中国生态仪器网是泽泉科技主办的网上生理生态仪器与应用信息交流中心 , 在这里您可以交流各种生理生态方面的技术，产品与应用信息

1. 中国生态仪器网是泽泉科技主办的网上生理生态仪器与应用信息交流中心,在这里您可以交流各种生理生态方面的技术，产品与应用信息中国生态仪器网是泽泉科技主办的网上生理生态仪器与应用信息交流中心,在这里您可以交流各种生理生态方面的技术，产品与应用信息 • 多位国内外优秀专家学者,多位泽泉科技的技术工程师,以及德国WALZ等国外厂商的技术人员分别主持各个栏目，希望能够共同建立一个交流的平台

2. 不仅仅是一个……产品的平台不仅仅是一个……技术的平台不仅仅是一个……服务的平台不仅仅是一个……资源的平台不仅仅是一个……应用的平台不仅仅是一个……不仅仅是一个……产品的平台不仅仅是一个……技术的平台不仅仅是一个……服务的平台不仅仅是一个……资源的平台不仅仅是一个……应用的平台不仅仅是一个…… • 中国生态仪器网……网上生理生态技术交流中心，期待您的参与！ • 网络由主要由AndyZhang负责技术维护,若您有意主持栏目或有什么建议，请联系：cong@zealquest.com 张聪 • 泽泉科技有限公司 • 中国 上海市定西路710弄16号鸿申大厦24楼A座 • 邮编：200052 • 电话：021-62837346，62837347，62837349，62838513 • 传真：021-62838512 sales@zealquest.com

3. Topics on PAM fluorometry Tuesday, 29.06.2004 14:00-15:30 Introduction to PAM fluorometry PAM-2100 and PamWin 15:45-17:15 Introduction to Imaging-PAM Standard-, Maxi-, Mini- and Micro-Heads Wednesday, 30.06.2004 9:00-10:30Hands on Imaging-PAM 10:45-12:15 Flow-Through Water-PAM (Rolf Gademann) Thursday, 1.07.2004 10:45-12:15 Monitoring-PAM (Rolf Gademann) 14:00-15:30 Hands on Monitoring-PAM

4. Progress in chlorophyll fluorescence

5. Recent stimulation by: Progress in LED-Technology Lumileds-Luxeon LEDs: up to 1000 mA; providing pulse-modulated light at intensities comparable to those obtained by halogen lamps. Use in new PAM Measuring Heads: Micro-, Mini- and Maxi-Head of IMAGING-PAM Flow-Through Water-PAM Monitoring-PAM

6. Basics of Chlorophyll Fluorescence Methods PAM fluorometry and the saturation pulse method What actually is measured with PAM fluorometers? What kind of information is provided? What does that tell us about the plants?

7. Chlorophyll fluorescence is measured to obtain information on the efficiency of photosynthetic energy conversion in plants

9. Chlorophyll fluorescence: small fraction of light absorbed by plant which is re-emitted in form of light • Chlorophyll fluorescence yield (F): • Measured with constant amplitude pulses of measuring light • Generally varies between 2-10% • Controlled by the rate of energy conversion at PS II reaction centers (P) • and by the rate of energy dissipation into heat in PS II (D) Ideally, the PAM measuring light does not change the state of the investigated plant sample.

10. P + D + F = 1 F = 1- P - D photochemical and dissipative fluorescence quenching

11. Before introduction of PAM fluorometry and of the Saturation Pulse method no distinction between fluorescence quenching by photochemical utilization and by heat dissipation (loss) was possible.

12. Quenching analysis by the saturation pulse method The rationale of this method is simple: Short pulse of saturating light PS II reaction centers are transiently fully blocked Photochemical quenching is fully suppressed The remaining quenching is nonphotochemical

13. Dissipative fluorescence quenching, generally referred to as non-photochemical quenching Indicative for the transformation of light energy into heat Non-photochemical quenching has evolved as a very useful indicator forthe regulated annihilation of potentially harmful excess light energy into harmless heat.

14. Fluorescence yieldand the Saturation Pulse method: 1) In any given state fluorescence yield is quenched with respect to its maximal value (Fm) by photochemical energy conversion (photochemical quenching) and heat-dissipation (non-photochemical quenching). 2) Fm is defined for a state of minimal non-photochemical quenching normally corresponding to the dark-state. 3) PAM Fluorometry allows to assess fluorescence yield under practically all physiologically relevant conditions, including strong background illumination (special modulation technique) 4) Photochemical quenching can be transiently completely suppressed by a short (ca. 1 s) pulse of saturating light (Saturation Pulse). The remaining quenching is non-photochemical quenching.

15. Application of the Saturation Pulse method requires a special measuring technique of modulated fluorescence which is not disturbed by extremely strong background light fluorescence excitation AC coupled fluorescence signal selective window 1 selective window 2

16. chloroplasts 5000 μE/m²s ethanolic extract 100 μE/m²s 0.1 μE/m²s

17. Quenching analysis by the Saturation Pulse method SP

19. Fluorescence parameters defined by Kramer et al.: Y(II) = quantum yield of Photosystem II Y(NPQ) = quantum yield of regulated dissipation of excitation energy Y(NO) = quantum yield of other non- regulated dissipation processes Y(II) + Y(NPQ) + Y(NO) = 1

21. Some important aspects

22. Fo and Fo’ Fluorescence yield observed when all PS II centers open: Fo, dark-adapted (no nonphotochemical quenching) Fo’, illuminated (more or less nonphotochemical quenching), determination requiring far-red illumination (PS I activity Fm and Fm’ Fluorescence yield observed when all centers are closed Fm, dark-adapted (no nonphotochemical quenching) Fm’, illuminated (more or less nonphotochemical quenching)

23. Function of far-red light (λ > 700 nm) • 1) Selective excitation of PS I • Intersystem electron transport chain emptied • Acceptor side of PS II free • PS II reaction centers open • 2) Major practical applications • a)Assessment of Fo’, • i.e. minimal fluorescence yield of illuminated sample; • non-photochemically quenched • For correct determination of quenching coefficients • (qP and qN) knowledge of Fo’ is required • b) Oxidation of P700, • important tool in P700 absorbance measurements

24. Blue versus red excitation light 1) When using blue excitation, fluorescence can already be measured at wavelenghts λ > 600 nm, i.e. also the short-wavelength peak of fluorescence is seen. 2) This leads to a considerable signal increase (ca. factor 3) in all types of samples in which the short wavelength emission peak (685 nm) is not reabsorbed, i.e. particularly in dilute suspensions. 3) Blue light has become a feasible alternative since very strong blue LEDs became available. 4) Blue LEDs can be applied without optical filtering • Problematic aspects with blue excitation: • Blue light is very poorly absorbed by cyanobacteria • Many organisms show “blue-light effects”, e.g. on stomates

25. Some practical examples of the use of the Saturation Pulse method in applied photosynthesis research

26. Saturation Pulse quenching analysis revealing use of transmembrane proton gradient for ATP synthesis linked to Calvin cycle activity

27. Typical example of stress induced suppression of Calvin cycle activity: Heat treatment of Arbutus unedo

28. The Kautsky-effect Characteristic changes of chlorophyll fluorescence yield upon actinic illumination after dark-adaptation (dark-light induction curves).

29. Light-response curves of sun and shade adapted leaves

30. Rapid Light Curves Using PAM fluorometry, Light Response Curves normally are measured with much shorter illumination periods than in the case of classical P-I curves (measured via gas exchange). Note: Rapid Light Curves measured by PAM fluorometry contain somewhat different information than classical P-I curves.

31. PAM-2100 Chlorophyll Fluorometer Major applications: 1) Ecophysiology and stress physiology 2) Plant molecular biology 3) Photosynthesis research 4) Horticulture and agriculture • Featuring far-red light source • Halogen as well as LED actinic light sources • Rapid induction and relaxation kinetics • Stand-alone operation under DA-2100 • PC-operation under PamWin

32. The peculiarities of the PAM-2100 are due to the fact that it was directly derived from the original PAM-2000 Major differences: 1) The external MS-DOS PC of the PAM-2000 is integrated within the PAM-2100 (panel PC, data acquisition under MS-DOS) 2) Alternatively, the PAM-2100 can be also operated via an external Windows-PC under the new windows software PamWin

33. The PAM-2100 features two completely independent modes of operation: Operation via internal panel PC (MS-DOS); DA-2100 Data Acquisition System; present software version 2.37; mainly for field work. Operation via external PC (Windows); PamWin software; present software version 1.24; for laboratory work

34. For operation under PamWin the panel PC must be switched off External Windows-PC and internal PC are linked in two occasions only: 1) For data transfer from the PAM-2100 to PamWin using the Transfer.exe software 2) Update of DA-2100 program using the Update.exe software

35. Data transfer from PAM-2100 to Windows-PC 1) Connect PAM-2100 with PC via RS-232 2) Switch on PAM-2100 (start of DA-2100 program) 3) Select Menu/Data/Transfer Files 4) Start Fileserver via „Return“ on PAM-2100 5) Start Trans2100.exe file in PamWin directory on external Windows-PC. 6) Select file to be transferred 7) Start transfer to PamWin data directory via “doubleclick“ on Windows-PC 8) Repeat 6-7 until all files are transferred.

36. Update of PAM-2100 firmware (DA-2100) 1) Connect PAM-2100 with PC via RS-232 2) Turn on external Windows-PC and select Update.exe in PamWin directory 3) Make sure that PamWin program is not running 4) Switch on PAM-2100 (start of DA-2100 program) 5) Select Menu/Data/Update DA-2100 6) Start Update.exe in PamWin directory on external Windows-PC 7) Finalize Update via „Return“ on PAM-2100 8) For the updated program version to become effective, the program has to be first quit and then started again

37. IMAGING-PAM Major fields of application: 1) Plant pathology 2) Plant molecular biology 3) Plant physiology 4) Eco- and Stress Physiology 5) Horticulture 6) Teaching labs • Images of fluorescence parameters • Multiple site measurements • Assessment of absorbed PAR The IMAGING-PAM has opened a new dimension in chlorophyll fluorescence studies and applied photosynthesis research. It can be particularly recommended for leaf measurements.

38. IMAGING-PAM Chlorophyll Fluorometer: Assessment of local heterogeneities in fluorescence yield and photosynthetic parameters determined by the Saturation Pulse method.

39. Components and Technical Data • Measuring Heads for different sample areas • Standard: up to 24 x 32 mm • Maxi: up to 95 x 125 mm • Mini: up to 30 x 40 mm • Micro: 3 x 4 mm • Epifluorescence Microscope: down to µm scale • Power-and-Control Unit • except for Maxi-Head battery powered; • for laboratory and field applications • CCD Video Camera • featuring IEEE 1394 interface (Firewire); no framegrabber required; • digitization within camera; rapid data transfer to PC; • 1/3” (640 x 480 pixel) or 1/2” CCD-Chip (with Maxi-Head); • different objective lenses for various Measuring Heads • PC with dedicated ImagingWin Windows-software

40. Special features of the new Mini-Head 1) Relatively small size 2) Well suited for application in conjunction with GFS-3000 3) Relatively large imaged area (30 x 40 mm)

41. Mini-Head mounted on GFS-3000

42. Special features of the new Max-I-PAM 1) Particularly large imaged area (95 x 125 mm) 2) Compatible with imaging of standard multiwell plates 3) Important tool for plant molecular biologists and toxicologists

43. Imaging of 24-well plate with green algae suspension 8 center wells with Diuron at concentrations increasing from 1.5x10-8 to 3x10-5M

44. Penetration of DCMU into leaf via petiole

45. Penetration of DCMU into leaf via petiole

46. Penetration of DCMU into leaf via petiole

47. Penetration of DCMU into leaf via petiole

48. Penetration of DCMU into leaf via petiole

49. Penetration of DCMU into leaf via petiole

50. Penetration of DCMU into leaf via petiole