Fluorescence

Fluorescence Reading Light Source Excitation Filter Sample Emission Filter PMT

Fluorescence Reading

The Lamp Spectral range 340 – 700 nm Light output less in the lower range Use appropriate filters with higher transmission or larger bandwidth

What is an optical filter? An optical filter is a passive component which basic function is to improve the performance of an optical system. With filters you select the (optimal) excitation and emission wavelengths to be used in measurements of a certain label.

Common types of filters Bandpass filters (e.g. interference filters) coated filters (alternately materials with high refractive index and materials with low) their operating principle is based on interference in and between the different coated layers very steep passband edges sometimes combination between coloured glass and coatings

Common types of filters Edge filters (e.g. long or shortpass filters) can be coated filters or color glass filters color glass filters are absorptive filters their operating principle is based on molecular absorption of certain wavelengths

Filters - Terminology Blocking level: the transmission level outside of the passband. Usually, the blocking is better than 10-5, i.e. only 1/1000 of a percent passes through the filter.

Filters - Terminology Center wavelength (0): the center wavelength is the middle wavelength for a passband. Mathematically it is defined as:

Filters - Terminology Cut-off (c): the wavelength specifying the location of the transition from a region of high transmission to a region of low transmission. Usually refers to the 5 % absolute transmission point. Rays must enter the filter surface at an angle close to 90°

Filters - Terminology Cut-on (c): the wavelength specifying the location of the transition from a region of low transmission to a region of high transmission. Usually refers to the 5 % absolute transmission point. Rays must enter the filter surface at an angle close to 90°

Filters - Terminology Full width at half maximum (FWHM): also called bandwidth (BW). The width of a bandbass filter between specific absolute transmission points, i.e. 0.5 × Tpk.

Filters - Terminology Passband (or bandpass): the range of wavelengths that will pass through a filter.

Filters - Terminology Peak transmission (Tpk): the maximum transmission of the filter expressed in percent. Not necessarily at 0.

Selecting filters Does the filters’ transmission spectra fit with the label/fluorophore in question? Should the filter be broad or narrow? as a general rule, narrow filters should be used in photometric applications (absorbance measurements) otherwise, broad filters can/should be used with broad filters the background level can increase and destroy the results ! Is the filters’ blocking level good enough? the blocking level should be at least 10-4

FITC excitation spectra…

FITC emission spectra…

FITC excitation & emission

Selecting filters Excitation and emission filter pairs are not to overlap with their transmission spectra!

Selecting Filters Rule of thumb: CWL >= 0.85 * (FWHMex + FWHMem)

Selecting filters Twinkle eliminates scattered light by second lens and aperture Angular light can pass interference filters -> cut-off shifted to lower wavelengths ! Select emission filter in Mithras with a CWL at even higher wavelenghts Missing in Mithras

Fluorescence Principle Jablonski - diagram: Excitation 1 Excited State 2 Emission 3

Excitation / Emission Difference in ex and em: Stoke`s Shift The excitation of a fluorophore at different s influences emission intensity, not emission wavelength

Common Fluorophores

Common Fluorphores Wavelength (nm) 300 400 500 600 700 BODIPY FL BFP YFP Cy5 Cy3 EGFP Cy2

Fluoreszierende Proteine Common Fluorphores Fluoreszierende Proteine Y66F 360 508   Y66H 442 EBFP 380 440  (Clontech) Quantum yield 0.18 Wild Type 396,475 508,503 GFPuv 385  (Clontech) ECFP 434 477  (Clontech) Quantum yield 0.40 Y66W 436 485 S65A 471 504 S65C 479 507 S65L 484 510 S65T 488 511 EGFP 489  (Clontech) Quantum yield 0.60 EYFP 514 527  (Clontech) Quantum yield 0.61 DsRed 558 583  (Clontech) Quantum yield 0.29

Common Fluorphores Calcium Fluo-3 506 526 * Fura 2 335 512-518 0.23 33 360 390-310 0.49 27 Indo 1 330 390-410 0.56 34 350 482-485 0.38

Tetramethylrhodamine ethylester Common Fluorphores Membrane potential Thiazole Orange 453 480 0.08 26 diO-Cn-(3) 485 505 0.05 149 diI-Cn-(3) 548 567 0.07 126 diI-Cn-(5) 646 668 0.4 200 diI-Cn-(7) 740 770 0.28 240 diBA-Isopr-(3) 493 517 0.03 130 diBA-C4-(5) 590 620 * 176 Tetramethylrhodamine ethylester 549 Rhodamine 123 511 534 0.9 85

Common Fluorphores Coumarine-phalloidin 387 470 * Phycoerytrin-R(PE) 480-565 578 0.68 1960 APC Allophycocyanine 650 660 700 Hoechst33258 346 460 0.83 47.0 Hoechst33342 120 Dapi (4',6diamidino-2-Phenylindolo) 350 450 0.83/td> Ethidium Bromide 526 604 3.2 Propidium Iodide 536 620 6.4 YOYO-1 491 509 DNA / RNA

Common Fluorphores TOTO-1, TO-PRO-1 509 533 * TOTO-3, TO-PRO-3 642 661 Acridine Orange DNA binding 480 520 Acridine Orange RNA binding 440-470 650 Pyronine Y ds DNA bindig 549-561 567-574 0.04-0.26 67-84 Pyronine Y ds RNA bindig 560-562 565-574 0.05-0.21 70-90 Pyronine Y ss RNA bindig 497 563 low 42 DNA / RNA

Common Fluorphores FITC (Fluorescein Isothiocyanate) 490 520 0.71 67 TRIC-amines 554 573 0.28 85 Texas Red 596 620 0.51 Lissamine Rhodamine Sulfonamide 570 590 * CY5.8-ITC-amine 664 663 0.09 129 CY7.8-amine 550 777 100 CY5.4IA-actin 652 672 0.15 140 CY3.12-OSu-amine 556 574 0.05 130 CY5.12-OSu-amine 650 674 0.13 200 CY3.18-OSu-Ab 568 0.14 CY5.18-OSu-Ab 552 CY7.18-OSu-Ab 755 778 0.02 Cascade Blue 378-399 423 26 Reactive Dyes

Common Fluorphores Enzyme substrates Product of rxn. (Rodamine) 495 523 0.91 67 Coumarin-glucoside 316 395 * 13 Product of rxn. (hydroxycoumarin) 370 450 17 Monochlorobimane (for GST measurements) 350 440-520 pH BCECF (2',7'-bis-2-carboxyethyl)- 5-(and-6)carboxyfluorescein) 505 530 SNARF-1 (seminaphtorodafluor) 518-548 587

Common Fluorphores Fluorophores CD

GFP & co. Use Luciferase Use black plates Lyse cells before reading Check homogeneity of growth in the well Use scanning mode in case of inhomogeneous growth Use high lamp energies (>25,000) Select appropriate filters: you may need to try a few with every new customer first

GFP limitations Low extinction coefficient Low solubility at 37 °C Poor expression at 37 °C pH <7 - excitation shift and drop in quantum yield Elevated temperature - loss of fluorescence Locally high GFP concentration - excitation shifts and changes in extinction coefficient increase @ 395 nm, decrease @ 475 nm !!! Low polarity environment - excitation shifts and changes in extinction coefficient

GFP limitations 2 Autofluorescent compounds in the cell Flavins 380 – 490 520 – 560 NADH 360 – 390 440 – 470 Lipofuscins 360 – 490 430 – 670 Glycation prod. 320 – 370 385 – 450 Elastin & Collagen 440 – 480 470 – 520 Lignin 488 530 Chlorophyll 488 685 (740) Publication of Billington & Knight, 2001

GFP limitations 3 Maturation & turnover times: 1 – 24 hours 1 – 24 hours

detection limits Microscope 103 – 104 per cell Flow cytometer 103 per cell Microplate reader 108 per ml