Fluorescence Imaging Filters

Figure 1.2  Dichroic filters have (a) an engraving on or (b) a carat pointing to the primary reflective surface. Light should be incident on this side for best performance.

Figure 1.1  
Excitation and emission filters are marked with an arrow that shows the recommended direction of light propagation.

Features

• Excitation, Emission, and Dichroic Filters for Fluorescence Imaging
• >90% Transmission over Desired Wavelength Band
• Sharp Cutoff (Up to T < 0.001%) Outside the Transmission Band
• Matched Filter Sets Designed to Target Many Common Fluorophores
  

  - BFP - CFP - WGFP - GFP - FITC -Alexa Fluor®∗ 488

  - YFP - tdTomato - TRITC - Texas Red®* - mCherry - Cy®†3.5

  - Cy5 - Cy5.5 - Cy7 - LI-COR IRDye®‡ 800CW

• Excitation and Emission Filters are Mounted in Black-Anodized Housings
• Dichroic Filters are Unmounted
• Filter Sets are Available at a Savings Over Individual Filter Prices

These excitation, emission, and dichroic filters are designed specifically for use in fluorescence imaging applications. They are fabricated at industry-standard dimensions that make them compatible with filter cubes from all major manufacturers. We offer filters designed to target the following common fluorophores: BFP, CFP, WGFP, GFP, FITC, Alexa Fluor 488, YFP, tdTomato, TRITC, Texas Red, mCherry, Cy3.5, Cy5, Cy5.5, Cy7, and LI-COR IRDye 800CW. While many of the filters are offered individually, some are only offered in a three-piece set containing the excitation, emission, and dichroic filters; see the Specs tab for details. A selection of these filters are also available pre-installed into our microscope filter cubes.

Filter Design
Our filters are manufactured to high-performance optical specifications and designed for durability. They are produced via multiple dielectric layers deposited on a high-precision, fused silica substrate. The substrate is ground and polished to ensure that the highest possible image quality is maintained. The resulting hard-coated optics consist of filter layers that are denser than those obtained from electron beam deposition techniques, and which reduce water absorption while greatly enhancing durability, stability, and performance of the filter. Each filter layer is monitored during growth to ensure minimal deviation from design specification thickness, ensuring overall high-quality filter performance.

Each excitation or emission filter is housed in a Ø25 mm black anodized aluminum ring which makes handling easier and enhances the blocking OD by limiting scattering. These filters can be mounted in our extensive line of filter mounts and wheels. As the aluminum rings are not threaded, Ø1" retaining rings will be required to mount the Ø25 mm filters in one of our internally-threaded SM1 lens tubes. For customers who wish to use these filters in Thorlabs, Olympus, or Nikon fluorescence microscopes, Thorlabs manufactures a family of Drop-In Microscope Filter Cubes. Thorlabs also manufactures a family of 30 mm Cage Cubes to integrate these filters into a 30 mm cage system. Additionally, the unmounted dichroic filters can be mounted in the KM2536 kinematic mount, which is designed to secure a 1 mm thick rectangular optic with minimal stress.

^∗Alexa Fluor and Texas Red are registered trademarks of Molecular Probes, Inc.
^†Cy is a registered trademark of Global Life Sciences Solutions Germany GmbH
^‡IRDye is a registered trademark of LI-COR Biotech, LLC 

Item #	Kit Item #	Fluorophore	Filter Type	Center Wavelength	FWHM	Reflection Band	Transmission Band	AR Coatinga	Transmission Datab
MF390-18	MDF-BFP	BFP	Excitation	390 nm	18 nm	-	-	-
MF460-60			Emission	460 nm	60 nm	-	-	-
MD416			Dichroic	-	-	Ravg > 90% 360 - 407 nm	Tabs > 90% 425 - 575 nm	Rabs < 2% from 400 to 800 nm
MF434-17	MDF-CFP	CFP	Excitation	434 nm	17 nm	-	-	-
MF479-40			Emission	479 nm	40 nm	-	-	-
MD453			Dichroic	-	-	Ravg > 90% 423 - 445 nm	Tabs > 90% 460 - 610 nm	Rabs < 2% from 400 to 800 nm
MF445-45	MDF-WGFP	WGFP	Excitation	445 nm	45 nm	-	-	-
MF510-42			Emission	510 nm	42 nm	-	-	-
MD480			Dichroic	-	-	Ravg > 90% 415 - 470 nm	Tabs > 90% 490 - 720 nm	Rabs < 2% from 400 - 800 nm
MF469-35	MDF-GFP	GFP	Excitation	469 nm	35 nm	-	-	-
MF525-39			Emission	525 nm	39 nm	-	-	-
MD498			Dichroic	-	-	Ravg > 90% 452 - 490 nm	Tabs > 90% 505 - 800 nm	Rabs < 2% from 400 to 800 nm
MF475-35	MDF-FITC	FITC	Excitation	475 nm	35 nm	-	-	-
MF530-43			Emission	530 nm	43 nm	-	-	-
MD499			Dichroic	-	-	Ravg > 90% 470 - 490 nm	Tabs > 90% 508 - 675 nm	Rabs < 2% from 400 to 800 nm
-	MDF-GFP2	GFP Alexa Fluor® 488	Excitation	482 nm	18 nm	-	-	-
-			Emission	520 nm	28 nm	-	-	-
-			Dichroic	-	-	Ravg > 93% 350 - 488 nm	Tavg > 93% 502 - 950 nm	-
MF497-16	MDF-YFP	YFP	Excitation	497 nm	16 nm	-	-	-
MF535-22			Emission	535 nm	22 nm	-	-	-
MD515			Dichroic	-	-	Ravg > 90% 490 - 510 nm	Tabs > 90% 525 - 700 nm	-
-	MDF-TOM	tdTomato	Excitation	531 nm	40 nm	-	-	-
-			Emission	593 nm	40 nm	-	-	-
-			Dichroic	-	-	Ravg > 98% 350 - 555 nm	Tavg > 93% 569 - 950 nm	-
MF542-20	MDF-TRITC	TRITC	Excitation	542 nm	20 nm	-	-	-
MF620-52c			Emission	620 nm	52 nm	-	-	-
MD568			Dichroic	-	-	Ravg > 90% 525 - 556 nm	Tabs > 90% 580 - 650 nm	Rabs < 2% from 400 to 800 nm
MF559-34	MDF-TXRED	Texas Red®	Excitation	559 nm	34 nm	-	-	-
MF630-69			Emission	630 nm	69 nm	-	-	-
MD588d			Dichroic	-	-	Ravg > 90% 533 - 580 nm	Tabs > 90% 595 - 800 nm	Rabs < 2% from 400 to 800 nm
-	MDF-MCHC	mCherry	Excitation	562 nm	40 nm	-	-	-
-			Emission	641 nm	75 nm	-	-	-
-			Dichroic	-	-	Ravg > 98% 350 - 585 nm	Tavg > 93% 601 - 950 nm	-
MF565-24	MDF-CY3.5	Cy®3.5	Excitation	565 nm	24 nm	-	-	-
MF620-52c			Emission	620 nm	52 nm	-	-	-
MD588d			Dichroic	-	-	Ravg > 90% 533 - 580 nm	Tabs > 90% 595 - 800 nm	Rabs < 2% from 400 to 800 nm
-	MDF-MCHA	mCherry	Excitation	578 nm	21 nm	-	-	-
-			Emission	641 nm	75 nm	-	-	-
-			Dichroic	-	-	Ravg > 98% 350 - 588 nm	Tavg > 93% 603 - 950 nm	-
-	MDF-CY5	Cy5	Excitation	619 nm	60 nm	-	-	-
-			Emission	700 nm	71 nm	-	-	-
-			Dichroic	-	-	Ravg ≥ 98% 500 - 648 nm	Tavg ≥ 95% 666 - 890 nm	-
-	MDF-CY55	Cy5.5	Excitation	650 nm	44 nm	-	-	-
-			Emission	720 nm	59.5 nm	-	-	-
-			Dichroic	-	-	Ravg > 98% 520 - 674 nm	Tavg ≥ 95% 692 - 1000 nm	-
-	MDF-CY7	Cy7	Excitation	709 nm	75.5 nm	-	-	-
-			Emission	813 nm	96 nm	-	-	-
-			Dichroic	-	-	Ravg > 98% 570 - 746 nm	Tavg ≥ 95% 766 - 1100 nm	-
-	MDF-NIR	LI-COR IRDye® 800CW	Excitation	741 nm	41 nm	-	-	-
-			Emission	N/Ae	-	-	-	-
-			Dichroic	-	-	Ravg > 98% 580 - 760 nm	Tavg ≥ 95% 780 - 1100 nm	-

• Select dichroics have an AR coating designed for 45° AOI on the back surface. 
• Click on for a plot and downloadable data.
• Designed to work with both TRITC and CY3.5.
• Designed to work with both CY3.5 and TXRED.
• This emission filter has a cut on wavelength of 780 nm with average transmission ≥94% for 790 - 1000 nm

Click to Enlarge
Figure 3.1  Schematic of light passing through a fluorescence microscope.

Click to Enlarge
Figure 3.2  MDF-YFP filter set transmission graph. Note the dichroic mirror (green) reflects light in the excitation wavelength range (blue), and transmits light in the emission wavelength range (green).

Filters for Fluorescence Microscopy

Fluorophores
A fluorophore is a molecule or portion of a molecule that is capable of producing fluorescence. When light of the appropriate frequency necessary to excite a molecule from its ground state to an excited state is present, excitation will occur. However, once in an excited state, the molecule will be unstable. After some short period of time (typically 10^-15 to 10^-9 s), a photon will be released, thereby enabling the molecule to return to a lower energy state. The emitted radiation will be at a longer wavelength (lower energy) than the absorbed radiation due to the loss of energy through various mechanisms such as vibrations, sound, and thermal energy. 

A single fluorophore can be continually excited unless it is destroyed by photobleaching (i.e. the nonreversible destruction of a fluorophore due to photon-induced chemical damage or covalent modification). The average number of excitation and emission cycles that a particular fluorophore can undergo prior to photobleaching depends on its molecular structure and the local environment; some fluorophores bleach quickly after emitting only a few photons while others are far more robust and can undergo thousands or even millions of cycles before bleaching occurs.

Filters for Fluorescence Microscopy
The experimental setup in Figure 3.1 shows the typical filters used for epi-fluorescence microscopy, a form of microscopy in which both the excitation and emission light travel through the microscope objective. By carefully choosing the appropriate filters and mirrors for a given application, the signal-to-noise ratio can be maximized. As shown in Figure 3.1, three types of filters are used to maximize the fluorescence signal while minimizing the unwanted radiation. Each optical element is discussed below.

Excitation Filter
The excitation filter only allows a narrow band of wavelengths to pass through it, around the peak fluorophore excitation wavelength. For example, as shown in Figure 3.2, the bandpass region corresponding to greater than 90% transmission for the Yellow Fluorescent Protein (YFP) Excitation Filter (MF497-16) is 489 - 505 nm; incident radiation outside of this range is either partially (for regions near the transmission region) or totally (for regions further from the bandpass region) blocked by the filter.

Dichroic Mirror
Dichroic mirrors are designed to reflect light whose wavelength is below a specific value (i.e. the cutoff wavelength) while permitting all other wavelengths to pass through it unaltered. In a microscope, the dichroic mirror directs the proper wavelength range to the sample as well as to the image plane. The cutoff wavelength value associated with each mirror indicates the wavelength that corresponds to 50% transmission. For example, as shown in Figure 3.2, the cutoff wavelength for the Yellow Fluorescent Protein (YFP) Dichroic Mirror (MD515) is ~515 nm. The Specs tab provides information on the reflectance and transmission for each type of dichroic mirror.

By placing one of these mirrors into the experimental setup at 45° with respect to the incident radiation, the excitation radiation (shown in blue in Figure 3.1) is reflected off of the surface of the dichroic mirror and directed towards the sample and microscope objective, while the fluorescence emanating from the sample (shown in red in Figure 3.1) passes through the mirror to the detection system.

Although dichroic mirrors play a crucial role in fluorescence microscopy, they are not perfect when it comes to blocking unwanted light; typically, ~90% of the light at wavelengths below the cutoff wavelength value are reflected and ~90% of the light at wavelengths above this value are transmitted by the dichroic mirror. Hence, some of the excitation light can be transmitted through the dichroic mirror along with the longer wavelength fluorescence emitted by the sample. To prevent this unwanted light from reaching the detection system, an emission filter is used in addition to the dichroic mirror.

Emission Filter
An emission filter serves the purpose of allowing the desirable fluorescence from the sample to reach the detector while blocking unwanted traces of excitation light. Like the excitation filter, this filter only allows a narrow band of wavelengths to pass through it, around the peak fluorophore emission wavelength. For example, as shown in Figure 3.2, the bandpass region corresponding to greater than 90% transmission for the Yellow Fluorescent Protein (YFP) Emission Filter (MF535-22) is 524 - 546 nm; incident radiation outside of this range is either partially (for regions near the transmission region) or totally (for regions further from the bandpass region) blocked by the filter.