At Omega Optical, we supply a range of precision filters and dichroic mirrors designed exclusively for flow cytometers. In this blog post, we’ll explore the basic working principles of flow cytometry to explain how our optics are increasingly important for high throughput, multiplexed flow cytometry.

Flow Cytometry: Fluidics

All flow cytometers are based on central fluidics systems known as flow cells which contain a continuous liquid stream traveling through the cytometer. Samples in suspension are injected into the center of the sheath fluid as it passes through the nozzle which ‘focuses’ the liquid stream according to the cells’ hydrodynamic radii. This causes them to flow through the system in single file.

At the heart of the flow cytometer is a laser, or multiple lasers, that is/are pointed through the liquid stream. Several different optical detectors obtain the signals generated by light interacting with the flowing cells. These include forward scattered and side scattered light, alongside characteristic fluorescence.

Measuring Scattered Light & Fluorescence

One cell at a time, the sample suspension passes through the laser/s and alters the beam. Scattered light indicates the granularity and size of the cell. Forward-scattered light, sometimes abbreviated to FSC, represents the cell-surface area or overall size. Side-scattered light, or SSC, provides information about the cell’s internal composition and structure. Fluorophores bound to labeled cells will also fluoresce when excited by incoming laser light. Each of these signals is generated simultaneously, providing rich detail about the cellular population, cell size, composition, and structure.

If you would like to learn more about the optimal filter layout, take a look at our flow cytometry application page

Engineers continue to push the boundaries of possibility in flow cytometry through multicolor detection. Fluorophores are sensitive to specific wavelengths of light, emitting characteristic signals when excited by specific wavelengths. For example, dyes based on fluorescein (FITC) have excitation and emission peak wavelengths in the region of 495 and 519 nanometres (nm). Using one laser and a single fluorescent reagent only gives limited insights into the type of cells within a population.

In a multichannel system, cell suspensions are stained with multiple fluorescent dyes and the flow cell is intersected with a sequence of laser beams. Precision optical filters designed to acquire signals of specific spectral bandwidths while avoiding cross-talk between channels are absolutely essential. Further to that, flow cytometry is rapidly moving into the realms of big data, leaving traditional histograms and scatter graphs behind in favor of advanced data analysis and visualization software - though this deserves an article of its own to fully do the subject justice.

Contact us today if you would like to learn more about our range of dichroic filters and mirrors for flow cytometers.

Interference filters are expensive primarily because there are a large number of competing requirements when manufacturing them. Most often, it is not a single parameter that moves the part from one camp to the other but instead a combination of requirements. On their own, these requirements don’t create significant hurdles. In concert, however, these can result in difficult manufacturing processes, low yields, and high prices. There are two areas where challenges can occur, the manufacturing process and the filter’s specification.

1. Making an abrupt transition from deep blocking to high transmission requires hundreds of interfering layers, requiring deposition times on the order of days.

Not only do long coating runs have a higher probability of failure, they also have a high consequence of that failure. Consider the impact of a system issue that aborts a coating 20 hours into the run and then requires new substrates followed by a rerun of the coating.

2. The portion of the coating run that simultaneously meets multiple requirements (transmission, blocking, transition slope, surface, and cosmetic requirements, etc) can be low, reducing the yield and driving up the price.

This is because the coating performance must thread the needle between the blocking and transmission requirements and because every coating deposition shows some level of spectral non-uniformity across the coated surface.

With this in mind, let’s take a look at some of the specifications that can contribute to a high filter price.

1. Surface Quality and Dimension

Since there will always be some level of mechanical imperfections in any coating process, a very tight surface quality specification will disqualify some portion of the coated material. This limitation can be dealt with by coating oversized plates and then avoiding the imperfections when configuring the material down to the final size. These oversized plates can also cause an increased price. If, however, the final size is particularly large, the number of parts that can be cut while avoiding the imperfections can be very low. In the extreme, there may be no parts that can be produced from a plate.

2. Aspect Ratio and Coating Specification

The aspect ratio is the ratio of the longest surface dimension to the thickness. The difficulty in producing a precision substrate increases significantly when the aspect ratio surpasses 6:1. The addition of a thick an optical coating compounds the difficulty. Coating stress will deform a substrate. Thicker coatings inherently introduce more stress. While design steps can be taken to mitigate this stress, it is extremely difficult to eliminate it entirely. If a thick optical coating is applied to a thin substrate, the resulting deformation will be extreme. Deep blocking over an extended range or an abrupt transition from rejection to transmission requires a thick coating. These spectral requirements on a large thin part combined with a stringent flatness requirement will be nearly impossible to achieve.

3. Tight Tolerance

Tight spectral tolerances will limit yield. Something to remember is that in optical coatings everything scales with wavelength. The thin-film design that produces a 10nm wide passband in the mid visible, will give a 30nm wide band in the near IR. Similarly, spectral non-uniformity across a coated surface also scales with wavelength. A well-tuned coating chamber can be expected to yield less than 1% spectral variation across a 200mm plate. In the visible, this amounts to a few Angstroms over a 1" part. Consequently, a spectral tolerance of less than a nanometer on a small part in the visible is tight but achievable. If, however, the part is large or it is at a long wavelength, the difficult tolerance can become unachievable, or at best extremely expensive.

In optical thin films, nearly anything can be accomplished given enough time and money. Given that neither of these is in infinite supply, it’s wise to consider not only the impact of any requirement , but also the interaction between all the requirements.