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Feature Articles

Lights! Camera! (microscopic) Action!

by Winn Hardin, Contributing Editor - AIA

Microscopy in its many variations is one of the most used, most powerful tools for understanding life, how it works when it does, and identifying problems when it goes wrong. Microscopy determines blood sugar levels for people with diabetes, explores DNA sequences and proteins that cause infectious disease, and even helps keep troops safe on the field from biological agents.  But despite all these strengths, microscopy has its challenges.

While cellular interactions may happen in quantity every few microseconds, their faint optical signals are very hard to detect and even harder to image. Plus, the sheer number of assays a microscopist may perform to learn one invaluable fact can number in the thousands or more. Automation is a key component to many commercially viable microscopy systems and that’s where machine vision helps.

Bright field microscope image of H&E stained tissue, Courtesy of DVC, Co.Whether it’s bright field or fluoroscopy, machine vision technology offers tools to help microscopists, but it requires new knowledge of light sources, life sciences, and how the two interact.

A Tale of Two Houses
“The most commonly used techniques in light microscopy, fall into two categories: bright field and fluorescence,” explains Ash Prabala, President and Founder of DVC Company, a specialist in life sciences imaging. “Our value add is in our low-noise, scientific-grade cameras, imaging software API and system level design experience. Although the reagents and cells that are being imaged vary widely, we find ourselves solving similar imaging problems for most Life Sciences Imaging OEMs.”

Bright field microscopy is the more commonly understood technique. Light reflects or transmits through a sample, a microscope collects the light and a camera (typically) collects an image. The microscope typically comes with a broadband illumination source, and the imaging is relatively straightforward.

Triple probe fluorescence image (captured via DVC 1500M camera on a Nikon microscope). Courtesy of DVC, Co.“In Fluorescence Microscopy,” adds Prabala, “there can be various excitation sources. Ultraviolet [UV] tends to be the excitation of choice, but these days there are more fluorophores and excitation wavelengths stretch through the near infrared [NIR]. Given that there are so many fluorophores with different excitation wavelengths, it’s an opportunity and a challenge at the same time. You might say there’s a demand for agile light sources can apply illumination of any desired wavelength, or even complex spectra, to the specimen.”

One agile source that DVC Company has used is the Optronic Labs DLP-based, OL490 Agile Light Source. The light source uses high energy, 500W broadband xenon arc lamp. The light passes through a diffraction grating, which spectrally disperse the light onto the DLP array, which is a semiconductor filled with moveable micro-mirrors. By directing individual mirrors or rows of mirrors, the OL490 can produce one or more narrow spectral illumination bands as narrow as 5 nm between either 480-780 nm, or 760-1600 nm and control the intensity and illumination time of individual bands through operator set programs. Other systems from other manufacturers use combinations of LEDs, LCDs, and acoustically tunable optical elements among other methods to achieve similar results.

“It’s a great source for research projects where you don’t know exactly what you’re looking for,” says Prabala. “You can scan the samples with different spectral bands quickly, look for fluorescence, and based on the emissions, determine what the sample contains. It works well on the research side, but for commercial applications, there is a need for less expensive, tunable light sources.

Developers of commercial OEM applications for specific reagents know in advance what fluorophores they will use, the relevant fluorescence excitation wavelengths and emission spectra.  Commercial applications typically choose lower-cost LEDs or laser diodes that have just the illumination wavelengths needed for that particular application. But these sources can also present challenges.

Calibration and Drift
As you can see, light sources, filters, and other optical elements are critical to microscopy. And whether the arc lamp is part of a bright field microscope, or an agile light source for fluoroscopic applications, these sources still face the challenges of time and heat.

“Illumination sources based on plasma discharge such as arc lamps (Mercury, Xenon and Metal Halide) require a considerable warm up period before reaching a stable operating state.” explains Markus Tarin, Founder and CEO of MoviMED (Irvine, California) and developer of custom machine vision solutions.

“Compared to Mercury or Xenon arc lamps, Metal Halide light sources, especially the once specifically designed for microscopy offer a far superior temporal stability after the initial warm up period. Metal Halide lamps usually offer a longer life time of 2,000 hours versus only 200 for Mercury arc lamps. Arc lamps exhibit multiple wavelength peaks in their emission spectrum ranging from the UV into the visible region. Metal Halide lamps however; tend to have up to 50% higher intensity output in the off-peak region compared to their Mercury counterpart, resulting usually in a brighter image when used with fluorophores with absorption bands falling in between typical mercury emission lines. The spatially and temporally more uniform Metal Halide lamps are more suitable for quantitative assay imaging than other arc lamps.”

“LEDs and lasers are alternatives to arc lamp sources. LEDs have life expectancies of 10,000 to 50,000 hours without a significant decrease in light output. The spectral drift is current/temperature dependent – hence a very stable power supply or driver is essential for microscopy applications. Compared to the excessive heat that arc lamps produce, LEDs run much cooler and are much smaller. Their broadened emission waveband is very suitable to excite a multitude of fluorescent probes, compared to the much narrower waveband of a laser source, making the LED more versatile in microscopy applications. LEDs have evolved in their ability to output higher intensities over the years and, although they have not quite reached the output levels of arc lamps, they are becoming a serious contender for illumination choices in microscopy applications. Their rapid response time (µsec) makes this device sensitive to small changes in the power supply”, Tarin adds.

Both LEDs and laser diodes also drift over time and as thermal conditions change. Many of these systems include feedback systems where the light is sampled inside the device so that electronic drivers can adjust the input power and cooling elements to keep the light source peak emission at the desired wavelength.

Depending on the application, speckle can also be an issue with lasers. Speckle is a random intensity pattern within the light distribution that can adversely affect experiments that need uniform, tightly controlled illumination. Finally, the stronger the light source the greater the chance of sample damage or photo bleaching of the fluorophores, resulting in less emissions and even weaker fluorescence. While laser diodes put out less power than arc lamps, they contain the optical power in a very narrow spectral range, which results in higher intensity or flux at the sample.

Growing Opportunities
In AIA’s recent article, “Machine Vision Market Speeds Ahead of Overall Economy,"  many respondents pointed to the biomedical field as a near-term growth area. MoviMED, for example, has created several biomedical imaging systems in recent years including polymerase chain reaction (PCR) systems for analyzing DNA and pathogens, high throughput biomedical assay systems that test up to 96 samples in one session, an evanescent wave analysis systems capable of testing up to 5000 samples in a session. DVC Company, which specializes in Life Sciences Imaging, has recently combined Fluorescence Microscopy and scientific-grade cameras to help a leading Life Sciences OEM develop breakthroughs in the detection and quantification of breast cancer by imaging Circulating Tumor Cells (CTCs) in blood.

The opportunities for machine vision are there and growing as the average age of world’s population increases. As volumes grow, component suppliers in lighting and high-sensitivity, silicon-based cameras will be able to drop their prices, pushing the technology further out to the edge of the customer base for point of care testing in markets around the world. “Today, the most sensitive instruments are very expensive, so only well-funded laboratories can afford these things. There’s pressure to make technologies more affordable so smaller labs, point of care testing, and other markets open up,” Tarin concludes.

 

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