endothelial cell surface. The surface area was measured by automatically analysing the area enclosed by a ROI drawn freehand around single endothelial cells. Calcium pulsars were analysed using a ROI defined by a 5×5 pixel box positioned at a point corresponding to peak pulsar amplitude. Line-scan analysis occurred offline.

Cells from non-GCaMP2-expressing mice were used as a control. Their endothelial cells were preferentially loaded with the calcium fluorescent indicator Fluo-4 (10 µm) for 45 minutes at 30°C in the presence of pluronic acid (2.5 µg ml⁻¹) before imaging. 12% laser power (3 mW) was used on these cells.

RESULTS

Individual endothelial cells with an average visible surface area of 737±35 µm² were clearly distinguishable using the SDLCM system (Figures 3 and 4). Holes in the autofluorescence of the internal elastic lamina (IEL) represented projections of the endothelial membrane towards the smooth muscle membrane. The IEL separates the cells (endothelial), which line arteries from the contractile smooth muscle, surrounding smooth muscle cells. There were 5.6±0.4 holes per thousand µm², with a mean surface area of 4.43±0.09 µm².

Figure 3.

Ca²⁺ pulsars colocalized with IEL holes. (A) (a) IEL autofluorescence shows the presence of ‘holes’ in the IEL. (b) Initiation sites of Ca²⁺ pulsars from the composite image correspond to holes in the ILE (red arrows). The yellow arrows indicate pulsar sites not associated with detectable IEL holes. (Scale bar = 10 µm.) (c) Histogram illustrating the distance between Ca²⁺ pulsar initiation sites and IEL holes in endothelium (n = 357 pulsar sites). (B) Time course of a three-dimensional Ca²⁺ pulsar originating from within an IEL hole (white circle) shown in the leftmost image. (Scale bar = 5 µm.) (C) Ca²⁺ pulsars from a pressurized artery (80 mm Hg) expressing GCaMP2. (Ca) An endothelial cell and its nucleus are outlined (dotted lines), with the initiation sites (Cb and Cc) indicated by red arrows. (Scale bar = 10 µm.) Reproduced with permission from Ledoux et al. PNAS 105(28):9627–9632, July 15, 2008. © 2008 The National Academy of Sciences of the USA.

Figure 4.

Kinetics and repetitive occurrences of Ca²⁺ pulsars. (A) Average of 10 images of a field of endothelial cells from mesenteric arteries of a GCaMP2-expressing mouse. The red arrow indicates the initiation site of Ca²⁺ pulsars shown in B and C. (Scale bar = 10 µm.) (B) Life span of a Ca²⁺ pulsar is shown. The field of view corresponds to the green square in A (Scale bar = 10 µm.) (C) Repetitive occurrence of Ca²⁺ pulsars at one site expressed as a line-scan analysis along the yellow line in A. (Scale bar = 5 s.) (D) Representative traces illustrating Ca²⁺ pulsar kinetics originating from two different sites (red and blue lines). Reproduced with permission from Ledoux et al. PNAS 105(28):9627–9632, July 15, 2008. © 2008 The National Academy of Sciences of the USA.

In unstimulated arteries from GCaMP2-expressing mice, calcium spark-like events were observed close to the IEL holes (Figure 3). Most occurred in or within 2 µm of the holes and occurred repeatedly at the same site. These events were termed ‘calcium pulsars’ because their mean frequency was similar to celestial pulsars (9.8±1.0 s) and they occurred near ‘black’ holes. The pulsars had a mean duration of 270 ms and a mean area of 14 µm².

Treatment with various inhibitors showed that calcium ‘pulsars’ are localised calcium release events mediated by inositol trisphosphate (IP3), a secondary messenger molecule used for signal transduction and lipid signalling in cells. During these calcium release events, IP3 must bind to and activate the IP3Rs (receptors) in the endothelial endoplasmic reticulum, opening a calcium channel. The electron multiplying CCD camera enabled the detection of these localised, dim calcium signals within the blood vessel wall.

The calcium-sensitive protein biosensor, GCaMP2, did not distort the kinetics of pulsars. Pulsar properties (amplitude, spatial spread, kinetics and frequency) were essentially the same when measured using the calcium-sensitive fluorescent dye Fluo-4 in arteries from wild-type mice [4]. The calcium biosensor also was expressed evenly throughout the endothelium (Figure 4).

DISCUSSION

Using a dual disk laser scanning microscopy system with EMCCD camera, evidence was collected supporting the existence of an IP3R signalling structure with profound significance for current understanding of communication between endothelial and smooth muscle cells. In a process reminiscent of neuronal projections to target cells, vascular endothelial cells have projections through the IEL that contact smooth muscle cells. IP3Rs local to these membrane projections mediate local calcium release events (pulsars), demonstrating a mechanism for calcium-dependent signalling between these cell types.

Ca²⁺ pulsars may be a fundamental endothelial Ca²⁺ signalling system whose activity is likely finely regulated by flow through the blood vessels. Disruption of endothelial function, by extension calcium pulsars, is a hallmark of virtually all cardiovascular diseases.

Studying Ca²⁺ pulsars in whole tissue requires an imaging system with high SNR, good spatial resolution, and high frame rates (10 to 100 frames per second). Dye concentrations or expression levels of genetically coded sensors must also be minimal. The specimen excitation dose should also be kept to a minimum to ensure it remains healthy and displays physiologically-relevant behaviours. Unfortunately, sensitivity can be limited by optical, mechanical or electronic noise.

The confocal system used in this study addresses these factors by including a spinning disk scanner with a low background orthogonal detection channel; high performance dichroic mirrors; matched achromatic camera coupling optics; a highly-sensitive EMCCD detector; highly stable and efficient laser delivery system; microsecond laser synchronization with AOTF blanking; and easy-to-use system control software.

CONCLUSIONS

This study shows that a spinning disk laser confocal microscopy system can be used to successfully observe fast-developing events occurring in a small volume. Calcium pulsars, which likely modulate blood vessel diameter, and hence blood flow, have a duration of <500 ms and occur in structures (endothelial projections in the blood vessel wall) smaller than 4 µm in diameter.

Many other whole tissue and live cell applications can benefit from the use of this instrumentation. It combines extreme sensitivity and high frame rate with high levels of instrumental stability and synchronisation. Applications include the study of intracellular signalling, protein translocation, membrane trafficking [9], cytoskeletal dynamics [10], nuclear organization and embryogenesis. Further with the addition of targeted laser photobleaching, activation and ablation tools, it is possible to isolate or disrupt cellular processes to extract quantitative information about these processes.

Biography

Mark Nelson graduated in mathematics and biology in 1976, completed a PhD in neural sciences at Washington University in St Louis and did postdoc research in the USA (ion transport) and Germany (biophysics). He is now University Distinguished Professor and Chair of the Department of Pharmacology at the College of Medicine, University of Vermont, USA. Nelson’s current interests are calcium signalling, ion channels in vascular function and neurovascular coupling; his expertise is live-cell imaging using confocal, TIRF and multiphoton microscopes.