Figure 1.2  Dichroic filters have (a) an engraving on or (b) a carat indicating 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 Aluminium Housings
• Dichroic Filters are Unmounted
• Compatible with Filter Cubes Designed for 12.5 mm, 25 mm, or 32 mm Optics
• 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 or with our cage cubes. 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 Ø12.5 mm, Ø25 mm, or Ø32 mm black anodized aluminum ring which makes handling easier and enhances the blocking OD by limiting scattering.

Click to Enlarge
Figure 1.3  The 32 mm MDF32-TOM filter set being installed in a DFM32T1 cage cube insert.

Compatibility
Thorlabs offers mounting solutions for the products sold here. The Ø12.5 mm filter sets are compatible with our 16 mm Cage Cubes for 12.5 mm filters while the Ø25 mm filters and filter sets are compatible with our 30 mm Cage Cubes for 25 mm filters. For customers who wish to use the Ø25 mm filters in Thorlabs, Olympus, or Nikon fluorescence microscopes, Thorlabs manufactures a family of Drop-In Microscope Filter Cubes. Finally, the Ø32 mm filter sets are compatible with our 30 mm Cage Cubes for 32 mm filters.

The excitation and emission filters can be mounted in our extensive line of filter mounts and wheels. As their aluminum rings are not threaded, compatible retaining rings will be required to mount the filters in one of our internally-threaded lens tubes. Additionally, the unmounted dichroic filters can be mounted in a compatible rectangular kinematic mount, which are 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 

Fluorophore	Filter Size	Set Item #	Filter Item #	Filter Type	Center Wavelength	FWHM	Reflection Band	Transmission Band	Transmission Dataa
BFP	12.5 mm	MDF05-BFP	-	Excitation	395 nm	25 nm	-	-
			-	Emission	460 nm	49 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 350 - 418 nm	Tavg ≥ 95% 433 - 750 nm
	25 mm	MDF-BFP	MF390-18	Excitation	390 nm	18 nm	-	-
			MF460-60	Emission	460 nm	60 nm	-	-
			MD416b	Dichroic	-	-	Ravg > 90% 360 - 407 nm	Tabs > 90% 425 - 575 nm
	32 mm	MDF32-BFP	-	Excitation	395 nm	25 nm	-	-
			-	Emission	460 nm	49 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 350 - 418 nm	Tavg ≥ 95% 433 - 750 nm
CFP	25 mm	MDF-CFP	MF434-17	Excitation	434 nm	17 nm	-	-
			MF479-40	Emission	479 nm	40 nm	-	-
			MD453b	Dichroic	-	-	Ravg > 90% 423 - 445 nm	Tabs > 90% 460 - 610 nm
WGFP	25 mm	MDF-WGFP	MF445-45	Excitation	445 nm	45 nm	-	-
			MF510-42	Emission	510 nm	42 nm	-	-
			MD480b	Dichroic	-	-	Ravg > 90% 415 - 470 nm	Tabs > 90% 490 - 720 nm
GFP	12.5 mm	MDF05-GFP	-	Excitation	469.5 nm	40 nm	-	-
			-	Emission	524.5 nm	48 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 442 - 488 nm	Tavg ≥ 95% 502 - 552 nm
	25 mm	MDF-GFP	MF469-35	Excitation	469 nm	35 nm	-	-
			MF525-39	Emission	525 nm	39 nm	-	-
			MD498b	Dichroic	-	-	Ravg ≥ 90% 452 - 490 nm	Tabs ≥ 90% 505 - 800 nm
	32 mm	MDF32-GFP	-	Excitation	469.5 nm	40 nm	-	-
			-	Emission	524.5 nm	48 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 442 - 488 nm	Tavg ≥ 95% 502 - 552 nm
FITC	12.5 mm	MDF05-FITC	-	Excitation	479 nm	30 nm	-	-
			-	Emission	537 nm	40 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 94% 380 - 497 nm	Tavg ≥ 92% 514.5 - 900 nm
	25 mm	MDF-FITC	MF475-35	Excitation	475 nm	35 nm	-	-
			MF530-43	Emission	530 nm	43 nm	-	-
			MD499b	Dichroic	-	-	Ravg > 90% 470 - 490 nm	Tabs > 90% 508 - 675 nm
	32 mm	MDF32-FITC	-	Excitation	479 nm	30 nm	-	-
			-	Emission	537 nm	40 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 94% 380 - 497 nm	Tavg ≥ 92% 514.5 - 900 nm
GFP Alexa Fluor® 488	25 mm	MDF-GFP2	-	Excitation	482 nm	18 nm	-	-
			-	Emission	520 nm	28 nm	-	-
			-	Dichroic	-	-	Ravg > 93% 350 - 488 nm	Tavg > 93% 502 - 950 nm
YFP	25 mm	MDF-YFP	MF497-16	Excitation	497 nm	16 nm	-	-
			MF535-22	Emission	535 nm	22 nm	-	-
			MD515	Dichroic	-	-	Ravg > 90% 490 - 510 nm	Tabs > 90% 525 - 700 nm
tdTomato	12.5 mm	MDF05-TOM	-	Excitation	540 nm	25 nm	-	-
			-	Emission	605 nm	55 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 94% 450 - 556.5 nm	Tavg ≥ 92% 575 - 900 nm
	25 mm	MDF-TOM	-	Excitation	531 nm	40 nm	-	-
			-	Emission	593 nm	40 nm	-	-
			-	Dichroic	-	-	Ravg > 98% 350 - 555 nm	Tavg > 93% 569 - 950 nm
	32 mm	MDF32-TOM	-	Excitation	540 nm	25 nm	-	-
			-	Emission	605 nm	55 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 94% 450 - 556.5 nm	Tavg ≥ 92% 575 - 900 nm
TRITC	25 mm	MDF-TRITC	MF542-20	Excitation	542 nm	20 nm	-	-
			MF620-52c	Emission	620 nm	52 nm	-	-
			MD568b	Dichroic	-	-	Ravg > 90% 525 - 556 nm	Tabs > 90% 580 - 650 nm
Texas Red	12.5 mm	MDF05-TXRED	-	Excitation	562 nm	42 nm	-	-
			-	Emission	635.5 nm	58 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 94% 460 - 587 nm	Tavg ≥ 92% 608 - 900 nm
	25 mm	MDF-TXRED	MF559-34	Excitation	559 nm	34 nm	-	-
			MF630-69	Emission	630 nm	69 nm	-	-
			MD588b,d	Dichroic	-	-	Ravg > 90% 533 - 580 nm	Tabs > 90% 595 - 800 nm
	32 mm	MDF32-TXRED	-	Excitation	562 nm	42 nm	-	-
			-	Emission	635.5 nm	58 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 94% 460 - 587 nm	Tavg ≥ 92% 608 - 900 nm
mCherry	12.5 mm	MDF05-MCHC	-	Excitation	560 nm	40 nm	-	-
			-	Emission	629 nm	71 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 450 - 578 nm	Tavg ≥ 95% 593 - 700 nm
	25 mm	MDF-MCHC	-	Excitation	562 nm	40 nm	-	-
			-	Emission	641 nm	75 nm	-	-
			-	Dichroic	-	-	Ravg > 98% 350 - 585 nm	Tavg > 93% 601 - 950 nm
	32 mm	MDF32-MCHC	-	Excitation	560 nm	40 nm	-	-
			-	Emission	629 nm	71 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 450 - 578 nm	Tavg ≥ 95% 593 - 700 nm
Cy3.5	25 mm	MDF-CY3.5	MF565-24	Excitation	565 nm	24 nm	-	-
			MF620-52c	Emission	620 nm	52 nm	-	-
			MD588b,d	Dichroic	-	-	Ravg > 90% 533 - 580 nm	Tabs > 90% 595 - 800 nm
mCherry (578 nm Excitation)	25 mm	MDF-MCHA	-	Excitation	578 nm	21 nm	-	-
			-	Emission	641 nm	75 nm	-	-
			-	Dichroic	-	-	Ravg > 98% 350 - 588 nm	Tavg > 93% 603 - 950 nm
Cy5	12.5 mm	MDF05-CY5	-	Excitation	619 nm	60 nm	-	-
			-	Emission	700 nm	71 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 500 - 648 nm	Tavg ≥ 95% 666 - 890 nm
	25 mm	MDF-CY5	-	Excitation	619 nm	60 nm	-	-
			-	Emission	700 nm	71 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 500 - 648 nm	Tavg ≥ 95% 666 - 890 nm
	32 mm	MDF32-CY5	-	Excitation	619 nm	60 nm	-	-
			-	Emission	700 nm	71 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 500 - 648 nm	Tavg ≥ 95% 666 - 890 nm
Cy5.5	12.5 mm	MDF05-CY55	-	Excitation	650 nm	44 nm	-	-
			-	Emission	720 nm	59.5 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 520 - 674 nm	Tavg ≥ 95% 692 - 1000 nm
	25 mm	MDF-CY55	-	Excitation	650 nm	44 nm	-	-
			-	Emission	720 nm	59.5 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 520 - 674 nm	Tavg ≥ 95% 692 - 1000 nm
	32 mm	MDF32-CY55	-	Excitation	650 nm	44 nm	-	-
			-	Emission	720 nm	59.5 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 520 - 674 nm	Tavg ≥ 95% 692 - 1000 nm
Cy7	12.5 mm	MDF05-CY7	-	Excitation	709 nm	75.5 nm	-	-
			-	Emission	813 nm	96 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 570 - 746 nm	Tavg ≥ 95% 766 - 1100 nm
	25 mm	MDF-CY7	-	Excitation	709 nm	75.5 nm	-	-
			-	Emission	813 nm	96 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 570 - 746 nm	Tavg ≥ 95% 766 - 1100 nm
	32 mm	MDF32-CY7	-	Excitation	709 nm	75.5 nm	-	-
			-	Emission	813 nm	96 nm	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 570 - 746 nm	Tavg ≥ 95% 766 - 1100 nm
IRDye® 800CW	12.5 mm	MDF05-NIR	-	Excitation	741 nm	41 nm	-	-
			-	Emission	N/Ae	-	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 580 - 760 nm	Tavg ≥ 95% 780 - 1100 nm
	25 mm	MDF-NIR	-	Excitation	741 nm	41 nm	-	-
			-	Emission	N/Ae	-	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 580 - 760 nm	Tavg ≥ 95% 780 - 1100 nm
	32 mm	MDF32-NIR	-	Excitation	741 nm	41 nm	-	-
			-	Emission	N/Ae	-	-	-
			-	Dichroic	-	-	Ravg ≥ 98% 580 - 760 nm	Tavg ≥ 95% 780 - 1100 nm

• Click on for a plot and downloadable data.
• These dichroics have an AR coating designed for 45° AOI on the back surface with R_abs < 2% from 400 to 800 nm. 
• 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.