Cost-Effective, High-Performance Confocal Microscopy

Mark Browne, Andor Technology PLC

Since the first commercial confocal laser scanning microscope (CLSM) was introduced more than 20 years ago, it has become an essential tool for life sciences research, contributing to many discoveries about cell and tissue structure and function. The key feature that sets a confocal microscope apart from a conventional epifluorescence microscope is its ability to acquire images of optical sections from the specimen. A series of optical section images can be manipulated with computer software to produce high-resolution 3-D images. Computer analysis of these images reveals spatial and functional relationships between discrete elements of the specimen. Sections can be very thin (approximately 1 to 5 µm), but because the technique interrogates optically, it is essentially nondestructive.

During the same period, and spurred on by the availability of increasingly sophisticated instrumentation and computing, scientists have invented many techniques to label specific cellular components with fluorescent probes. Examples include immunolabeling and genetic transfection, in which genes expressing fluorescent proteins can be introduced into the DNA of host cells and organisms. These transfected genes can be used as “optical reporters,” lighting up when the host is “expressing” a specific genetic function. When this is done with multicolored fluorescent probes, the confocal microscope can acquire data with exquisite detail in 3-D, revealing structural and functional information. An example of such a 3-D image is shown in Figure 1.

Figure 1. This example of a 3-D data set (and zoomed view, right) was obtained with a confocal microscope. The raw data consists of optical sections from a zebra fish embryo, which is used to study tumor cell morphology and metastasis. Blood vessels are shown in green, while tumor cells are in red. The optical sections have been rendered with Bitplane Imaris software to provide a surface rendering; when time-series data is available, cell morphology and movements can be quantified. Data courtesy of professor R. Klemke and colleagues, University of California, San Diego.

The CLSM has been especially useful in the study of fixed specimens. However, in the past 12 to 15 years, research focus has widened to include the study of living cells. Studying time-sensitive events in live specimens requires monitoring environmental conditions related to cell health as well as limiting light exposure, to avoid phototoxicity and cell death. While CLSM has been applied to live cells, its sequential scanning of the specimen is slower and more phototoxic than the parallel illumination and detection approaches available with laser spinning disk confocal (LSDC) microscopy,¹ a technique that has been developed more recently.

CLSM and LSDC instruments both rely on high-performance laser illumination, which is expensive and requires regular maintenance. This may make it difficult for individual users and smaller laboratories to justify having it, yet the need still exists for these labs to have direct access to equipment for their studies outside of the “central imaging facility” model. Fortunately, a recent development is set to change the cost-performance balance in this space as manufacturers develop fast confocal instruments for rapid fixed and live-cell imaging.

One such system is the first to successfully deliver high-performance confocal imaging using a white-light source rather than lasers. The differential spinning disk (DSD) scanner can provide multifluorophore imaging from a single light source at a cost substantially lower than CLSM or LSDC instruments. However, unlike previous white-light systems, a DSD offers performance that is comparable to that of a point scanning system, but with higher speed and lower photobleaching.

The new Andor Revolution DSD, a structured illumination spinning disk confocal scanner, uses a patented optical detection principle known as “aperture correlation” to reject out-of-focus light.²

Key benefits of the DSD scanner include frame rates up to 10 times those of point scanning systems; optical sectioning with objectives ranging from 10x to 100x; usability with many fluorophores by selection of appropriate filters; and, as will be discussed, simultaneous acquisition of confocal and epifluorescence images.

In a spin

DSD scanners differ from traditional CLSM and LSDC microscopes because they scan a “structured illumination” pattern instead of one or more laser beams (Figure 2). Structured illumination microscopy has been applied before but has not been integrated into a commercial spinning disk system.

Figure 2. A solid model of a differential spinning disk confocal scanner shows a schematic of the optical path. The excitation beam is green, while the emission fluorescence beams are shown in orange and yellow to illustrate the different optical paths of, respectively, the reflected and transmitted signals. The two signals are combined at the prism and projected onto two sides of the camera.

Spinning disk systems in general are much faster than point scanners because they scan the specimen illumination pattern in parallel. This illumination pattern induces in the specimen fluorescence that is captured by a sensitive CCD or electron multiplying CCD camera system. Further, CCD and electron multiplying CCD detectors commonly have photon conversion (quantum) efficiencies on the order of 60 to 90 percent.

In contrast, CSLM point scanners are sequential illumination and detection systems usually employing photomultiplier tubes, whose quantum efficiencies are in the range of 20 to 30 percent. Moreover, sequential beam scanning means that, because the beam is scanned faster, less time is spent on each part of the sample. Thus, for the fluorophore to return enough signal to form an image, illumination intensity must be increased.

Initially, intensifying the light excites more fluorescent labels and increases the amount of fluorescence emitted. However, at a certain point, all the fluorescent labels are in their excited state, and intensifying the light further will not generate any additional fluorescence. This means that the laser scanning speed cannot be increased further without decreasing the image quality.

In a DSD system, the spinning element comprises a single synthetic quartz disk supporting a thin layer of aluminum in which the structured illumination pattern (SIP) is created by photolithography. The aluminum SIP has a 1:1 mark to space ratio (half metal and half space), which means that approximately half of the light falling upon it is reflected (R), while half is transmitted (T) (Figure 3).

Figure 3. The differential spinning disk is manufactured with two structured illumination patterns, radially disposed. In the Andor differential spinning disk, the inner pattern, referred to as the “high signal” region, is designed with a 320-μm pitch and a 1:1 mark-space ratio. The outer pattern, referred to as the “high sectioning” pattern, has a 160-μm pitch. The smaller the pitch, the sharper the axial response or “optical sectioning” capability. These patterns have been found to deliver a good compromise between optical sectioning and signal-to-noise ratio.

The disk is located at an image plane of the microscope optical system, so that an image of the SIP is projected into the specimen, and about half of the illumination intensity arrives at the specimen, while the remainder is reflected back into the illumination pathway, where it is baffled to minimize background. In the detection pathway, the resulting fluorescence signal comprising what is in focus (confocal, or C) and out of focus (wide field, or WF) is imaged back onto the disk, where we make use of its transmissive and reflective properties.

Getting into focus

The fluorescent light transmitted by the DSD comprises the C signal plus about half of the WF signal, while the light reflected from the disk comprises about half of the WF minus the C signal. In confocal terminology, the SIP is located in a conjugate image plane and hence acts as both the confocal source and the detection apertures. However, as it is not a pinhole, we must undertake some further image processing to separate confocal and wide-field signals. The optical path is illustrated in Figure 2.

From the description above, we can see that the transmitted and reflected signals can be approximated as follows:

T = 0.5WF + C; R = 0.5WF — C (Equation 1)

and simple algebra shows us that

2C = T — R; WF = T + R (Equation 2)

As Equation 2 highlights, we need to collect both transmitted and reflected light signals (images) to compute the confocal signal. Furthermore, we can easily compute the wide-field (conventional epifluorescence) signal as well as the confocal.

Bear in mind that the simple mathematics of Equations 1 and 2 hides one complexity in the principle of the DSD; i.e., the T and R images must be well-registered for the calculations to provide high-quality images. Any misalignment between the two will result in registration noise, so an essential feature of image processing for the DSD is a high-performance, real-time registration algorithm.

The SIP pitch is comparable to the pinhole size in a conventional confocal scanner. Its dimension (as well as the microscope objective properties and imaging wavelength) determines the resulting optical section thickness, but the similarity ends there, because the DSD confocal image results from an image processing operation that actively subtracts the wide-field signal. The result is that the DSD achieves high contrast even at low magnification with relatively thick optical sections. In addition, it should be pointed out that, although the SIP has been simplified to an array of linear features (as shown in Figure 3), the DSD performance is far superior to that of a slit scanning confocal. A recent publication shows that active rejection of wide-field signal delivers a sharper roll-off in the axial response compared to a point scanning instrument.³

DSD compares well to CLSM with respect to photobleaching, a nonlinear effect in which high illumination power has the greatest effect. Photobleaching leads to toxicity in live specimens and loss of signal over time. LSDC is known to provide lower bleaching by virtue of parallel illumination and detection: The laser is expanded to create about 1000 individual beams for scanning. DSD yields bleaching performance similar to that of LSDC with roughly 100 SIP lines being scanned in parallel with a similar illumination power per unit volume.

On the other hand, because DSD must detect confocal signal in the presence of the much larger wide-field signal, its signal-to-noise ratio is constrained by the wide-field shot noise. Shot noise-limited signal-to-noise ratio means that signal must be traded with optical sectioning. Very thin optical sections result in a poor signal-to-noise ratio; hence DSD SIP design is intended to optimize the trade-off (Figure 3).

Figure 4. Illustrated is the image processing flow used to register and combine the T and R images as defined in Equations 1 and 2. On the top left of the figure is the raw image collected by the camera. On the top right are the flipped WF — C and WF + C images that must be registered to high precision. Addition of the resulting image pair yields the WF image, while subtraction produces the C image. Because addition and subtraction are very fast compared to the registration step, both C and WF images can be obtained for little overhead and almost simultaneously.

Because the DSD system collects and separates wide-field and confocal light, it is possible to switch between the two with a simple click in Andor’s iQ control software (Figure 4). This can be very helpful when bringing the specimen into focus – a major advantage because focusing on a specimen can be difficult with a conventional spinning disk or point scanner. An image of a fixed specimen acquired with an Andor Revolution DSD system is shown in Figure 5, where wide-field and confocal images are shown side by side.

Many factors must be considered when choosing an instrument for scientific research. DSD-based systems will not replace CLSM or LSDC in all conditions, but they do represent a viable alternative in many applications ranging from analysis of fixed tissue to live-cell imaging. DSD can be tuned to a broad range of fluorophores, operates at high and low magnification, and acquires full-resolution images at frame rates on the order of 1 to 10 Hz.

Figure 5. Three-color images of a fixed specimen were acquired with an Andor Revolution DSD system. On the left is an extended-focus confocal image exhibiting high clarity and spatial resolution, low background and lack of out-of-focus blur. On the right is the equivalent wide-field image (acquired simultaneously) with the characteristic limitations of epifluorescence imaging.

Developing a high-quality white-light confocal system has been a goal for many companies because of cost and maintenance benefits over laser-based instruments. However, until recently, such systems delivered unacceptable image quality for all but a few applications.

DSD scanners deliver a solution to these limitations and change the confocal landscape from the user’s perspective. DSD provides image quality comparable to that of laser point scanners at moderate light levels.

Meet the author

Mark Browne is director of the systems division at Andor Technology plc; e-mail: m.browne@andor.com.

References

1. E. Wang et al (May 2005). Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems. J Microsc, pp. 148-159.

2. R. Juskaitis et al (October 1996). Efficient real-time confocal microscopy with white light sources. Nature, pp. 804-806.

3. V. Poher et al (August 2008). Improved sectioning in a slit scanning confocal microscope. Opt Lett, pp. 1813-1815.