Detailed Explanation of Fluorescent Filter Examples for Quantitative PCR Instruments

Fluorescence filter is a key optical component used in life science instruments and biomedical applications. Its main function is to select and separate the excitation light and emission fluorescence characteristic spectral bands of substances in fluorescence inspection and analysis systems such as biology and medicine, and then observe them through a fluorescence microscope. Fluorescence filter generally consists of three combinations, namely excitation filter, dichroic mirror, and emission filter.

Fluorescence excitation filter, also known as Exciting Filter, Exciter Filter, Excitation Filter in English, refers to a filter that allows only the wavelength of excitation fluorescence to pass through in fluorescence microscopy or fluorescence imaging systems. Of course, there are also filters that directly use laser as excitation light.

Dichroic Beamslit, also known as Dichroic Beamslit, Dichroic Mirror, Dichrotic Beamslit, and Chinese name Dichroic Filter, Dichroic Mirror, Dichroic Mirror, etc. Usually, the optical path of a dichroic mirror and a microscope is at a 45 degree angle; Angle placement, the function of this filter is to reflect one wavelength band of light (i.e. excitation light) and transmit another wavelength band of light (i.e. emission light). Of course, our company also has customers whose structural components use the opposite direction, i.e. transmission of excitation light and reflection of emission light. The principle is the same. The dichroic mirror used here requires at least a reflectivity of the excitation light to be greater than 90%, a transmittance of the emitted light to be greater than 90%, and the parts of the spectrum that cannot be transmitted to be reflected instead of absorbed. The colors of the filter under transmitted and reflected light complement each other, so it is called a dichroic mirror.

Emission filter, also known as Barrier Filter or Emission Filter, refers to a filter used to select and transmit the fluorescence emitted by the detected substance. Except for the spontaneous fluorescence of the detected substance, the light in other ranges is cut off, and the wavelength of the emitted light is usually longer than that of the excitation light. When developing a system, narrowband filter, band-pass filter, or long wavelength filter can be selected as the emission filter.

The quantitative PCR instrument produced by Ruicheng Optoelectronics commonly uses 4-channel and sometimes 2-channel detection filters. Let's take a look at the parameters of each channel:

excitation wavelength
First channel: 450nm-490nm
Second channel: 500nm-535nm
Third channel: 555nm-585nm
Fourth channel: 620nm-650nm

emission wavelength
First channel: 515nm-530nm
Second channel: 560nm-580nm
Third channel: 610nm-650nm
Fourth channel: 675nm-730nm

This four channel instrument can detect the following fluorescent dyes and fluorescein:
First channel: FAM, SYBR Green I
Second channel: VIC, HEX, JOE, TAMRA, TET, Cy3
Third channel: TEXAS Red, ROX
Channel 4: Cy5

Correspondingly, our company has chosen the following solutions:
FAM channel: corresponding to excitation filter BP470-30k, emission filter BP525-20k, and matching dichroic mirror
HEX channel: corresponding to excitation filter BP523-20k, emission filter BP564-20K, and matching dichroic mirror
ROX channel: corresponding to excitation filter BP571-20k, emission filter BP612-20k, and matching dichroic mirror
CY5 channel: corresponding to excitation filter BP635-30k, emission filter BP683-15k, and matching dichroic mirror

In addition, some customers choose the fifth channel to detect the corresponding excitation filter BP678-12k and emission filter BP718-25k of cy5.5 dye.

Please find attached the fluorescent filter combinations corresponding to commonly used fluorescent dyes. If needed, please consult our business personnel. The attachments are as follows:

Fluorescent dye or fluorescein	excitation wavelength peak	emission wavelength peak	Excitation filter		Emission filter		Dichroic mirror
			wavelength	bandwidth	wavelength	bandwidth	Reflection range	Through scope
Alexa Fluor® 350	342	442	350	50	460	50	330-380	430-700
Alexa Fluor® 488	499	520	480	40	535	50	400-500	520-700
Alexa Fluor® 514	518	543	500	40	560	50	450-510	530-650
Alexa Fluor® 532	531	554	530	30	575	40	450-540	560-650
Alexa Fluor® 546	556	572	545	25	590	30	450-540	570-700
Alexa Fluor® 555	553	565	545	25	590	30	450-540	570-700
Alexa Fluor® 568	579	603	570	25	615	40	500-580	600-700
Alexa Fluor® 594	590	618	580	25	625	30	500-590	610-700
Alexa Fluor® 633	631	647	605	50	670	50	550-620	645-750
Alexa Fluor® 647	653	668	640	30	690	50	600-650	665-730
Alexa Fluor® 680	679	702	650	45	720	60	600-660	690-800
Alexa Fluor® 700	696	719	650	45	720	60	600-660	690-800
Alexa Fluor® 750	751	776	710	75	810	90	600-730	770-900
AmCyan	458	489	435	20	480	40	425-445	460-600
APC (Allophycocyanin)	650	660	620	60	700	75	600-650	665-730
AsRed 2	576	592	545	30	620	60	530-560	580-700
ATTO 390	392	477	395	25	460	50	350-410	430-550
ATTO 425	436	484	445	30	500	40	400-460	480-600
ATTO 465	453	507	470	40	525	50	400-480	500-600
ATTO 488	500	525	480	40	535	50	400-500	520-600
ATTO 550	553	576	545	20	590	30	450-550	570-700
ATTO 633	629	658	605	50	670	50	550-620	640-750
ATTO 647N	645	668	620	60	700	75	590-650	665-735
ATTO 680	680	700	650	45	720	60	600-670	690-800
BCECF (pH 5.2)	482	520	480	40	540	40	350-500	520-650
BCECF (pH 9.0)	502	528	480	40	540	40	350-500	520-650
Biosearch Blue	352	447	350	50	460	50	330-390	430-550
BODIPY FL/pH7.2	505	512	480	40	535	50	400-500	520-600
Brilliant Violet™ 421	408	422	395	25	430	30	350-405	415-500
Brilliant Violet™ 480	437	478	435	20	480	30	425-445	460-600
CAL Fluor® Gold 540	522	544	530	30	575	40	450-540	555-650
CAL Fluor® Orange 560	538	559	530	30	575	40	450-540	555-650
CAL Fluor® Red 590	569	591	570	25	615	40	500-580	595-700
CAL Fluor® Red 610	590	610	580	25	625	30	500-590	605-700
CAL Fluor® Red 635	618	637	605	50	670	50	550-620	645-750
Calcein	494	517	480	40	535	50	400-500	515-600
Calcium Green™-1	507	529	480	40	535	50	400-500	515-600
Cerulean	434	473	435	20	480	40	425-445	460-600
CFP	433	475	435	20	480	40	425-445	460-600
Citrine	514	529	500	20	535	30	480-505	520-650
Coumarin	360	448	350	50	460	50	330-390	430-550
Cy2™	492	507	480	40	535	50	400-500	515-600
Cy3.5™	578	591	570	25	615	40	500-580	595-700
Cy3™	554	566	545	25	605	70	450-550	570-700
Cy5.5™	675	695	650	45	720	60	600-660	690-800
Cy5™	649	666	620	60	700	75	590-650	665-735
Cy7™	757	781	710	75	810	90	600-730	770-900
DAPI	359	461	350	50	460	50	330-390	430-550
Di-8-ANEPPS non-ratiometric	466	630	480	30	620	60	400-495	515-700
DiA	457	586	480	30	620	60	400-495	515-700
DiD	648	670	620	60	700	75	590-650	665-735
DiI	551	569	545	25	605	70	450-550	570-700
DiO	490	506	470	40	525	50	400-480	500-600
DiR	750	782	710	75	810	90	600-730	770-900
Draq5	647	683	620	60	700	75	590-650	665-735
DsRed	559	583	545	25	605	70	450-550	570-700
DyLight 350	351	434	350	50	460	50	330-390	430-550
DyLight 405	398	420	405	20	460	50	350-410	430-550
DyLight 549	553	569	545	25	605	70	450-550	570-700
DyLight 594	593	618	580	25	625	30	500-590	605-700
DyLight 633	620	649	605	50	670	50	550-620	645-750
DyLight 649	651	673	620	60	700	75	590-650	665-735
DyLight 680	676	705	650	45	720	60	600-660	690-800
DyLight 750	751	778	710	75	810	90	600-730	770-900
DyLight 800	770	795	740	40	800	40	650-750	780-900
EBFP2	385	448	395	25	460	50	350-410	430-550
ECFP	434	477	435	20	480	40	425-445	460-600
EGFP	488	507	470	40	525	50	400-480	500-600
Emerald GFP	489	510	470	40	525	50	400-480	500-600
Eosin	525	546	500	40	560	50	450-510	530-650
Ethidium bromide	518	603	545	30	620	60	530-560	580-700
Ethidium homidimer-1/DNA	527	617	545	30	620	60	530-560	580-700
EYFP/pH 7	514	527	500	20	535	30	480-505	520-650
FAM	492	518	480	40	535	50	400-500	515-600
FITC	495	519	480	40	535	50	400-500	515-600
FlAsH-CCPFCC	511	530	500	20	535	30	480-505	520-650
Fluo-3	506	527	500	20	535	30	480-505	520-650
Fluo-4	494	516	480	40	535	50	400-500	515-600
FM™ 1-43	473	579	470	30	580	60	400-495	515-700
FM™ 4-64	508	730	510	40	730	100	450-590	605-800
Fura-2/Ca2+ - free	366	515	365	50	510	80	330-390	430-700
Fura-2/Ca2+ - saturated	336	505	335	30	510	80	320-390	430-700
FusionRed	577	604	560	40	630	75	500-570	595-750
GFP	489	511	470	40	525	50	400-480	500-600
HEX,SE	533	559	530	30	575	40	450-540	555-700
Hoechst 33258	352	455	350	50	460	50	330-390	430-700
Indo-1/Ca2+ -free	346	480	350	50	460	50	330-390	430-700
Indo-1/Ca2+ -saturated	330	400	350	40	420	40	310-380	395-550
JOE	520	548	500	40	560	50	450-510	530-650
Killer Red	585	611	560	40	630	75	500-570	595-750
Li-Cor IRDye® 680LT	674	693	650	45	720	60	600-660	690-800
Li-Cor IRDye® 800CW	778	794	740	40	800	40	650-750	780-900
Lucifer yellow	428	544	420	40	535	40	390-445	460-600
LysoTracker Blue	373	422	395	25	460	50	350-410	430-550
LysoTracker Green	501	509	470	40	525	50	400-480	500-600
LysoTracker Red	573	592	560	40	630	75	500-570	595-750
LysoTracker Yellow HCK-123	488	565	500	40	560	50	450-510	530-700
mCherry	587	610	560	40	630	75	500-570	595-750
mCitrine	514	529	500	20	535	30	480-505	520-650
MitoTracker™ Deep Red	641	662	620	60	700	75	590-650	665-735
MitoTracker™ Green	490	512	470	40	525	50	400-480	500-600
MitoTracker™ Orange	551	575	550	20	590	30	450-550	570-700
MitoTracker™ Red	578	598	560	40	630	75	500-570	595-750
mKate2	588	633	560	40	630	75	500-570	595-750
mKO	549	559	530	30	575	40	450-540	555-700
mOrange2	551	565	530	30	575	40	450-540	555-700
mPlum	589	649	560	40	650	40	450-590	605-800
mRFP1	590	612	560	40	630	75	500-570	595-750
mTFP1	462	492	445	30	500	40	400-460	480-600
mWasabi	493	509	470	40	525	50	400-480	500-600
NBD-X (MeOH)	467	538	480	40	535	50	400-500	515-700
Nile blue	631	660	620	60	700	75	590-650	665-735
NirFP	604	669	600	40	670	40	580-620	650-800
Oregon Green™ 488	498	526	480	40	535	50	400-500	515-700
Oregon Green™ 514	513	532	500	20	535	30	480-505	520-650
Pacific Blue™	404	455	405	20	460	50	350-410	430-550
Propidium Iodide (PI)	305	617	545	30	620	60	530-560	580-700
Pulsar™ 650	460	650	440	40	630	75	400-480	500-600
Qdot® 525 Nanocrystals	300	525	425	50	525	30	370-470	490-700
Qdot® 545 Nanocrystals	300	543	425	50	545	30	370-470	490-700
Qdot® 565 Nanocrystals	300	564	425	50	565	30	370-470	490-700
Qdot® 585 Nanocrystals	300	588	425	50	585	30	370-470	490-700
Qdot® 605 Nanocrystals	300	602	425	50	605	30	370-470	490-700
Qdot® 625 Nanocrystals	300	621	425	50	625	30	370-470	490-700
Qdot® 655 Nanocrystals	300	654	425	50	655	30	370-470	490-700
Qdot® 705 Nanocrystals	300	702	425	50	705	30	370-470	490-700
Qdot® 800 Nanocrystals	300	792	425	50	800	40	370-470	490-700
Quasar® 570	548	566	530	30	575	40	450-540	555-700
Quasar® 670	647	670	620	60	700	75	590-650	665-735
Quasar® 705	690	750	650	45	720	60	600-660	690-800
ReAsH-CCPGCC	592	606	560	40	630	75	500-570	595-750
Resorufin	571	584	545	20	590	30	450-550	570-700
Rhod-2	553	577	545	25	605	70	450-550	570-700
Rhodamine 123	507	529	500	20	535	30	480-505	520-650
Rhodamine 6G	525	547	500	40	560	50	450-510	530-700
Rhodamine Red-X	573	591	560	40	630	75	500-570	595-750
ROX	578	604	560	40	630	75	500-570	595-750
R-phycoerythrin	565	578	545	10	585	40	500-550	570-800
SBFI/Na+ -free	340	560	350	40	560	100	330-390	430-700
SBFI/Na+ -saturated	337	527	350	40	560	100	330-390	430-700
Sulforhodamine 101-EtOH	578	593	560	40	630	75	500-570	595-750
SYBR® Green I	498	522	480	40	535	50	400-500	515-700
SYTO 60	650	681	620	60	700	75	590-650	665-735
SYTO 9	483	500	470	40	525	50	400-480	500-600
TagBFP	402	457	405	20	460	50	350-410	430-550
TagRFP	556	584	545	25	605	70	450-550	570-700
TAMRA	555	580	545	25	605	70	450-550	570-700
tdTomato	554	581	545	25	605	70	450-550	570-700
TET	521	542	500	40	560	50	450-510	530-700
Tetracycline	405	550	405	40	550	60	350-460	480-650
Tetramethylrhodamine dextran	554	582	545	25	605	70	450-550	570-700
Texas Red®	592	614	560	40	630	75	500-570	595-750
TO-PRO™-3	642	657	620	60	700	75	590-650	665-735
TRITC	552	578	545	25	605	70	450-550	570-700
TurboFP650	592	650	560	40	630	75	500-570	595-750
wtGFP	474	509	450	50	525	50	370-470	490-700
X-Rhod-1 Indicator	580	601	560	40	630	75	500-570	595-750
ZsGreen1	493	505	470	40	525	50	400-480	500-600
ZsYellow1	529	539	500	40	560	50	450-510	530-700