Fluorescence Filtering

Figure 3: A design for manufacturing (DFM) analysis helps to achieve system performance goals while keep manufacturing costs to a minimum.
Fluorescence filter sets are essential in separating the fluorescence emission photons at the detector from the more intense excitation photons from the source. It is necessary to reduce the excitation light intensity while maximizing the number of fluorescence emission photons. High capture efficiency enables reductions in overall excitation light levels and thus, reductions in dye photobleaching and photo-toxicity of the biological sample.

Filter selection is most often a complex analysis of the spectral relationships of fluorophores, optical filters, excitation sources, and detectors. Because off-the-shelf components offer several ways to minimize costs in product development, purchasing, manufacturing, quality, and reliability within the DFM process, off-the-shelf filters available from commercial suppliers such as Newport Corporation (Irvine, CA) and Omega Optical (Brattleboro, VT) were considered and modeled. Optical and mechanical characteristics of these off-the-shelf filters were imported from TracePro’s libraries and modeled in the system. Narrowband excitation and emission filters centered on the Alexa Fluor 488 dye’s absorption and emission peaks were selected to minimize spectral overlap of the emission signal with the excitation signal and increase signal isolation.

Filter selection was further complicated by fluorophores’ significantly different spectral properties in a particular application such as nucleic acid stains bound to RNA compared to fluorophores in aqueous solution. Complex and application-specific characteristics of the fluorophores’ sample preparation and biological target were then modeled in combination with the selected filter pair by iterating and analyzing quantum efficiency and fluorophore concentration values directly in the TracePro and SolidWorks model.

Simulation and Analysis

The complete opto-mechanical system, including the fluorescence-tagged biological sample, was modeled and documented in SolidWorks with the TracePro Bridge add-in. The single, archived SolidWorks file was then opened with TracePro for optical simulation and analysis. Source rays propagate through the model with portions of the flux of each ray allocated to absorption, specular reflection and transmission, fluorescence, polarization, and scattering.

From the simulation, contributions to sensitivity were analyzed, including system throughput at the detector, flux absorbed by surfaces and bulk material, and stray and scattered light from mechanical and optical surfaces. Contributions to dynamic range were analyzed by changing the concentration and quantum efficiency of the dye to simulate the breadth of addressable sample preparations.

Based on the TracePro irradiance map, designers concluded that the sensitivity specification was not achieved. From the TracePro flux report, it was concluded that there were insufficient fluorescence emission photons captured, and unwanted stray light from optical and mechanical components reached the detector.

Design for Manufacturability

Component-level improvements affecting system level sensitivity were identified relative to driving DFM factors such as component cost, number of components, and manufacturing tolerances (see Figure 3).

From the TracePro and DFM analyses, the design team concluded that the sensitivity specification may be achieved by increasing the f/# on both the LED and detector collection optics and modifying the mechanics to reduce stray light at the detector. These design modifications offered the largest improvement in performance at orders of magnitude less expense.

The design of the new collection lenses was realized and toleranced in commercially available lens design software and translated to SolidWorks with LensWorks into the single archived SolidWorks file. Mechanics were modified and a single set of documentation was updated in SolidWorks. The same SolidWorks file was then opened by TracePro for design validation.

The development of the fluorescence spectroscopy product platform demonstrates that with an integrated set of software design tools, a disciplined DFM product development process can be executed effectively and efficiently within a multidisciplinary design team. System-level performance and component-level specifications can be communicated across technical disciplines while data and design integrity can be insured by documenting in compliance with cGMP. Product cost and time to market are minimized with improved product quality and reliability.

This article was written by Eric Heinz of Heinz Optical Engineering Co. (San Diego, CA), and Edward Freniere, Ph.D., Richard Hassler; and Linda Smith from Lambda Research Corporation (Littleton, MA), with acknowledgement to Iain Johnson at Invitrogen Corporation (Carlsbad, CA). For more information, contact Ms. Smith at This email address is being protected from spambots. You need JavaScript enabled to view it., or visit http://info.hotims.com/10960-202.