Upgrading to five-color imaging

We’ve recently been working on upgrading one of our microscopes to do five-color imaging, using a DAPI / FITC / Cy3 / Cy5 / Cy7 filter set. To do so, we replaced our Sutter Lambda XL with a Sutter Lambda LS xenon arc lamp, containing a special 1100 nm cold mirror (thanks Sutter!) so that more of the infrared emission makes it to the output. The spectra for both lamps, measured at the liquid light guide output, are shown below.new LS

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Theatrical gels as color filters

A recent chat with a colleague about cheap sources for optical filters and a similar conversation on facebook reminded me of an old trick for getting cheap fluorescence filters: theatrical gels. These are the filters used to give theatrical lights their color and they are sheets of dyed plastic. While they don’t have the performance of dichroic filters (or even absorptive glass filters), they are much cheaper – a 2 foot square piece costs less than $10. Conveniently, Rosco, one of the major manufacturers of gels, provides absorption spectra for all of them. They also have an easy-to-use web tool for browsing through different color filters.

For example, here’s the spectrum of Roscolux #15, which makes a not bad long pass filter with a cut-on wavelength of about 550nm:

Rosco15crop

Transmission spectrum of Roscolux #15.

Their low cost makes them useful for situations where you need a large area filter and don’t need the performance of a dichroic filter. For instance, I originally discovered them for screening GFP libraries on a 22 cm plate – we plated out thousands of colonies, illuminated them with a filtered arc lamp, and then covered a pair of goggles with gel filters to see the fluorescence of each colony. ¬†They’ve also been used for building cheap blue transilluminators for visualizing DNA gels.

These gels aren’t going to replace dichroic filters, but have their place. If you have a clever use for them, post a comment.

Using Matlab to Find an Optimal Filter

I ran into a problem today – I’m putting together a quote for a new spinning disk confocal, and I want to find optimal emission filters for the dyes I want to image. For example, my confocal will have a 647 nm excitation laser that I want to use to excite both Alexa 647 and the infrared proteins iFP1.4 and iRFP. So, I need to find an appropriate emission filter. If I go to Semrock, I can look up the filter they recommend for Alexa 647, but not the infrared proteins. If you do this, you will find a large number of filter sets recommended for Alexa 647, of which this one seems to be the most appropriate for laser excitation.¬† It uses a 676/29 emission filter, which seems pretty narrow for Alexa 647, and certainly doesn’t extend far enough to the infrared to collect fluorescence from the infrared proteins.

I can look through Semrock for a better filter, but what I’d really like to do is automate this process. Ideally, I’d check each filter to see that it blocked the 647 nm laser excitation, and then find the one that has maximal transmission of the dyes I want to image. It turns out that with some computer tricks, this is not that hard to do. Continue reading