In just a few years, flow cytometry panels have jumped from 18-25 colors to beyond 40, making multicolor detection and specification of the optics system even more complex. With a standard 12-fluorophore flow cytometer often hosting over 40 optical filters, the number of components in the optical train scales accordingly, and each component – and optical coating – needs to be designed to optimize signal integrity.
In immunophenotyping, oncology diagnostics and life sciences research, the difference between resolving a rare cell population and missing it can come down to the optical coatings used within a flow cytometer.
Across several distinct stages in the light path, each optic must be specified appropriately – from excitation and beam shape to fluorescence separation:
Three coating parameters directly govern flow cytometry detector sensitivity: reflectivity, wavelength selectivity and environmental stability.
Steering mirrors demand reflectivity above 99% at the operating wavelength. For every fraction of a percentage lost at the mirror surface, less excitation power reaches the fluorescently labeled cells, resulting in weaker fluorescence signals at the optical detector. If working with multiple mirrors in flow cytometry, this loss multiplies along the beam path.
To avoid overlap between adjacent fluorophore labels, dichroic mirrors require sharp spectral transitions; the sharper the edge, the lower the spillover across fluorescence channels. When edges aren’t steep enough, fluorescent emissions from neighboring fluorophores can create crosstalk, resulting in signal bleed.
In order to capture as many photons as possible, bandpass filter transmission should be above 90% at the center wavelength.
Porous coatings can absorb moisture from the air, which, in turn, alters their spectral position. This is of particular concern in clinical or cleanroom environments where flow cytometers run continuously, and coatings that shift with temperature and humidity are unacceptable. To counter this, dense films are applied – such as dielectric coatings produced by ion beam sputtering (IBS) – that are non-porous and exhibit minimal thermal shift, less than 0.5 pm/ºC.
Delivering flow cytometry coating specifications consistently – and for biomedical-grade applications – requires controlled fabrication, in-house metrology capabilities and the right
At Torrent Photonics, we manufacture laser optics - including optical filters and dichroic mirrors - for flow cytometry applications using IAD, magnetron sputtering, and IBS coating methods. These techinques produce dense, low-absorption films that improve signtal-to-noise and crosstalk suppression, and are explored further in our guide to advanced laser optics.
Additionally, we fabricate micrpatterened optical filters for multi-channel detector configurations. With spectral flow cytometers driving more demand for compact, high-density arrays, we expect these filters to become particularly relevant to the cytometry field.
All coating is carried out in our Class 100 and Class 1000 cleanrooms – where particulate contamination would otherwise become embedded scatter sites – and validated through interferometry and spectrophotometry before shipping.
To discuss your flow cytometer optical path requirements or request a custom coating quote, contact our engineering team.