1. Fluorescence

2. Fluorescence Reading Light Source Excitation Filter Sample
Emission Filter
PMT

3. Fluorescence Reading

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

5. 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.

6. 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

7. 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

8. 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.

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

10. 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°

11. 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°

12. 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 × Tpk.

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

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

15. 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

16. FITC excitation spectra…

17. FITC emission spectra…

18. FITC excitation & emission

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

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

21. 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

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

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

24. Common Fluorophores

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

26. 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

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

28. 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

29. Common Fluorphores Coumarine-phalloidin 387 470 * Phycoerytrin-R(PE)
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

30. 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
650
Pyronine Y ds DNA bindig
67-84
Pyronine Y ds RNA bindig
70-90
Pyronine Y ss RNA bindig
497
563
low 42
DNA / RNA

31. 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
423 26
Reactive Dyes

32. 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 pH
BCECF (2',7'-bis-2-carboxyethyl)- 5-(and-6)carboxyfluorescein)
505
530
SNARF-1 (seminaphtorodafluor)
587

33. Common Fluorphores
Fluorophores CD

34. 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

35. 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 395 nm, 475 nm !!!
Low polarity environment - excitation shifts and changes in extinction coefficient

36. GFP limitations 2 Autofluorescent compounds in the cell
Flavins – – 560
NADH – – 470
Lipofuscins 360 – – 670
Glycation prod – – 450
Elastin & Collagen 440 – – 520
Lignin
Chlorophyll (740)
Publication of Billington & Knight, 2001

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

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