Brian's Technical FAQ
Burning questions? Just sit back and let Dr. Manning help...
Do you sell a filter set for fluorochrome X, Y or Z?
Yes, we do. Most filter sets are named for the fluorochrome they are used most appropriately with. However, with thousands of possible fluorochromes available, and more being generated daily, filter set choice may not be obvious. Please contact us to discuss the possibilities, or gather our recommendations if you are unsure.
Do you sell this filter set for my microscope? Will set # 12345 fit into my scope?
Yes, we do and, yes, it will. We manufacture all of our cataloged filter sets at sizes appropriate for every make and model of microscope (or other commercially-available or home built apparatuses). All we need to know is the model of apparatus you’ll be using to ensure that we get you the appropriate optics.
Does the list price for filter set #12345 include the filtercube for my microscope?
No, it does not. The list price printed with each set would include all listed excitation and emission filters, as well as the di/polychroic mirror. Filter cubes, sliders and holders are a separate part and are listed in our 91000 series. If you do not see an appropriate filter cube or holder for your apparatus, contact us.
What are your standard filter and dichroic sizes?
Unfortunately, there is no “standard” size, as many microscopes use different-sized optics. However, our most common filter size is 25mm diameter (~1”), whereas our most common dichroics are 25.5x36mm or 26x38mm. Rest assured, though, that we can produce optics of almost any physical specification that is required. Contact us for more information.
I see three different filter sets appropriate for FITC: one that contains D-filters, another that contains HQ-filters, and a third that contains ET-filters. What is the difference (beyond the price)?
Very briefly, all three filter sets will give you a perfectly suitable image of your FITC-labeled samples. The major difference between the D-type and HQ-type filters are the slopes of the bandpass’ cut-ons and cut-offs. The D-filters have a shallower slope to their cut-on and –offs, whereas the HQ-filters’ are steeper. In other words, it takes more of the spectrum to go from low blocking to high blocking with the D designs, than the HQ. This allows us to widen the bandpasses of the HQ-filters, and move the excitation and emission bandpasses closer together in “spectral space”.
The ET-filters possess similar blocking properties to the HQ-filters, though the ET-filters’ average transmission across the bandpass is 95-98% (see Figure 1).
Differences between D, HQ and ET-style filters.
A) Typical transmission for D480/40x, HQ480/40x, ET480/40x,
B) Typical out-of-band blocking for same. Notice higher average bandpass transmission
of ET480/40x in A) and shallower slope of blocking ability in B)
I’d like to design a custom filter set using filters I don’t see listed in the catalog. What do you recommend?
This can be a tricky endeavor for a number of reasons. We recommend that you please contact us for assistance.
I need a new emission filter for my application. What information do I/you need to find an appropriate optic?
We need to know the excitation filter or excitation wavelength range to insure that the emitter in question is capable of blocking/attenuating these wavelengths appropriately. The type of light source (mercury or xenon lamp, laser or diode, etc.) can’t hurt to mention, either.
I’d like to use a specific excitation and emission filter together, but their transmission curves are separated by 10nm. Is this enough?
Why do you need to know about my excitation wavelength(s) whenever I request a new emission filter?
In order to determine the suitability of an excitation/emission filter pairing, we do not look at transmission of the requested filters. We first determine the blocking/attenuation abilities of both filters (OD or optical density = -[log(%T)]). For typical epifluorescence or wide-field microscopy, we recommend an excitation/emission filter pair with NO LESS than an OD5 blocking/attenuation (see Figure 2). For more information about blocking/attenuation, please see “Handbook of Optical Filters for Fluorescence Microscopy” pg. 8.
Figure 2. Appropriate and inappropriate blocking/attenuation characteristics of excitation and emission filter pairs.
A) Typical transmission of HQ480/40x and HQ535/40m. B) Blocking/attenuation abilities for same combination (please note ~OD 6.0 blocking/attenuation of emitter for entire excitation filter range). C) Typical transmission for HQ480/40x and HQ525/40m. D) Blocking abilities for same combination (note excitation light from 500-505nm will not be blocked sufficiently, e.g. to OD 5.0 or better).
Why is blocking/attenuation so important?
If excitation wavelengths are not properly blocked/attenuated by the emission filter, stray excitation light can and will be transmitted by the emitter. Keep in mind that the excitation light source intensity of is orders-of-magnitude greater than the intensity emitted from an excited fluorochrome. Any stray excitation light transmitted by the emitter would appear as an excitation light-colored haze present anywhere in the visible field and would ruin signal-to-noise ratios of the images. Furthermore, in certain instances, unblocked excitation light can lead to destruction of equipment or damage to the user’s eyesight.
I use an excitation filter wheel, and the excitation filter I use for DAPI/Hoechst is beginning to deteriorate/burn. What can I do to minimize this damage in future?
These filters (Chroma part #D350/50x) are known to be sensitive to ill effects generated by their proximity to the light source, under certain circumstances. We recommend a piece of KG1 IR-absorbing Schott glass to be mounted into the filter wheel between the light source and the filter itself. We are constantly attempting to re-design filters that are not prone to this type of damage, though the work proceeds apace.
I need a dichroic mirror to reflect 300-650, and transmit 650 and higher. I don’t see an appropriate optic listed in the catalog. Does a suitable mirror exist?
Yes, it does, with caveats. For 45 degree angle-of-incidence (AOI)optics , such as dichroic mirrors, the transition from maximum reflection to maximum transmission can take from anywhere from 10-30nm or more. We have many, many optical designs that are capable of UV reflection or IR transmission or any other specifications you may have. Please contact us for more information.
I would like to order a dichroic or emission filter I see listed on your webpage, what information do I need to do so?
In order to ensure that we provide you with the most appropriate precision optic for your application, we need the following:
- Size of the optic (if known) or make and model of apparatus.
- Wavelength range to be reflected or transmission range of the filter to be reflected (R specification).
- Wavelength range to be transmitted or transmission range of the filter to be transmitted (T specification).
- Size of the optic (if known) or make and model of apparatus.
- Wavelength range to be blocked or transmission range of the excitation filter (blocking specification).
The examples of optics shown on the webpage are intended for informational purposes only. The final optic may differ slightly from what is pictured. If you need further information or have questions, please contact us.
Why do you not ask about all of this information for your excitation filters?
Our excitation filters are manufactured to block all out-of-band wavelengths from UV to near-IR. This is to ensure that your sample will not be excited by inappropriate wavelengths. All we need to provide you with an excitation filter is the appropriate size for the filter.
I am looking for the typical transmission properties for part XYZ that I already own, yet I can’t find it listed on the webpage. What should I do?
We currently have approximately 12,000 optical designs to choose from. Putting them all (particularly custom optics) onto a webpage would prove unwieldy, though we are trying our best to list a significant number. If you don’t see what you need listed on the webpage, please contact us with as much relevant information as you can (part number, lot number, etc.), so that we can better assist you.
I’m concerned about the ability of filter set # 12345 to transmit the fluorescence of both fluorochromes I will have present in my sample (e.g. bleed-through). What do you recommend?
If you have these types of concerns, please contact us to discuss them. The use of multiple fluorochromes in a given sample, and the choice of appropriate optics, can be tricky. Two fluorescent molecules that exhibit absorption and emission peaks that are different may still overlap to a significant degree.
Do you sell fluorochrome X, Y or Z?
No, we do not.
What is the function of the plastic slides I got when I visited your booth at last year’s meeting?
These are intended to determine the consistency and evenness of illumination of excitation light on a fluorescent sample. However, we cannot make any guarantees about the substances these slides are manufactured with, nor what their emission properties are.
I would like to use 14 Qdots simultaneously in my samples? What do you recommend?
I’d like to separate CFP from GFP in one sample and GFP from YFP in another using a wide-field scope? What solution can you recommend?
I’d like to begin experimenting using underwater 3p-TIRF-FRET-CARS microscopy in my bathyscaphe located in the tropics. What can you recommend?
We recommend that you contact us.
How can I contact you?
There are many way we can be contacted, including:
- Call us at 802-428-2500 (or 800-824-7662 within the US), and follow the prompts to Technical Support.
- Send your request via email to tech [at] chroma [dot] com
- Fax in an information request to 802-428-2525.