Illumination in microscopy often overlooked

BioPhotonics
Sep 2019

JASMIN SCHAEFER, IAIN JOHNSON, AND CLAUDIA JAFFE, LUMENCOR INC.

As scientists engage in the development and manufacture of high-performance lighting, we find it frustrating to encounter a lack of appreciation for the illumination source in the successful application of light microscopy. Like that other pervasive illumination source, the sun, microscope light sources are commonly taken for granted. It often seems they only draw attention when they are absent. This is unfortunate, as it not only leaves many users unaware of the benefits of modern solid-state illumination technologies, but it leaves them reliant on archaic incandescent bulbs and mercury arc lamps.

We cannot help but observe how stark the contrast is with the technologies on the detection side of the microscope. During the last 30 years, emulsion film recording has been completely displaced by solid-state digital cameras. While solid-state lighting has been around for more than 50 years, we have yet to see its full appreciation or similarly broad adoption in academic spheres, biotech laboratories, hospitals, and clinical labs, or by biotech equipment manufacturers.

The benefits of solid-state light sources are significant. Their nonreliance on mercury and other hazardous substances and their minimal operation and maintenance costs are well documented. A typical HBO100W bulb used in fluorescence microscopy contains about 20 mg of mercury and has a surface temperature of 800 °C and an internal pressure of 30 to 75 bar (400 to 1100 psi) under typical operating conditions. Under such challenging conditions, operational lifetime is only 200 to 300 hours. In stark contrast, LEDs used in lighting systems with adequate heat dissipation operate at ambient temperature and pressure and are capable of more than 10,000 hours of user operation (equivalent to 5 years of 40-hour weeks). Solid-state illuminators also provide superior light output stability with respect to wavelength and power, and on both a frame-to-frame and day-to-day basis. This is of value for quantitative comparisons where images are acquired at various intervals in a time-lapse sequence. Solid-state light sources facilitate application-specific customization of illumination. The traditional “top-down” illumination approach starts with a spectral distribution that is physically invariant (e.g., the atomic emission of mercury vapor). Such output spectra are adapted to application requirements by selective blocking and attenuation of the superfluous wavelengths and optical power. Yet the more modern approach assembles discrete solid-state light sources such as LEDs and laser diodes within an electronic control framework. Such contemporary designs enable the user by optimally matching application requirements in terms of the spectral distribution, angular distribution, and radiant flux (power).

The electronic control systems of today’s solid-state illuminators are increasingly sophisticated. They provide not only the basic output control functions (color band selection, output on/off, and intensity adjustment) but also real-time performance monitoring and feedback control. These control systems can now incorporate Ethernet connectivity, allowing performance data to be accessed remotely for diagnostic purposes. This will facilitate improved troubleshooting, providing a benefit to users and service providers alike. We commonly observe that users of fluorescence microscopes evaluate the performance of a light source based on the camera exposure time required to acquire an image of a fluorescent specimen. Yet this is a fundamentally unsound practice, because many factors determine the exposure time and only some of them directly relate to the output of the light source. Onboard monitoring systems allow the performance of the light source to be evaluated directly and independently of the microscope, camera, and specimen.

In conclusion, it is our opinion that users of fluorescence microscopy and other optical imaging techniques should keep in mind that although the light source may be hidden in plain sight, overlooking its importance may result in missed opportunities. The potential exists today for improved data quality, better operational reliability and throughput, cleaner techniques, cost-effective operation, and illumination that is customized to the requirements of the application. To fully employ today’s solid-state illumination, users need to pay mindful attention to the uniqueness these state-of-the-art products offer versus their all-too-familiar and pervasive bulb-containing predecessors.

Meet the authors

Jasmin Schaefer, technical support specialist at Lumencor Inc., earned her Master of Science degree in biomedical engineering from Oregon Health and Science University in Portland, Ore. There she developed a fluorescence imaging method for rapid intraoperative tumor margin assessment. At Lumencor, she works under Iain Johnson to aid researchers and internal sales staff with technical support. Her background in fluorescence imaging has been an asset when it comes to understanding customer applications and while troubleshooting issues that may crop up with Lumencor light engines.

Iain Johnson, Ph.D., director of technical support at Lumencor, is a biochemist with expertise in the biophysics of fluorescence microscopy. With numerous patents and publications to his credit, he is steeped in the physical chemistry of fluorescent and luminescent probes. For many years he has been a faculty member at the annual University of British Columbia 3D Microscopy course as well as with Molecular Probes/Invitrogen/Life Technologies and his own consulting firm. He is responsible for technical support of both internal sales staff and cutting-edge researchers around the world.

Claudia Jaffe, Ph.D., is co-founder and executive vice president at Lumencor. She earned her doctorate in bioanalytical chemistry from the University of Pittsburgh. She has developed, published, and patented a variety of electrochemical and photoelectrochemical sensors and bioanalytical chips, focusing her efforts on high throughput analyses employing enzymology, immunology, and genomics. At Lumencor, she is an inventor in nearly all of the company’s patents and the leader of new business development. She supervises sales and marketing as well.

The views expressed in ‘Biopinion’ are solely those of the author and do not necessarily represent those of Photonics Media. To submit a Biopinion, send a few sentences outlining the proposed topic to [email protected]. Accepted submissions will be reviewed and edited for clarity, accuracy, length, and conformity to Photonics Media style.

More news & features