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Filter economicsWhen we look at a fluorescence imaging system the choices of light sources, objectives and cameras are given a great deal of attention, and rightly so – they are critical components and can be very expensive.  Fluorescence filters, on the other hand, are often taken somewhat for granted if their specifications approximately match up with excitation and emission spectra of the fluorophore being observed.  The fact is, filters bring a lot more to the party than simply filtering light.  They can play a significant role in the final image (signal) intensity and image contrast, two parameters that are paramount especially when viewing dim images. Let's take a brief look at some of these components and their contribution as a function of cost.

First, of course, is the objective lens, and its numerical aperture (NA) is the key specification that impacts light throughput.  Let's consider a couple of high-end Planapo objectives:   a 40X, 0.85 NA and a 40X, 1.25 NA.  For the objective, brightness or intensity can be defined with the proportionality:

I ~ NA4/Magnification2.

Doing the math we can see that the 1.2 NA objective captures and transmits almost 4.7X as much light as its 0.85 NA counterpart.  Considering that you can buy the better objective for well less than a $1000 premium over the 0.85 version, upgrading your objective is hands down the least expensive way to get more light through the system.

The next two components, the fluorescence filters and the camera, are not quite as obvious.  Given the significant difference between the price of a camera and of optical filters, the economics of signal throughput versus cost for each of these components is worth considering. A good scientific-grade camera ($5,000 – $10,000) used in a fluorescence microscope system provides about 50-70% quantum efficiency (QE). This means that 30 – 50% of the photons striking the camera are lost during the process of converting photons into electrons, for the purpose of visual display.  Of course, it is possible to replace such cameras with higher (say > 90%) quantum efficiency but this typically comes at a high price – high-end premium cameras can cost considerably more.

Optical filters, on the other hand, cost well under a $1,000 to upgrade.  A set of premium optical filters can provide much higher transmission (typically > 97%) compared to average performance filters (75 – 85%).  But how do these options for upgrading your system really compare?  Consider a fluorescence microscope with average performance filters and a scientific-grade camera – call this the starting point.  Upgrading the filters alone costs ~ $1,000, and yields a relative increase in throughput of almost 50% (assuming both excitation and emission filters limit the total throughput).  Upgrading the camera alone, which costs tens of thousands of dollars nets about the same relative performance increase.  Clearly, the first and most economical step to improving system throughput or sensitivity is to upgrade the filters.  Of course, upgrading both the filters and the camera results in the best possible performance – over 120% improvement.

 

Upgrade Cost

Filter
Transmission

Camera
QE

Total
Throughput

Relative
Increase

Starting Point

$0

80%

60%

38%

0%

Upgrade Filters Only

~ $1,000

97%

60%

56%

47%

Upgrade Camera Only

~ $20,000

80%

90%

58%

50%

Upgrade Filters & Camera

~ $21,000

97%

90%

85%

121%

 

Until now we have restricted the discussion to throughput or image brightness.  Without sufficient contrast however, the signal will be difficult to distinguish from the background.   Simply put, contrast is the difference in intensity between the image and the adjacent background relative to the overall background intensity.  It is actually contrast, and not brightness, that allows the fine details of a sample to be visualized.

Obviously people don't pay 10's of thousands of dollars for higher-end, premium cameras just to increase quantum efficiency – these cameras also improve contrast by reducing various noise sources.   However, the role of optical filters in contrast improvement is often overlooked, despite the fact that the impact of filters on contrast can rival that of cameras in many systems and experiments.  Filters impact contrast by blocking unwanted excitation energy (including UV and IR) or sample and system autofluorescence to ensure the darkest background.   This blocking is expressed as optical density (OD) and high quality filters generally have an OD of 6 or greater which translates to a transmission of only 0.0001%, which is a very small value indeed.  Furthermore, the best optical filters provide blocking that is optimized to be deepest at the wavelengths that need it most, and provide very steep transitions from deep blocking to high transmission.  Filters also influence  contrast by positioning the edges at the most optimal wavelengths for your typical sample and imaging conditions.  For example, a sample with very low background autofluorescence can benefit from wider passbands, whereas narrower passbands that allow only the strongest portion of the fluorophore emission spectrum to be transmitted reduce autofluorescence noise and thus improve the signal-to-noise ratio in high background autofluorescence samples.


So when choosing components for your fluorescence imaging system don't overlook the filters.  In many cases they represent the most performance for the dollar of the entire system.