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  • Method developed for testing and comparing microscope performance

BioPhotonics
Sep 2006
Enables calibration for quantitative microscopy

Gary Boas

Optical microscopes can be found in all areas of biological research: from studies monitoring translational movement of proteins with fluorescence recovery after photobleaching to those probing deep into tissue with two-photon excitation and beyond. In all cases, changes in any number of parameters can result in less-than-optimal performance. Indeed, performance might differ significantly from microscope to microscope, in different configurations of the same microscope and even in the same device from one imaging session to the next.

Edward H. Cho and Stephen J. Lockett of the Imaging Analysis Laboratory at the National Cancer Institute in Frederick, Md., were keenly aware of how even slight changes can affect the performance of an optical microscope. They knew this from their own experiences and because the laboratory is a core facility with users from throughout the institute. Changes in microscope performance might come from a smudge on the lens, from dirt left in the microscope, or from normal wear and tear.


Researchers have developed a means to assess the performance of optical microscopes quantitatively. Subsequent comparisons of detection efficiency with GFP-tubulin expressing MCF-7 cells yielded a number of interesting findings — indicating, for example, that high dynamic range can be achieved by reducing the gain (from 950 V in the left image above to 650 V in the right) and thus the amplification noise. The cell line was created by Janis Bunker and provided by Mary Ann Jordan and Kathy Kamath. Specimen preparation and fixation of cells was by M. Katherine Jung.


The investigators searched the literature for a simple method to assess the performance of a microscope but did not find one that could provide an absolute measure or that could be used to compare one microscope with another, “at least not in a convenient way,” Lockett said. So they developed a methodology, which they described in the July issue of the Journal of Microscopy.

Assessing detection efficiency

They chose not to evaluate all of the parameters that factor into a microscope’s performance, but rather focused on the one they considered to be the most important: the fraction of emitted photons counted in the image, which they refer to as detection efficiency. The best way to assess this, they decided, was to place on the stage a light source that emits a defined number of photons and compare that with the number of photons that contribute to the image.

They focused on the detection of the microscope and not on the excitation provided by the light source because, although the efficiency of the excitation is important, the lasers that come with microscopes have an enormous excess of power, so any loss there can be offset, Lockett said.

For the method to work, though, they had to approximate the isotropic emission of fluorescent molecules. They initially considered placing an extremely small pinhole in front of a conventional light source, assuming that the light would be diffracted in such a way as to mimic the emission.

Computer simulations showed, however, that the pinhole would have to be tremendously small and would require an especially intense source of light. In short, they confirmed that the approach was not a viable option.

The researchers, therefore, used a material that diffuses light, eventually choosing a near-lambertian diffuser from Edmund Optics of Barrington, N.J. The light source they made consisted of this and an LED emitting at 565 ±30 or 587 nm in a 35-mm glass-bottom dish. Combined with a mathematical model of the light path for a confocal microscope and detection electronics, this yielded an excellent approximation of the fluorescence emission.

They used the method to characterize the performance of two inverted confocal microscopes made by Carl Zeiss Inc. of Thornwood, N.Y., each outfitted with a 40x, 1.3-NA oil-immersion objective lens, a dichroic mirror and an emission filter (they varied these to determine how they affected the efficiency). They acquired images with either a Hamamatsu photomultiplier tube or the Meta detector that came standard with the microscopes.

They knew the intensity of the light from the source and, based on the distribution of pixel intensity, determined the number of photons that contributed to the acquired image. They calculated the efficiency by dividing the former by the latter, and showed that it fluctuated with the different mirrors and filters. The relative efficiency was 1.00 with an 80/20 neutral mirror, 1.16 with an HFT458 filter and 0.93 with an HFT458/561 filter. When they switched to a 63x, 1.4-NA objective, the efficiency was 0.84.

This technique enables comparison of a microscope’s performance under a number of other configurations and settings, including pinhole size, detector gain and pixel dwell time. Indeed, experiments with various settings yielded a range of findings, some confirming existing assumptions, others providing new insights.

Insights gained

The scientists noted, for example, that the ratio of the amplification noise to the gain was always 1:4. This told them that increasing the gain would not produce more efficiency. The other insight is more interesting, Lockett added.

It is well-known that one can capture a very large range of intensities within a single image because of the high dynamic range of the digital image. However, because the achievable dynamic range is drastically reduced by the amplification noise, it is desirable to decrease this noise. “So how does one do that?,” he asked. “Well, since the noise is proportional to the gain, reducing the noise can be achieved by reducing the gain.”

The absolute measurements of detection efficiency also provide quantitative measurements of biological samples as opposed to the relative measurements more commonly performed with today’s microscopes. Thus, they contribute to significant advances in high-throughput imaging, for example, or in vivo fluorescence imaging of protein dynamics.

All of which is good news for the Lockett lab, which is generally interested in developing and using quantitative microscopy to understand both intracellular and intercellular processes.

Currently, the investigators are using laser scanning confocal microscopy to understand the dynamics and interactions of proteins in living cells by measuring ensembles of single fluorescently labeled molecules. This requires detecting the molecules many times over, with the accuracy of the results increasing with the number of times the measurements are performed. “Thus, it is vital to operate the microscopes at maximum detection efficiency,” Lockett explained.

The researchers also are performing quantitative comparisons of fluorescence from samples imaged in various sessions and on various microscopes. Such comparisons are possible only when the absolute detection efficiency of the microscopes is known.

As for the method itself, Lockett noted that it could be extended to include multiple LEDs in the light source, allowing measurements at different wavelengths. This would provide for comparison of a microscope’s performance with different fluorescent dyes, for example.


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