International Carl Zeiss Workshop on International Carl Zeiss ...

<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on 

Schedule 

<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on 

Fluorescence Correlation 

Spectroscopy and Related Methods 

March 30, 2000, 10:00 to March 31, 16:00 

in Jena / Germany 

<strong>Carl</strong> <strong>Zeiss</strong> Jena GmbH 

<strong>Carl</strong> <strong>Zeiss</strong> Promenade 10 

07745 Jena 

Germany

<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on Fluorescence Correlation Spectroscopy and Related Methods 

March, 30 to March 31, Jena / Germany 

1 WELCOME TO JENA 2 

2 LOCATION 2 

3 TRANSPORTATION 2 

4 PARKING LOTS 3 

5 REGISTRATION DESK 3 

6 CONFERENCE OFFICE 4 

7 TALKS 4 

8 POSTER 4 

9 FACTORY TOUR 4 

10 DEMONSTRATION OF CONFOCOR 2 5 

11 AN EVENING IN THE OPTICAL MUSEUM 5 

12 PUBLICATION OF RESEARCH ARTICLES 5 

13 INTERNATIONAL SCIENTIFIC COMMITTEE 10 

14 SCHEDULE 12 

15 POSTER TITLES 16 

16 ABSTRACTS 21 

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1 WELCOME TO JENA 

Welcome to Jena, welcome at <strong>Carl</strong> <strong>Zeiss</strong>. On behalf of the organization committee of the 

“<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on Fluorescence Correlation Spectroscopy (FCS) and 

Related Methods”, we wish you a pleasant stay and a lot of fruitful discussions. 

Reviewing the program, we were impressed about the large amount of promising 

contributions to the workshop. The variety of subjects shows, that Fluorescence 

Correlation Spectroscopy is a well established method now and finds its way into lots of 

diverse research areas. 

The <strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on Fluorescence Correlation Spectroscopy (FCS) 

and Related Methods continues the series of ConfoCor User Clubs organized by 

<strong>Carl</strong> <strong>Zeiss</strong> (1997 Leuven/Belgium, 1998 Jena/Germany, 50 participants each). 

The change in name reflects the intention of the organizers to invite all scientists 

interested in FCS instrumentation, theory, applications and related techniques to 

participate the meeting, regardless whether they use a <strong>Carl</strong> <strong>Zeiss</strong> ConfoCor or not. 

The workshop will start Thursday, March 30th at 10:00 am at the Conference Room of 

<strong>Carl</strong> <strong>Zeiss</strong> Jena, <strong>Carl</strong>-<strong>Zeiss</strong>-Promenade 10 (formerly known as Tatzendpromenade 1a). The 

registration desk will open at 8:00. 

Dr. Winfried Wiegräbe 


Microscopy 

Advanced Imaging Systems 

2 LOCATION 


<strong>Carl</strong>-<strong>Zeiss</strong>-Promenade 10 

07745 Jena 

Germany 

The conference room is located at the large, flat building in front of the main 

entrance to <strong>Carl</strong> <strong>Zeiss</strong> Jena GmbH. This building houses the canteen of the 

“Fachhochschule”, too. 

3 TRANSPORTATION 

If you stay at the ibis or Esplanade hotel, we recommend to take the Bus starting at the 

city center named "Holzmarkt" with destination "Burgau" (lines 10,13), or 

"Goeschwitz" (line 10), bus stop "Fachhochschule" or “<strong>Zeiss</strong> Werk”. <strong>Zeiss</strong> is located 

between both stops on the right side, coming from the center. 

From the "Best Western Hotel", we have arranged a shuttle. It will start on Thursday, 

30 th at 9:00 am and on Friday, 31 st at 8:30 am. If you plan to use this service, please 

inform the reception desk of the hotel when arriving. 

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Fachhochschule 

<strong>Zeiss</strong> Werk 

4 PARKING LOTS 

Parking lots are available in front of the conference building exclusively for participants of the 

workshop. 

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5 REGISTRATION DESK 

The registration desk will be open on Thursday, 30 th March from 8:00 to 12:00 am. Later 

registration is possible at the conference office. 

Please prepare the conference fee (100,- DM, 50,- DM for students) in cash. Unfortunately, credit 

cards are not accepted. 

6 CONFERENCE OFFICE 

During the workshop a conference office is located at the “Innovationsraum”. Leave the 

conference room through the corridor at the west side. The conference office is located 

one floor below the conference room. 

At the conference office, you may receive messages by phone and FAX: 

Phone: ++49 – (0)3641 – 64 – 3254 

FAX: ++49 – (0)3641 – 64 – 3347 

At the office you can check your slides and settings of your computer when using a 

beamer. 

If you need any additional assistance, please feel free to contact the conference office or 

any member of the organization committee. 

7 TALKS 

Talks are scheduled to take 20 min plus 5 min for discussion. For your talk, there will be a 

slide projector (35mm), an overhead projector, a beamer to be used together with your 

laptop and a laser pointer available. 

If you need additional equipment (e.g. video), please contact the conference office. 

8 POSTER 

For your posters, we have prepared boards approximately 180 cm high and 90 cm wide. 

Posters should be mounted on Thursday, 30th March between 8:00 and 10:00 am. 

Posters will be on display during the whole workshop. 

All posters are listed stating at page 16. Posters are numbered. Please attach your poster 

at the board showing the appropriate number. 

9 FACTORY TOUR 

There will be factory tours to the optics production and the system integration of the ConfoCor 

2. The tours will be during the breaks. Each tour will last approximately half a hour. 

Because each tour is restricted to 25 persons, please put your name on the lists available at the 

registration desk. 

The tours will start in the conference office. 

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10 DEMONSTRATION OF CONFOCOR 2 

You might be interested in having a demonstration of the ConfoCor 2, the new fluorescence 

correlation microscope of <strong>Carl</strong> <strong>Zeiss</strong>. There are two instruments on display, one is located at the 

conference room, the other one is shown at the casino. The casino is approached through the 

corridor at the west end of the conference room. 

The ConfoCor 2 is demonstrated during the breaks and the poster session. Please feel free to 

schedule a private demonstration with one of the <strong>Carl</strong> <strong>Zeiss</strong> FCS experts. 

11 AN EVENING IN THE OPTICAL MUSEUM 

On Thursday evening there will be a get together at the optical museum of Jena. The optical 

museum is located near the city center, not far away from the Esplanade and ibis hotels. 

Between 18:00 and 19:00, there will be buses from the conference location to the optical 

museum. The buses start in front of the conference building. 

12 PUBLICATION OF RESEARCH ARTICLES 

Abstracts are available at www.zeiss.de/fcsevents. They will be on display some time after 

the workshop to. Additional abstracts are welcome. Please mail them to 

wiegraebe@zeiss.de, preferable as an one page Word 97 for Windows document or as 

plain text. You are free to include links to your own web page. Please use as subject 

"Abstract FCS workshop" and the name of the first author. 

The November 2000 issue of the journal "Biological Chemistry" will be published as 

highlight issue covering the topics of the workshop. "Research Articles" or "Short 

Communications" should be submitted directly to: 

Biological Chemistry 

Editorial Office 

attn. Dr. Ulrich Hartl 

Walter de Gruyter GmbH & Co. 

Genthiner Str. 13 

D-10785 Berlin 

Tel.: +49-30-26005-279 

Fax: +49-30-26005-298 

E-mail: biol.chem.editorial@deGruyter.de 

Internet: www.degruyter.de/journals/bc 

Deadline for submission: May, 31st, 2000. 

The articles will undergo the standard review process of the journal. You will find the 

instructions for authors at http://www.degruyter.de/journals/bc/bcins.html and at the 

conference site. 

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Scope and policy of the Journal 

Biological Chemistry provides rapid publication for reports on molecular studies of 

exceptional biological interest. The Journal publishes full length papers, short 

communications, reviews and mini-reviews. Reviews are published by invitation only, but 

suggestions to the Editor-in-Chief are welcome. 

Submission of a manuscript to Biological Chemistry implies that the work described has 

not been published before and is not under consideration for publication elsewhere. It is 

the corresponding author's responsibility to ensure that all authors see the manuscript 

and approve of its submission for publication. Once the manuscript is accepted, it must 

not be published elsewhere without the consent of the copyright holders. 

Submission of manuscripts 

One original and three high-quality copies of the manuscript, including all figures and 

tables, should be submitted to: 

Biological Chemistry 

Editorial Office 

attn. Dr. Ulrich Hartl 

Walter de Gruyter GmbH & Co. 

Genthiner Str. 13 

D-10785 Berlin 

Tel.: +49-30-26005-279 

Fax: +49-30-26005-298 

E-mail: biol.chem.editorial@deGruyter.de 

Each manuscript should be accompanied by a cover letter containing a brief statement by 

the authors as to the element of novelty upon which they base their request for 

publication in Biological Chemistry. The reviewing process may be expedited by indicating 

the names and full postal addresses (including phone and fax numbers) of at least four 

potential referees who are not editors or members of the Editorial Board of the Journal. 

Review of manuscripts and speed of publication 

Papers will be independently reviewed by at least two referees selected by the Editors of 

Biological Chemistry. On the average, decisions will be reached within three to four 

weeks of submission. When papers are accepted subject to revision, revision is allowed 

only once. The revised manuscript (one original and two copies) must be submitted 

within two months of the authors' notification of conditional acceptance. It is the aim of 

the Journal to publish papers within four months after their receipt by the Editor-in-Chief. 

Preparation of manuscripts 

Manuscripts must be written in clear and concise English and should be regarded as final 

texts. Illustrations must be submitted in original quality with all four copies. No changes 

may be made at the proof state other than correction of printer's errors. 

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General format and length 

Manuscripts (including table legends, figure legends and references) should be typed 

double-spaced with font size 12 letters on only one side of A4 or 8 1/2 x 11" paper. 

Pages should be numbered (with the title page as 1) and have margins of 2.5 cm (1 inch) 

on all sides. Footnotes in the text should be avoided; parentheses should be used instead. 

Full length papers and reviews should occupy no more than eight printed pages, short 

communications and mini-reviews should not exceed four printed pages. Each full page 

of printed text corresponds to approximately 1400 words. Please allow sufficient space 

for tables and illustrations within the page limit. 

Sections 

- Full length papers should be organized into Title page, Summary, Key words, 

Introduction, Results, Discussion, Materials and Methods, Acknowledgments, 

References, Tables and Figure legends. 

- Short communications should be subdivided into an Abstract, Key words, and a single 

section of main text without headings. Experimental procedures should be described 

in legends to figures or footnotes to tables. Acknowledgments and References should 

be presented as in full length papers. 

Title page 

The title page should include (a) a short and informative full article title (series titles are 

not accepted); (b) names of all authors (with one forename in full for each author), 

followed by their affiliations (department, institution, city with postcode, country); (c) the 

mailing address, fax and phone number of the corresponding author; (d) a running title 

of 50 characters or less. If there is more than one institution involved in the work, 

authors' names should be linked by superscript consecutive numbers to the appropriate 

institutions. If required, small letters should be used to indicate present addresses. 

Summary and key words 

The second page of the manuscript should contain the summary and the key words. The 

summary should be a single paragraph of no more than 200 words (full length paper or 

review) or 100 words (short communication or mini-review) which must be 

comprehensible to readers before they have read the paper. Abbreviations and reference 

citations should be avoided. Below the summary up to six key words, which are not part 

of the title, should be given in alphabetical order and separated by slashes (/). Together 

with the title the key words provide the basis for the annual Subject Index. 

Acknowledgments 

Acknowledgments should be placed at the end of the text. 

Names of funding organizations should be written in their entirety. 

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References 

For citations authors are encouraged to rely as far as possible upon articles published in 

primary research journals. 

Unpublished results and personal communications should be cited as such within the 

text; personal communications must be substantiated by a letter of permission Meeting 

abstracts may not be cited. 

Within the text references should be cited by author and date; et al. should be used if 

there are more than two authors. At the end of the text citations should be in 

alphabetical order and should contain complete titles. 

The number of references should not exceed 150 for reviews or 30 for mini-reviews. 

The names of Journals should be abbreviated according to the World List of Scientific 

Periodicals. 

The volume number of a journal should be given in italics. Citations should be in 

accordance with the following examples: 

Kyte, J., and Doolittle, R.F. (1982). A simple method for displaying the hydrophobic 

character of a protein. J. Mol. Biol. 157, 105-132. 

Khan, M.A. (1987). HLA and ankylosing spondylitis. In: Ankylosing 

Spondylitis. New Clinical Applications in Rheumatology, Vol. I, 

J.J. Calabro and C. Dick, eds. (Lancaster, England: MTP Press, 

Ltd.), pp. 23-45. 

Hogan, B., Costantini, F, and Lacy, E. (1986). Manipulating the 

Mouse Embryo: A Laboratory Manual (Cold Spring Harbor, New 

York: Cold Spring Harbor Laboratory Press). 

Tables 

Tables should be typed on separate pages and be numbered consecutively using Arabic 

numerals. If a table is exceptionally large or contains special symbols it should be 

submitted camera-ready. A short descriptive title, column headings and (if necessary) 

footnotes should make each table self-explanatory. Please indicate in the manuscript the 

approximate position of each table. 

Illustrations 

Illustrations will be reduced in size to fit, whenever possible the width of a single column, 

i.e. 80 mm, or a double column, i.e. 168 mm, of text. Ideally, single column figures 

should be submitted with a width of 100 mm, double column figures with a width of 

210 mm. Lettering of all figures within the article should be uniform in style (preferably a 

sans serif typeface) and of sufficient size (so that the final height will be approximately 2 

mm). Uppercase letters A, B, C etc. should be used to identify individual parts of multipart 

figures. On the back of each figure the name of the first author the figure number 

and the top margin should be indicated, preferably with a soft pencil. All figures must be 

cited in the text in numerical order and the approximate position of each should be 

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indicated in the margin. Reference to figures is to be made in the text as Figure 1 etc., 

Fig. 1 etc. should be used in the figure legends. 

Photographs 

Photographs should be provided as high-quality glossy prints in order to withstand the 

inevitable loss of contrast and detail inherent in the printing process. 

Color plates 

To partially offset the cost of production, color figures will be printed with the following 

charges to the author: 

DM 1200.- for the first illustration and DM 600.- for each subsequent illustration in one 

article. The total cost for color figures will be reduced if several color illustrations appear 

on one printed page. 

Line drawings 

These should be provided as high-quality prints. No additional artwork, redrawing or 

typesetting will be done by the publisher. Note that faint shading or stippling may be lost 

upon reproduction, heavy staining or stippling may appear black. 

Figure Legends 

Theses should be provided on separate, numbered manuscript pages. All symbols and 

abbreviations used in the figures must be explained, except for standard abbreviations or 

others defined in the preceding text. 

Nomenclature 

With respect to biochemical terminology the Journal will follow the rules laid down by 

the IUPAC-IUB Commission on Biochemical Nomenclature as given in Biochemical 

Nomenclature and Related Documents, published by the Biochemical Society, U.K. 

Enzyme names should be in accordance with the recommendations of the IUPAC-IUB 

Commission on Biochemical Nomenclature, 1978, as given in Enzyme Nomenclature 

published by Academic Press, New York, 1992. Genotypes should be given in italics, 

phenotypes should not be italicized. Nomenclature of bacterial genetics should follow 

Demerec et al. (1966). Genetics, 54, 61-76. 

Abbreviations 

The Journal accepts standard Journal of Biological Chemistry abbreviations. Uncommon 

abbreviations should be defined parenthetically within the text upon first appearance. 

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Cover illustrations 

Each cover of Biological Chemistry will have an illustration selected from one of the 

articles published in the Issue. Authors who wish to have a color photograph considered 

for the cover should submit a glossy print, 18cm by 24cm or 8 inches by 10 inches, 

and/or a slide. A short descriptive summary of what the picture shows should be 

included. 

Offprints 

For each article 20 offprints are supplied free of charge. An offprint Order Form will 

accompany the page proofs and should be completed and returned immediately. 

13 INTERNATIONAL SCIENTIFIC COMMITTEE 

- Chair: Prof. Rudolf Rigler 

Department of Medical Biochemistry and Biophysics 

The Karolinska Institute 

The Laboratory of Medical Biophysics, MBB 

S-171 77 Stockholm, 

SWEDEN 

- Univ. Doz. Dr. Manfred Auer 

Novartis Leading Scientist 

Research Programme Head: "Fluorescence based HTS Technology" 

Associate Professor, University of Salzburg, 

Novartis Forschungsinstitut, NFI 

Allergic diseases 

Brunner Strasse 59 

A-1235 Vienna 

AUSTRIA 

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- Prof. Manfred Eigen 

Arbeitsgruppe 081 (Biochemische Kinetik) 

MPI für biophysikalische Chemie 

Am Faßberg 

37070 Göttingen 

Germany 

- Prof. Enrico Gratton 

Department of Physics 

University of Illinois 

at Urbana-Champaign 

1110 West Green Street 

Urbana, IL 61801-3080 

USA 

- Prof. Antonie Visser 

Microspectroscopy Centre 

Wageningen University 

Dreijenlaan 3 

6703 Ha Wageningen 

The Netherlands 

- Prof. Horst Vogel 

Institut of Physical Chemistry 

Swiss Federal Institute of Technology 

1015 Lausanne 

Switzerland 

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14 SCHEDULE 

Thursday, 30 th March 

Time Authors Title Abstract 

(page) 

08:00- 

10:00 

Registration 

Setting up Posters 

4, 2 

10:00- 

10:15 

F.v.Falkenhausen 

President 

<strong>Carl</strong> <strong>Zeiss</strong> Jena GmbH, 07745 

Jena, Germany 

Welcome Address 

10:15- 

10:30 

W. Wiegräbe 


Jena, Germany 

Organizational Remarks 

10:30- 

11:00 

R. Rigler 

Karolinska Institute, Dept. of 

Medical Biophysics, 17177 

Stockholm, Sweden 

Introduction into FCS 

11:00- 

12:00 

Posters 

Factory Tour 

4, 5 

12:00- 

12:25 

A. Casoli and M. Schönhoff 

MPI for Colloids and Interfaces, 

14476 Potsdam/Golm, Germany 

Fluorescence Correlation Spectroscopy as a Tool to 

investigate Single Molecule Probe Dynamics in 

Ultrathin Polymer Films 

27 

12:25- 

12:50 

Z. Földes-Papp and R. Rigler 

Karolinska Institute, Dept. of 

Medical Biophysics, 17177 


Fluorescent Nucleotide Tagging, Bead Loading and 

Exonucleolytic Degradation of DNA for Sequencing 

of Single DNA Molecules 

12:50- 

14:30 

Lunch 

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Thursday, 30 th March (continued) 


(page) 

14:30- 

15:00 

15:00- 

15:25 

15:25- 

16:10 

16:10- 

16:35 

16:35- 

17:00 

17:00- 

18:00 

18:00- 

21:00 

E. Gratton 

Department of Physics, University 

of Illinois at Urbana-Champaign, IL 

61801-3080 Urbana, USA 

K. Gall, K. Palo, and P. Kask 

Evotec BioSystems AG, 22525 

Hamburg, Germany 

G. Jung, A. Zumbusch, and C. 

Bräuchle 

Inst. f. Phys. Chemie, LMU 

München, 81377 München, 

Germany 

A. Koltermann, U. Kettling, T. 

Winkler, J. Stephan and M. Eigen 

Max Planck Institute for 

biophysical chemistry, 37077 

Goettingen, Germany 

to be announced 

FIDA, more than a glimpse beyond the FCS horizon 

Posters 

Factory Tour 

Two-Colour Fluorescence Correlation Spectroscopy 

of the Green Fluorescent Protein 

Dual-color fluorescence cross-correlation 

spectroscopy and its application in biotechnology 

4, 5 

Poster session 16 

An Evening at the Optical Museum 5 

33 

35 

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Friday, 31 st March 


(page) 

09:00- 

9:30 

09:30- 

9:55 

09:55- 

10:20 

10:20- 

11:00 

11:00- 

11:25 

11:25- 

11:50 

11:50- 

12:15 

12:15- 

12:40 

T. Visser 

University of Wageningen, 

Netherlands 

R. Brock 

Institute of Organic Chemistry, 

72076 Tübingen, Germany 

A. Pramanik and R. Rigler 

Department of Medical 

Biochemistry and Biophysics, The 

Karolinska Institute, 17177 


P. Schwille 

Max-Planck-Institut für 

biophysikalische Chemie, AG 

Experimentelle Biophysik; Turm IV, 

081, 37077 Göttingen, Germany 

T. Wohland, R. Hovius and H. 

Vogel 

EPFL, 1015 Lausanne, Switzerland 

R. Jordan* and J. Klingauf 

Max-Planck-Institute for 

Biophysical Chemistry / 

Department of Membrane 

Biophysics, 37077 Göttingen, 

Germany 

N. Petersen 

Univ. Western Ontario, 

Department of Chemistry, N6A 

5B7 London, Canada 

FCS of flavins and flavoproteins 50 

Fluorescence Correlation Microscopy - Perspectives 

for Intracellular High-Throughput Sceening 

FCS-analysis of ligand-receptor interactions in the 

membranes of cultured cells. 

Posters 

Factory Tour 

Molecular dynamics in cells and on membranes: new 

challenges for FCS 

The Characterization of a Transmembrane Receptor 

Protein by Fluorescence Correlation Spectrosocpy 

Synaptic Vesicle Dynamics Studied by Fluorescence 

Correlation Spectroscopy 

24 

43 

4, 5 

Spatial Correlation Spectroscopy 42 

48 

51 

32 

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Friday, 31 st March (continued) 


(page) 

12:40- 

13:30 

13:30- 

14:00 

14:00- 

14:25 

14:25- 

15:10 

15:10- 

15:35 

15:35- 

16:00 

M. Auer 

(1) Novartis Pharma Research, 

4002 Basel, Switzerland, (2) 

Massachusetts Institute of 

Technology, Cambridge, MA 

02139, (3) Novartis 

Forschungsinstitut, 1235 Vienna, 

Austria 

S. Ashman, U. Haupts, M. 

Ruediger, S. Turconi, and A. Pope 

SmithKline Beecham, CM19 5AW 

Harlow, United Kingdom 

Ch. Buehler(1), J. Dreessen(1), K. 

Mueller(1), P.T.C. So(2), C.Y. 

Dong(2), A. Schilb(1), U. 

Hassiepen(1), K. Stoeckli(1), and 

M. Auer(3) 

(1) Novartis Pharma Research, 

4002 Basel, Switzerland, (2) 

Massachusetts Institute of 

Technology, Cambridge, MA 

02139, (3) Novartis 

Forschungsinstitut, 1235 Vienna, 

Austria 

J. Bieschke 

MPI f. biophys. Chemistry, 37077 

Göttingen, Germany 

Lunch 

Confocal fluorescence spectroscopy and 

nanoscanning in drug discovery 

Single Molecule Detection in uHTS 21 

Posters 

Factory Tour 

Multi-photon Excitation of intrinsic Protein 

Fluorescence has the potential to dramatically 

improve Pharmaceutical Drug-Screening 

Detection of amyloid aggregates by SIFT 

4, 5 

26 

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15 POSTER TITLES 

No. Authors Title Abstract 

(page) 

4 G. Bohnert, S.O. Kirschstein and 

L. Kittler 

Institut für Molekulare 

Biotechnologie Jena e.V., 7745 

Jena, 

DNA-Polymerase proof-reading activities monitored 

by fluorescence-correlation-spectroscopy 

15 L. Cognet, P.H.M. Lommerse, Individual eGFP and eYFP fusion proteins studied in 

G.S. Harms, G.A. Blab, H.P. cell membranes 

Spaink and Th. Schmidt 

Leiden University, 2333 AL 

Leiden, Netherlands 

1 P. Czerney 

Dyomics GmbH, 07743 Jena, 

Germany 

14 F. Delie(1), R. Gurny(1) and A. 

Zimmer(2) 

(1) Department of Pharmceutical 

Technology and 

Biopharmaceutics, 1211 Geneva, 

Switzerland, (2)Biocenter, Johann 

Wolfgang Goethe-Universität, 

Institut for Pharmaceutical 

Technology, 60439 Frankfurt 

(Main), Germany 

6 H. Häberlein 

Dept. of Pharmaceutical Biology, 

35032 Marburg, Germany 

2 A. Hascher, K. Korn, H.-H. 

Foerster, U. Hahn and F. Bordusa 

Universtität Leipzig; Institut fuer 

Biochemie; Fakultaet fuer 

Biowissenschaften, Pharmazie & 

Physiologie, 4103 Leipzig, 

Germany 

Tailor-made dyes for Fluorescence Correlation 

Spectroscopy (FCS) 

Fluorescence Correlation Spectroscopy for the 

Characterisation of Drug Delivery Systems 

Aspects in the development and application of small 

ligands in FCS. 

Chymotrypsin-catalyzed fluorescence labeling of 

ribonuclease T1 

23 

28 

29 

30 

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Poster titles (continued) 


(page) 

16 M.A. Hink, J.W. Borst, G.N.M. Intracellular Fluorescence Correlation Spectroscopy: 

van der Krogt J. Goedhart, T.W.J. towards monitoring Signal Transduction Processes in 

Gadella Jr.,and A.J.W.G. Visser vivo. 

University of Wageningen, 6703 

HA Wageningen, Netherlands 

22 A.Digris, V.Skakun, E.Novikov, 

M.A.Hink and A.J.W.G.Visser 

K.U.Leuven, 3001 Heverlee, 

Belgium 

23 T. Jankowski, V. Jüngel, and R. 

Janka 


Jena, Germany 

Software tools for global analysis in Fluorescence 

Correlation Spectroscopy 

Optimizing Opticts for Cross Correlation 

7 R. Kiehl 

Glutathione: The essential factor for mitochondrial 

energy-linked funktions 

Reinhold Kiehl Institute for 

Molecular Medicine/Biology, 

93437 Furth, Germany 

8 T. Kobayashi and J. Gruenberg 

Riken Frontier Research System, 

351-0198 Wako-shi, Saitama, 

Japan 

17 K. Korn, H.-H. Foerster, R. Rigler 

and U. Hahn 

Universtität Leipzig; Institut fuer 

Biochemie; Fakultaet fuer 

Biowissenschaften, Pharmazie & 

Physiologie, 4103 Leipzig, 

Germany 

FCS Analysis of an Organelle Specific Antibody 34 

FCS measurements of phages displaying RNase T1 or 

RNase A 

21 N. Kunst 

MicroSpectroscopy 

Development of a piezo driven xyz-translation stage 

for intracellular FCS 

Center, 

Dreijenlaan 3, 6703 HA 

Wageningen, The Netherlands 

31 

37 

38 

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(page) 

3 E. Lopez-Calle, J. R. Fries*, D. A Strategy for Highly Parallel Synthesis of Tyrosineand 

Histidine-Reactive Labeling Reagents 

Riester, D. Winkler, A. Wiesner, S. 

Dröge, H. Knorr, S. Viebrock, C. 

Winkler, G. Mielck, M. Wrobel 

39 

EVOTEC BioSystems AG, Life 

Science Technologies -Chemistry-, 

22525 Hamburg, Germany 

18 T. Neumann, S.O. Kirschstein, G. Measurement of the dynamic instability of 

Bohnert, J.A. Camacho Gomez, L. microtubules by FCS experiments 

Kittler and E. Unger 

40 

Institut für Molekulare 

Biotechnologie Jena e.V., 7745 

Jena, Germany 

9 O. Pänke, K. Häsler and W. Junge On the Stator of Rotary ATP Synthase: The Binding 

Strength of Subunit Delta to (Alpha Beta) 3 As 

Universität Osnabrück; FB 

Determinated by Fluorescence Correlation 

Biologie/Chemie; Abt. Biophysik, 

Spectroscopy 

49069 Osnabrück, Germany 

10 J. Rao, A. Nicol, P. Jordan, D. 

Zicha 

Imperial Cancer Research Fund, 

WC2A 3PX London, United 

Kingdom 

11 J. Schell, O. Schäfer, J. Tatzelt* 

and D. Riesner 

Heinrich-Heine-Universität, Inst. 

für Physikalische Biologie, 40225 

Düsseldorf, Germany 

Interactions of mutant EGF receptors analysed by 

fluorescence correlation spectroscopy (FCS) 

Influence of solvent conditions and chaperones on 

Prion Protein aggregation 

41 

45 

46 

- 18 -





(page) 

12 H. Schürer(1), A. Buchynskyy(2), 

K. Korn(1), P. Welzel(2) and U. 

Hahn(1) 

Investigation of aptamer/moenomycin interaction by 

FCS 

46 

(1)Institut für Biochemie, Fakultät 

für Biowissenschaften, (2)Institut 

für Organische Chemie, Fakultät 

für Chemie und Mineralogie, 

Universität Leipzig, Germany 

13 G. Stanciu 

Department of Physics, University 

"Politehnica" of Bucharest, 7206 

Bucharest, Romania 

24 K. Starchev, J. Ricka and J. Buffle 

Universite de Geneve, CH 1211 

Geneva, Switzerland 

25 J. Toivola 

University of Jyväskylä, 40351 

Jyväskylä, Finland 

Investigations on new semiconductor materials (HgBr 

and HgBrI) 

Noise on Fluorescence Correlation Spectroscopy 

Fluorescence Correlation Spectrosocpy 

5 E. Van Rompaey 

FCS, a technique to study characteristics of gene 

complexe 

Ghent University-General 

Biochemistry and Physical 

Pharmacy, 9000 Ghent, Belgium 

26 E. Van Craenenbroeck, G. A statistical analysis of fluorescence fluctuation data 

Matthys, J. Beirlant and Y. with rare events 

Engelborghs 

Laboratory of Biomolecular 

Dynamics, 3001 Leuven, Belgium 

49 

- 19 -





(page) 

27 S. Wennmalm, H. Blom and R. 

Rigler 

Medical Biophysics, Karolinska 

Institutet, 112 60 Stockholm, 

Sweden 

19 O. Zschörnig, J.Pittler, and G. 

Bohnert 

University of Leipzig, Institute for 

Medical Physics and Biophysics, 

4103 Leipzig, Germany 

Fluorescence Correlation Spectroscopy in the 

Ultraviolet 

Different modes of Ca 2+ 

mediated interaction of 

annexin V with phospholipid vesicles 

- 20 -



16 ABSTRACTS 

Single Molecule Detection in ultra-High Throughput Screening 

Steve Ashman 

Molecular Interactions & New Assay Technologies, SmithKline Beecham, New Frontiers Science Park (North), 

Third Avenue, Harlow, Essex CM19 5AW, UK 

Homogeneous fluorescence techniques are now established as the most important 

readout strategy for miniaturised HTS in the future 1 . However, this encompasses a wide 

range of different approaches, each with particular advantages, limitations and 

applications. In this talk, we discuss a mixture of theoretical and practical aspects aimed 

at identifying the techniques of choice for robust miniaturised (e.g. 1536-well of greater 

densities) screening of different classes of drug targets. Where possible the single 

molecule detection techniques explored are compared to a macroscopic (ensemble) 

fluorescent equivalent. The latter monitor the time and assay volume weighted-average 

ensemble signal from the well and include most of those techniques currently used in 

HTS 1 (fluorescence intensity, prompt/time-resolved energy transfer, anisotropy). 

3,4 

Single molecule detection techniques (e.g FCS ) monitor the optical/biophysical 

properties of individual molecules within the assay well in a stochastic fashion by using a 

tightly focussed (typically 10 -15 

L) volume element. Traditionally, FCS has been used to 

analyse binding reactions via differences in the diffusion rates of fluorescently-labelled 

analytes undergoing binding reactions 4 . However, a number of recent developments 

have opened the way to considerably broader applications including the discrimination of 

molecular brightnesses ("FIDA", ref. 5, 6), distance-independent formation of labelled 

complexes 7,8 

(analogous to energy-transfer techniques) and a range of 2-dimensional 

techniques in which a combination of biophysical properties are analysed simultaneously 

(e.g. brightness and diffusion rates ("FIMDA") or anisotropy). 

Cont. 

- 21 -



Microscopic techniques, by definition, do not suffer from losses in signal quality as assay 

volumes are reduced. It remains to be seen whether this will turn out to be a decisive 

factor at the miniaturisation limit of discrete well MTP screening (probably around 1 µl, 

based upon liquid handling, evaporation etc.). Although Microtiter plate readers to 

perform HTS assays in the 5-10µl range are now readily available, single molecule 

detection has the potential advantages of very low volume assays and the rich 

information content of the signal output. 

1. Pope, A.J and Moore, K.J.M (1999) Drug Discovery Today 4:350-362. 

2. Owicki, J (1998). Presentation at SBS Annual Meeting. Baltimore 

3. Maiti S, Haupts U and Webb WW (1997) Proc. Natl. Acad Sci. 94, 11753 

4. Auer, M., Moore, K.J.M., Meyer-Almes, F-J., Guenther, R., Pope, A.J., and Stoekli, K. (1998) Drug 

Discovery Today 3 : 457. 

5. <strong>International</strong> Patent WO 98/16814, published 23rd April, 1998 

6. Kaask, P., Palo, K., Ullmann, D., Gall, K. (1999). Fluorescence-intensity distribution analysis and its 

applications in biomolecular detection technology. Proc Natl Acad Sci (USA) 96:13756-13751 

7. Kettling U, Kolterman A, Schwille P, and Eigen M (1998) Proc Natl Acad Sci 95, 1416 

8. Winkle, T., Kettling, U., Koltermann & Eigen, M (1999) Proc Natl Acad Sci 96:1375 

- 22 -



DNA-POLYMERASE PROOF-READING ACTIVITIES MONITORED BY FLUORESCENCE- 

CORRELATION-SPECTROSCOPY 

G. BOHNERT, S.O. KIRSCHSTEIN AND L. KITTLER 

Institute of Molecular Biotechnology e.V., Department of Single Cell and Single Molecule Techniques, 

Beutenbergstr. 11, D-07747 Jena, Germany 

Confocal fluorescence correlation spectroscopy (FCS) is ideally suited for monitoring 

molecular interactions in solution. FCS allows the discrimination between bound and 

free states of the molecules based on the autocorrelation time of the temporal 

fluctuations in fluorescence. The fluctuations are induced by molecular diffusion 

through the small detection volume (in the femtoliter range). 

In our contribution the proof-reading activities of the thermophilic DNA-polymerases 

Pfu (proof-reading active) and Taq (proof-reading inactive) have been studied, 

employing the FCS-technique. To this end the 3‘-end of the primer exposed to proofreading 

is fluorescently labeld. Two different target DNAs are used. The first produces 

a perfect match of the primer with the target, whereas the second creates a 

mismatch at the 3‘-end of the primer. In the case of a perfect match incubation with 

Pfu results in an essentially unaltered correlation time. In the case of a mismatch, 

however, the average autocorrelation time is significantly decreased. A careful 

analysis shows the presence of two components. The first one is identical with the 

original diffusion time of the primer-target-hybrid. The second one corresponds to 

the faster diffusion of a single nucleotide generated by proof-reading activity. To 

confirm the experimental procedure, incubations with Taq instead of Pfu are 

performed. The autocorrelation time remained essentially unchanged for both 

targets, indicating that no significant proof-reading had taken place. The enzymatic 

proof-reading efficacy is significantly influenced by the spacer length between 

fluorescence label and 3‘-end of the primer. 

- 23 -



Fluorescence Correlation Microscopy – Perspectives for Intracellular High-Throughput 

Screening 

Roland Brock 

Institute of Organic Chemistry, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, 

Ph.: +49-7071-2977629, Fax: +49-7071-295560, roland.brock@uni-tuebingen.de 

Fluorescence Correlation Microscopy (FCM) is highly attractive for cellular highthroughput 

screening providing complementary information on molecular interactions 

and molecular distribution with high sensitivity within the context of an intact cell. 

Absolute numbers (N) of mobile molecules inside the cell as well as diffusion constants 

(D) are obtained directly from analysis of the autocorrelation function. The fluorescence 

per molecule (fpm), calculated by dividing the fluorescence signal through the number of 

molecules, yields information on homo-aggregation of fluorescently labeled molecules. 

However, the correct determination of N, D, and fpm as read-outs in screening, requires 

freely diffusing molecules, the absence of photobleaching of molecules inside the 

detection volume, and a fluorescence yield independent of the local environment, 

respectively. Transiently expressed free GFP as well as a fusion protein of the epidermal 

growth factor receptor (EGFR) with GFP (EGFP-variant in both cases) were employed as 

paradigmatic test-cases to investigate to which degree these prerequisites could be 

fulfilled and thereby identify potential applications of cellular FCM-based screening 

applications. 

For free GFP, measurement times of a few seconds were sufficient for obtaining 

autocorrelation functions. In contrast to this, for the EGFR-GFP fusion protein, diffusion 

constants longer by a factor of one hundred compared to free GFP, dictated 

measurement times for acquisition of autocorrelation functions incompatible with highthroughput 

applications. Furthermore, initial photobleaching occurred at laser powers as 

low as 

0.4 kW/cm 2 – the minimum laser power required for recording of autocorrelation 

functions. Correct determination of the fraction of mobile molecules would require 

protocols relating the integrated fluorescence signal originating from bleached molecules 

to the one measured for the mobile fraction during the autocorrelation measurement. At 

laser powers required for rapid autocorrelation measurements of free GFP, 

photodestruction occurred for the receptor-fusion protein. This observation demonstrates 

that the dynamic range over which diffusion autocorrelation times for different proteins 

or protein complexes versus free protein can be determined simultaneously, is 

significantly smaller than the total temporal dynamic range accessible to FCS. The fpm 

was determined for free nuclear and cytoplasmic GFP. While slightly longer diffusion 

autocorrelation times were found for cytoplasmic GFP, the fpm was identical for both 

subcellular locations, compatible with applications addressing oligomerizations of GFPfusion 

within both compartments. 


- 24 -



It can be concluded that because of the restraints imposed on throughput and the 

additional complications in determining molecule numbers, applications of FCM in 

cellular high-throughput screening should focus on small molecules and molecular 

complexes. Furthermore, automation of image recognition and placement of the 

measurement volume for FCS will be a more feasible task for these systems. For slowly 

diffusion molecules or molecular aggregates more heterogeneous distributions are 

expected that complicate the identification of a representative location for subsequent 

FCS measurements. 

- 25 -



MULTI-PHOTON EXCITATION OF INTRINSIC PROTEIN FLUORESCENCE HAS THE 

POTENTIAL TO DRAMATICALLY IMROVE PHARMACEUTICAL DRUG-SCREENING 

Ch. Buehler 1 , J. Dreessen 1 , K. Mueller 1 , P.T.C. So 2 , C.Y. Dong 2 , A. Schilb 1 , U. Hassiepen 1 , 

K. Stoeckli 1 , and M. Auer 3 

1 

Novartis Pharma Research, CTA/LFU/NAT, 4002 Basel, Switzerland 

2 

Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 

Cambridge, MA 02139 

3 

Novartis Forschungsinstitut, Dermatology, A-1235 Vienna, Austria 

Multiple-photon excitation (MPE) of intrinsic protein fluorescence is likely to become a 

very valuable tool for assay development and drug screening. Since numerous proteins 

contain intrinsic fluorophores (tryptophan or tyrosine) which naturally sense molecular 

structures, dynamics, and interactions, intrinsic fluorescence assays allow for a label-free 

and therefore rapid and unbiased study of protein ligand interactions. Conventional UVspectroscopy 

and UV-based drug screening are often hampered by the indiscriminate 

background, photo-bleaching, scattering, large signal variability across the sample carrier, 

and interfering assay compounds ( e.g. inner filter effect). These problems can be 

significantly minimized by means of molecular three-photon excitation (3PE) with infrared 

femtosecond light pulses. Since the non-linear nature of MPE limits the region of 

photo-interaction to a sub-femtoliter volume at the focal spot, out-of-focus photobleaching, 

background generation, and the inner filter effect are dramatically minimized. 

We demonstrate the general feasibility of 3PE for numerous proteins and illustrate the 

technique’s excellent potential for high-throughput screening (improved well-to-well 

accuracy, reduced inner filter). By using the (intrinsic) fluorescence intensity of a threephoton 

excited protein-ligand, we were able to discriminate between ligands of different 

affinity in binding assays. 

- 26 -



Fluorescence Correlation Spectroscopy as a Tool to investigate Single Molecule Probe 

Dynamics in Ultrathin Polymer Films 

Alain Casoli and Monika Schönhoff 

Dye molecules as tracers in polymer matrices can be used to investigate the local mobility 

in bulk polymer materials. Our interest is the investigation of ultrathin films, e.g. 

polyelectrolyte self-assembled multilayers (SAM), where a strong complexation of 

opposite charges leads to extremely slow polymer dynamics. The idea of our work is to 

apply FCS to dyes in polymer films, where especially in heterogeneous systems, such as 

SAMs, the local information is of great interest. 

To establish the procedure, first a reference system of Rhodamin 6G embedded in PDMS 

films of different molecular weight, spin cast onto glass, is investigated in a <strong>Zeiss</strong> 

Confocor spectrometer. In addition to ensemble bleaching, intensity fluctuations of the 

fluorescence signal are found. Single molecule fluctuations are identified, they occur on 

the time scale of seconds, no fast processes are detected. A large number of single 

fluctuation measurements is accumulated to calculate correlation functions. The 

correlation time shows a dependence on molecular weight. 

Several mechanisms such as lateral diffusion, rotational diffusion or bleaching are 

discussed as potential origins of intensity fluctuations. Control experiments and the 

functional dependence of the correlation function lead to the interpretation that the 

fluctuations are arising from rotational diffusion of surface bound dye molecules. The dye 

in the PDMS films is probably electrostatically bound to the glass surface, leading to 

much slower rotational processes than expected for probes in the bulk polymer matrix. 

The correlation time is dependent on the polymer molecular weight, but the variation of 

the local mobility at the surface is much lower than that of the macroscopic polymer 

viscosity. 

We have thus shown the applicability of FCS to slow probe molecule dynamics in 

ultrathin films. Further experiments are under way on dye bound to SAMs, where also 

very slow (sub-second), but no fast processes are found. 

- 27 -



Tailor-made dyes for Fluorescence Correlation Spectroscopy (FCS) 

P. Czerney 

Dyomics GmbH, Botzstraße 5, D-07743 Jena 

Tel.: (+49) 03641 948386; Fax.: (+49) 03641 948387; e-mail: p.czerney@dyomics.com 

Different innovative applications in „life science“ have led to a new dye chemistry. At 

present time the design of functional dyes is the most important and interesting field in 

dye chemistry. Needs by using these materials are focused on high thermal and 

photochemical stability in aqueous solvents, pH-undependent absorption and 

fluorescence, high quantum yield and different possibilities for covalent coupling to 

proteins or oligonucleotides. 

Additionally, development in laser technology has extended the applications of organic 

dyes in the near infrared. Specially developed NIR-dyes are currently used as effective 

photoreceivers for diode lasers, and become the key materials in different analytical and 

diagnostic applications. 

Our research group in Jena (www.dyomics.com) has experience in design, synthesis and 

covalent coupling of different fluorophores and efficient light emitting marker dyes for 

more than twenty years. 

This versatility permits the design of until now unknown red and NIR-absorbing indicator 

systems as well as new NIR-emitting markers for FCS and multi color assays useable in 

standard methods of molecular biology, for example PCR, sequencing and primer 

elongation. Most of them belong to the class of water soluble polymethines, showing 

good long term stability. 

- 28 -



Fluorescence Correlation Spectroscopy for THE characterisation OF drug delivery 

systems 

F. Delie 1 , R. Gurny 1 and A. Zimmer 2 

(1) Department of Pharmceutical Technology and Biopharmaceutics, 1211 Geneva, Switzerland, 

(2)Biocenter, Johann Wolfgang Goethe-Universität, Institut for Pharmaceutical Technology, 60439 Frankfurt 

(Main), Germany 

Fluorescence Correlation Spectroscopy (FCS) is a unique and very sensitive technique to 

dynamically characterise ligand-receptor interaction. The FCS principle is based on a time 

resolved analysis of the fluorescence intensity fluctuations. The sensitivity of such a 

system allows a single molecule detection. 

This technique has been used for drug characterisation, high-throughput screening in 

pharmaceutical drug discovery and, more recently, to measure the diffusion coefficient of 

drugs within living cells (1). 

Part of the effort in pharmaceutical development is based on modulating drug availability 

by the use of carriers that will either controlled the release of the compound or target the 

drug to the active site. The most commonly used systems are entrapment in liposomes or 

polymeric particles, and complexation with macromolecules. The nature of the 

association of the active compounds to the carriers is rather critical to improve the 

systems and to predict in vivo release kinetics. By providing a real time in situ 

characterisation, FCS may assist the design of new drug delivery systems. 

The utility of this method in the field of drug delivery systems was investigated with a 

special attention to oligonucleotide delivery systems. In the present study, we measured 

in a time resolved manner the interactions between a fluorescently labelled 

oligonucleotide and cationically charged peptides and polymeric nanoparticles 

instantaneously in solution. 

- 29 -



Chymotrypsin-catalyzed fluorescence-labeling of ribonuclease T1 

Antje Hascher, Kerstin Korn, Hans-Heinrich Foerster, Ulrich Hahn and Frank Bordusa 

Institut fuer Biochemie, Fakultaet fuer Biowissenschaften, Pharmazie und Psychologie Universitaet Leipzig, 

Germany 

Since proteins play an essential role in numerous biological processes, many attempts 

have been made to study their interaction with their environment. In this context, FCS 

becomes more and more important. Usually proteins are detectable by fluorescence 

spectroscopy, via the amino acids Trp and Tyr or after labeling with a fluorescent dye. 

One main problem appears using conventional labeling procedures due to the unselective 

coupling (e. g. lysine-moities) of the dye to the protein molecules. In the approach 

described here specific enzymes were used to link one dye per protein selectively to the 

N-terminus of the target. Since we used the back reaction of chymotrypsin for selective 

labeling, the target protein had to show an N-terminal Arg for specific substrate 

recognition [1]. The target protein - in our case ribonuclease T1 – was modified first by 

recombinant techniques by adding an Arg at the N-terminus yielding the variant RG- 

RNase T1. For labeling, the fluorescent dye 6-carboxy-tetramethylrhodamine (TAMRA) 

was chemically linked to the tripeptide ester Gly-Pro-Tyr-OMe resulting in TAMRA-GPY- 

OMe. The fluorescent dye labeled tripeptide ester was then enzymatically ligated to the 

N-terminal Arg of the RG-RNase T1 variant. Purified TAMRA-GPY-RG-RNase T1 could be 

proven by FCS measurements. Furthermore the selective N-terminal labeling of RNase T1 

could be verified by the protease mediated cleavage of the fluorescent dye from the 

labeled protein. 

[1] Schellenberger, V., Turck, C. W., Hedstrom, L., and Rutter, W. J. (1993) Mapping the S´-Subsites of 

Serine Protease Using Acyl Transfer to Mixtures of Peptide Nucleophiles. Biochemistry 32, 4349-4353. 

- 30 -



FLUORESCENCE CORRELATION SPECTROSCOPY: TOWARDS MONITORING SIGNAL 

TRANSDUCTION PROCESSES IN VIVO 

M.A. Hink, J.W. Borst, G.N.M. van der Krogt J. Goedhart, T.W.J. Gadella Jr.and A.J.W.G. Visser 

MicroSpectroscopy Centre, Laboratories for Biochemistry and Molecular Biology, Wageningen University, 

Dreyenlaan 3, 6703 HA Wageningen, The Netherlands 

(e-mail: mark.hink@laser.bc.wau.nl) 

We report on the application of fluorescence correlation spectroscopy to analyze the 

dynamical behaviour of fluorescent molecules in various living cells. Since autofluorescent 

components can severely disturb the experiments, special attention must be paid to the 

choice of cell type, fluorescent probe and excitation and detection conditions. 

Spodoptera frugiperda (insect) cells were used to observe the diffusion of recombinant 

proteins, which were fused to mutants of green fluorescent protein. In addition, we have 

measured the diffusion of several fluorescent phospholipids and receptor proteins in the 

plasma membrane of these cells. One example of a signal transduction process in plants 

is the formation of nodules in legume roots. Nod factors, which are secreted by 

Rhizobium bacteria, are the key signalling molecules in this process. Using fluorescence 

correlation spectroscopy we have determined the distribution and dynamics of a 

fluorescent Nod factor in root hairs of Vicia sativa plants. Other examples of diffusion of 

green fluorescent protein derivatives in special tobacco protoplasts and suspension cells 

having a low level of autofluorescence, are presented. 

- 31 -



Synaptic Vesicle Dynamics Studied by Fluorescence Correlation Spectroscopy 

Randolf Jordan*, Jürgen Klingauf 

Max-Planck Institute for Biophysical Chemistry, Department of Membrane Biophysics, 

37077 Göttingen, Germany 

Little is known about the dynamics of synaptic vesicles within small mammalian 

synaptic boutons. In order to visualize the movements of single vesicles in 

hippocampal synapses in culture we adopted fluorescence correlation spectroscopy 

(FCS). After staining approx. 30 % of the vesicle pool with FM 1-43 by electric field 

stimulation a small volume fraction (• 0.05 fl confocal detection volume) of single 

identified boutons was imaged. In resting boutons we observed large fluctuations in 

fluorescence due to single vesicle movements with apparent diffusion constants 

between 0.5·10 -3 and 5·10 -3 µm 2 /s, when analysed by autocorrelation or Fourier 

transformation. 

To probe the involvement of active transport we applied the actin filament inhibitors 

Cytochalasin D (10 µM) or Latrunculin B (1 µM), the microtubule blocker Colchicine 

(10 µM), or ML-7 (15 µM), a potent inhibitor of myosin light chain kinase. In the 

presence of ML-7 we found frequencies between 0.07 to 0.7 Hz to be suppressed 

significantly in power. Cytochalasin D, Latrunculin B as well as Colchicine did not 

have clearly detectable effects. 

The influence of kinases and phosphatases in vesicle mobilisation was tested by 

application of Forskolin (10 µM), an adenylyl cyclase activator, or ocadaic acid (5 µM), 

a phosphatase blocker. Both lead to an increased vesicle mobility. 

Thus, our results suggest that vesicle movement/mobilisation in small synaptic 

terminals is caused both by diffusion and myosin-based transport and is under 

control of a variety of kinases and phosphatases. 

- 32 -



Two-Colour Fluorescence Correlation Spectroscopy of the Green Fluorescent Protein 

G. Jung, A. Zumbusch, C. Bräuchle 

Inst. f. Physikalische Chemie, LMU München 

Fluorescence correlation spectroscopy (FCS) is widely used for the analysis of the 

photophysical and chemical behaviour of dye molecules in the microsecond time range 

[1]. In the present study, we apply FCS with two-colour excitation for the investigation of 

the photochemistry of the Green Fluorescent Protein. The mutants that we investigate 

exhibit a single absorption peak corresponding to the anionic chromophore(class II, 

following the nomenclature of Ref.[2]). From bulk saturation experiment it is known that 

excitation (with λ 1 

= 476 nm or 496 nm) of these mutants leads to a photoproduct that 

correspond to the neutral chromophore. This state can efficientely be depopulated with a 

second excitation at λ 2 

= 407 nm. This two-colour effect is also seen in FCS of mutant 

E222Q where the additional illlumination reduces the fraction of dark molecules. 

However, even in the case of saturation of λ 2 

, some molecules remain in the dark state. 

The photodynamics of the molecule are therefore described within the framework of a 4- 

level system with two dark states, where the saturation with λ 2 

reduces the rate equation 

system to a 3-level system with one dark state [3]. The complete analysis of the twocolour 

FCS data leads to rate constants that allow the explanation of the observed 

behaviour in the two-coluor bulk saturation experiments. First results on other mutants 

will be presented. An outlook on how the results of FCS can be directly exploited to 

fluorescence imaging in biology will be givven. 

[1] J. Widengren et al., J. Phys. Chem. 99 (1995), 13368. 

[2] R. Y. Tsien, Annu. Rev. Biochem. 67 (1998), 509. 

[3] G. Jung et al., J. Phys. Chem. A 104 (2000), 873. 

- 33 -



FCS Analysis of an Organelle Specific Antibody 

Toshihide Kobayashi and Jean Gruenberg 

RIKEN Frontier Research System, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan, Department of 

Biochemistry, Sciences II, 30 quai E. Ansermet, 1211-Geneva-4, Switzerland 

Little is known about the structure and function of membrane domains in the vacuolar 

apparatus of animal cells. Endosomes in particular contain in their lumen a complex 

system of poorly characterized internal membranes. We find that internal membranes of 

late endosomes contain high amounts of a unique phospholipid, lysobisphosphatidic acid 

(LBPA), thus forming specialized domains within endosomes. These domains are involved 

in sorting and transport of both proteins and lipids from late endosomes. In the present 

study, we have further characterized the distribution of LBPA using monoclonal antibody 

(6C4) which specifically recognizes LBPA. When the 6C4 antibody was pre-treated with 

intact late endosomes, immunoreactivity was unaffected. In contrast, immunoreactivity 

was abolished when using late endosomes pre-disrupted by a freeze-thaw and sonication 

protocol, which yields a homogenous population of small vesicles derived from both 

internal and external membranes. 6C4 antibody microinjected into the cytoplasm did not 

stain late endosomes, whereas the antibody added to the medium was accumulatedinto 

late endosomes. Direct evidence that the antibody could bind an internal epitope, 

accessible after late endosome disruption, was obtained by correlation fluorescence 

spectrometry. Approximately 45 % fluorescently labeled 6C4 antibody was able to react 

with sonicated vesicles obtained from late endosomes, but not from other membranes. 

Size predictions derived from the diffusion constants confirmed that the antibody was 

then associated with vesicles with Mr. bigger than one million kD, in contrastto the free 

antibody (predicted Mr. 200 kD). Altogether, these experiments suggest that the 6C4 

epitope is present on late endosome internal membranes. 

Kobayashi et al. (1998) Nature, 392, 193-197 

Kobayashi et al. (1999) Nature Cell Biol. 1, 113-118 

Kobayashi et al. (2000) Mol. Biol. Cell in press 

- 34 -



Dual-color fluorescence cross-correlation spectroscopy and its application in 

biotechnology 

Andre Koltermann * , Ulrich Kettling, Thorsten Winkler, Jens Stephan and Manfred Eigen 

Dept. Biochemical Kinetics and * Work Group Experimental Biophysics, Max Planck Institute for biophysical 

chemistry, Göttingen, Germany 

Fluorescence-based assay technologies play an increasing role for research and diagnostic 

applications in life sciences. Precise and sensitive detection and even real-time monitoring 

of biological reactions as well as large number screening with low analysis time at high 

sensitivity - so-called high-throughput screening (HTS) - are demands to modern 

fluorescence-based assay technologies. These can be classified into: Fluorescence 

polarization (FP), time-resolved fluorescence (TRF), fluorescence resonance energy 

transfer (FRET), and fluorescence correlation spectroscopy (FCS). Classical applications of 

fluorescence spectroscopy detect emission which is co llected from comparatively large 

ensembles of fluorescent particles, i.e. the signal is averaged over space and time. In 

contrast to this, confocal fluorescence methods such as FCS restrict the probe volume to 

a tiny spot of less than one femtoliter, which is the size of a typical bacterial cell. The 

high spatial resolution can be accomplished by epi-illumination of a microscope objective 

with appropriate laser beams and co nfocal imaging of the collected emission photons 

onto a detector. A sufficiently high temporal resolution (down to the range of 

nanoseconds) is enabled through the use of avalanche photo diodes as sensitive singlephoton 

detectors in comb ination with suitable data collection systems. Employing 

fluorophore-labeled biomolecules at concentrations of nanomole per liter and below, on 

average less than one emitter resides in the femtoliter focal volume. The temporal 

behavior of these single molecules can be followed and underlying biomolecular 

reactions can be analyzed at the single molecular level [1]. 

Recently, the single-color FCS has been extended to a dual-color cross-correlation scheme 

which shows several advantageous characteristics[1,2]. Dual-color FCS allows the tracing 

of two spectrally distinguishable fluorophores at the same time, and the cross-correlation 

analysis of the two signals gives access to number and time constants of correlated 

fluorescence fluctuations in the different emission ranges. Co mpared to single-color 

autocorrelation measurements, signal specificity and accuracy is strongly improved by this 

new method, i.e. even at large excess of background fluorescence in each channel, dualcolor 

FCS enables specific detection of double fluorescent molecules. 

The typical assay format investigated by the dual-color cross-correlation method is based 

on the distinction between double-labeled and single-labeled molecules. The technique 

enables to examine preferably reactions that can be attributed to either formation or 

destruction of linkages between two fluorophore-tagged (molecular) fragments or 

association or dissociation between two fluorophore-tagged biomolecules. 


- 35 -



Its universality was demonstrated by a variety of biochemical reactions, for example 

binding studies by annealing of oligomeric DNA [2], or enzyme kinetic studies of 

catalyzed cleavage of DNA and polypeptides [3]. Dual-color FCS was furthermore 

adapted to screening applications and was termed Rapid Assay Processing by Integration 

of Dual-color Fluorescence Cross-correlation Spectroscopy (RAPID FCS) [4]. While 

conventional autocorrelation methods identify molecules by their diffusion properties, 

therefore requiring a considerable amount of analysis time, RAPID FCS simply counts 

double-labeled molecules. Data collection times for precise determination of molecular 

concentrations lay in the range of one second and below, corresponding to a throughput 

of up to 10 5 samples per day. 

Besides cross-correlation FCS, alternative algorithms for data processing of multi-color 

fluorescence signals emanating from single molecules are currently examined. Special 

attention is given to the extraction of accurate signals at shor test analysis times possible, 

thereby enhancing the throughput rate. Confocal Flu orescence Coincidence Analysis 

(CFCA) uses a coincidence algorithm. This approach was combined with technical 

improvements, like modifications concerning the laser source and a controlled external 

enhancement of concentration fluctuation. As a result, sampling times in the range of 

100 ms were achieved [5]. The presented achievements break the ground for throughput 

rates as high as 10 6 

samples per day using small amounts of sample substance and 

therefore constitute a solid base for screening applications in drug discovery and 

evolutionary biotechnology. 

[1] Eigen, M. and Rigler, R. Sorting single molecules: application to diagnostics and evolutionary 

biotechnology. Proc.Natl.Acad.Sci.USA 91(13):5740-5747, 1994. 

[2] Schwille, P., Meyeralmes, F.J., and Rigler, R. Dual-Color Fluorescence Cross-Correlation Spectroscopy for 

Multicomponent Diffusional Analysis in Solution. Biophys.J. 72(4):1878-1886, 1997. 

[3] Kettling, U., Koltermann, A., Schwille, P., and Eigen, M. Real-time enzyme kinetics monitored by dualcolor 

fluorescence cross-correlation spectroscopy. Proc.Natl.Acad.Sci.USA 95:1416-1420, 1998. 

[4} Koltermann, A., Kettling, U., Bieschke, J., Winkler, T., and Eigen, M. Rapid assay processing by 

integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme 

activity. Proc.Natl.Acad.Sci.USA 95:1421-1426, 1998. 

[5] Winkler, T., Kettling, U., Koltermann, A., and Eigen, M. Confocal fluorescence coincidence analysis: an 

approach to ultra high-throughput screening. Proc.Natl.Acad.Sci.USA 96(4):1375-1378, 1999. 

- 36 -



FCS measurements of phages displaying RNase T1 or RNase A 

Kerstin Korn 1 , Hans-Heinrich Foerster 1 , Rudolf Rigler 2 and Ulrich Hahn 1 

1 

Institut für Biochemie, Fakultät für Biowissenschaften, Universität Leipzig, Germany 

2 

Department of Medical Biophysics, Karolinska Institutet, Stockholm, Sweden 

With the aim to develop a new screening procedure for finding ribonuclease (RNase) T1 

variants with altered specificities, we fused the RNase T1 or the RNase A gene, 

respectively, to the 5’-site of gene III of the filamentous bacteriophage M13. This resulted 

in the presentation of these enzymes on the exterior surface of the phage particle. The 

phages were prepared with high purity [1,2]. 

RNase T1 catalyses the hydrolysis of single stranded RNA specifically on the 3’-site of 

guanosin residues (G). Since RNase A has a specificity for pyrimidine residues (C, U), it 

can be used to simulate RNase T1 variants with altered specificities compared to RNase 

T1 wildtype. A substrate molecule (a gapped heteroduplex containing a single stranded 

G) has already been designed and proven to be useful for the determination of RNase T1 

activity in FCS measurements [3]. The substrate used in the experiments described here 

was changed like that C, U and A, but no G were placed in the single stranded part. 

Hydrolysis of the rhodamine B-labelled substrate was determined by autocorrelation 

analysis. Due to the enzymatic cleavage, the autocorrelation signal changed after adding 

phages displaying RNase A, whereas phages displaying RNase T1 did not change the 

signal. These experiments showed the possibility for distinguishing between these two 

different phage species just by yielding pure yes-or-no decisions. 

[1] Hubner, B., Korn, K., Foerster, H.-H. & Hahn, U. (1997) Display of ribonuclease T1 on the surface of 

bacteriophage M13. Nucleosides Nucleotides 16, 727-732. 

[2] Korn, K., Foerster, H.-H. & Hahn, (2000) U. Phage display of RNase A and improved method for 

purification of phages displaying RNases. Biol. Chem. 381, 197-181. 

[3] Korn, K., Wennmalm, S., Foerster, H.-H., Hahn, U. & Rigler, R. Analyzing the RNase T1 mediated 

cleavage of an immobilized gapped heteroduplex via fluorescence correlation spectroscopy. Biol. Chem., in 

press. 

- 37 -



Development of a piezo driven xyz-translation stage for intracellular FCS 

B.H. Kunst 

MicroSpectroscopy Center, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. 

A user-friendly scanning device has been developed to scan cells in one-, two- and in 

three dimensions. At each scanning point, a fluorescence correlation spectrograph is 

recorded using the Confocor I. 

The scanning device (Tritor 3D 101SG, Piezosystem, Jena) has a maximum 

displacement of 80 µm with a reproducibility better than 60 nm in the feedback 

mode (using the integrated strain gauges). The in-house developed software (based 

on Labview, National Instruments Inc., Austin, Texas, USA) sends a start signal to 

the fluorescence correlation spectrograph (Confocor I, <strong>Zeiss</strong> Inc., Oberkochen). 

When the measurement ends, the stage is moved to a new position and a new 

measurement starts. 

The modular sample holder allows for rapid interchange of samples. To prevent cell 

movement, samples are embedded in 0.3% agarose. Presently one- and twodimensional 

scans are made. 

Our homepage: http://gcg.tran.wau.nl/local/MSC/ 

The author acknowledges STW for financial support (WBI. 4797) and Prof. Dr. A.J.W.G. Visser for 

leading the project. 

- 38 -



A strategy for highly parallel synthesis of tyrosine- and histidine-reactive labeling 

reagents 

E. Lopez-Calle, J. R. Fries*, D. Riester, D. Winkler, A. Wiesner, S. Dröge, H. Knorr, S. Viebrock, C. 

Winkler, G. Mielck, M. Wrobel 

Evotec Biosystems AG, Schnackenburgallee 114, 22525 Hamburg, Germany, 

Tel. ++49 40 560810, Fax. ++49 40 56081222, Email: joachim.fries@evotec.de, www.evotec.com 

Due to the extreme sensitivity of fluorescence, the application of highly developed 

fluorescence-based techniques like EVOTEC’s FCS+plus have become a powerful tool in 

assay development and HTS applications. Biological probes generally exhibit no or a low 

auto-fluorescence and thus it is necessary to incorporate fluorescent labels into such 

systems. The most common method for labeling is the modification of lysines or 

cysteines. However, in cases where lysines and cysteines are essential for biological 

activity or sterically not available, alternatives are required. The labeling of 

tyrosines/histidines by diazonium salts has proven to be a versatile method for the 

derivatization of proteins. The availability of these reagents, however, is strongly limited. 

Here we describe a method for the parallel solid phase synthesis of tyrosine-/ histidinereactive 

labels. After diazotization the resulting diazonium salt reacts with the aromatic 

system. This approach is also suitable for the generation of reagents for detection, 

purification and characterization of proteins. 

- 39 -



Measurement of the dynamic instability of microtubules by FCS experiments 

Tobias Neumann, Steffen Omar Kirchstein, Georg Bohnert, Juan Alberto Camacho Gomez 

Leonhard Kittler, and Eberhard Unger 

IMB Jena 

In cells the microtubule cytoskeleton plays an essential role in cell division and cell 

morphogenesis. Microtubules are formed by polymerization of •/• tubulin dimers. 

Inhibition or promotion of microtubule formation can be determined by turbidity 

measurements up to drug concentrations of 10 -8 M. Pharmacologically relevant drug 

concentrations are some orders of magnitudes lower. 

Microtubules are dynamic polymers that change stochastically between periods of 

growth and shrinkage, a property known as dynamic instability. Drugs like Taxol or 

vinblastine interfere with dynamic instability, prevent cells from reaching the 

checkpoints in mitosis and lead cells to switch to apoptosis. Until now the 

measurement of microtubule dynamic instability was a time-consuming process 

difficult to manage in a cost-effective screening assay. 

Using FCS-measurements as a new approach we demonstrate the influence of Taxol 

and other compounds onto the equilibrium between microtubules and tubulin 

dimers. By addition of tetramethylrhodamin-labeled tubulin dimers onto microtubule 

suspensions beeing in an equilibrium between microtubules and tubulin dimers we 

can measure the exchange rate between polymer and dimer. This rate can be 

regarded as a measure of dynamic instability of microtubules. The effect of the 

microtubule drugs could be demonstrated down to concentrations of 10 -10 M, thus 

providing the basis for a fast in vitro screening assay for tubulin binders at 

pharmacologically relevant drug concentrations. 

- 40 -



On the stator of rotary ATP synthase: The binding strength of subunit δ to (αβ) 3 

as 

determined by fluorescence correlation spectroscopy 

Oliver Pänke, Katrin Häsler and Wolfgang Junge 

Div. of Biophysics, Dept. Biology/Chemistry, Universität Osnabrück, D-49069 Osnabrück, Germany 

ATP synthase is presently conceived as a rotary enzyme. Proton flow drives the rotor, 

namely subunits c 12 

εγ, relative to the stator, namely subunits ab 2 

δ(αβ) 3 

, and extrudes 

spontaneously formed ATP from three symmetrically arranged binding sites on ( αβ) 3 

into 

the solution. We asked whether the binding of subunit δ to (αβ) 3 

is of sufficient strength 

to hold against the elastic strain which is generated during the operation of this enzyme. 

According to current estimates the elastically stored energy is about 50 kJ/mol. 

Subunit δ was specifically labeled without impairing its function. Its association with 

solubilized ( αβ) 3 

γ in detergent-free buffer was studied by fluorescence correlation 

spectroscopy (FCS). A very strong tendency of δ to dimerize in detergent-free buffer was 

apparent ( K d 

≤ 0.2 nM). Taking the upper limit of this figure into account, the 

dissociation constant between monomeric δ and (αβ) 3 

γ was 0.8 nM if not smaller. It is 

equivalent to a free energy of binding of at least 52 kJ/mol and therewith sufficient for 

the assumed hold-function of δ in the stator. Our data were compatible with a single 

binding site for δ on the hexagon of (αβ) 3 

. 

- 41 -



Spatial Correlation Spectroscopy 

Nils O. Petersen 

Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7, Canada 

Spatial correlation spectroscopy provides an opportunity to measure the spatial frequency 

as well as the occupation number on a surface or through an object in which the 

dynamics is either slow or non-exiting. In this presentation, the focus will be on the 

interpretation of the amplitude of the correlation function in the context of aggregation 

phenomena. Examples will be drawn from fluorescence measurements of molecules on 

cell surfaces as well as analysis of secondary ion mass spectrometry (SIMS) images. The 

concept of cross-correlation spectroscopy can also be used in the spatial domain. In this 

case, the cross-correlation function becomes a sensitive and quantitative measure of the 

co-localization of different molecules. In this presentation, examples will be provided 

from co-localization of cell membrane components as well as from co-localization of 

molecular or atomic species measured by SIMS. While the spatial correlation 

spectroscopy does not have any inherent dynamic information, it is possible to study the 

kinetics of processes that occur between distinct spatial measurements. The timecorrelation 

of pairs of images can therefore provide measured of slow diffusion or flow 

processes and, in principle, slow intermolecular interactions. A few examples from work 

on cell surfaces will be presented. 

- 42 -



FCS-analysis of ligand-receptor interactions in the membrane of cultured cells 

Aladdin Pramanik and Rudolf Rigler 

Dept. of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden 

Receptor binding studies on peptide/hormone ligands most often require the use of 

radioactively labeled ligands and include several washing steps to remove unbound 

ligands. In certain cases, the numbers of receptors are few per cell and no specific 

binding is detected because of high background. In addition, the half-life of the receptorligand 

complex is often shorter or similar to the time required for separations of free and 

bound ligands, respectively. Specific interactions between certain ligands (e.g. peptides, 

hormones, natural products) and their different receptor subtypes are, therefore, often 

overlooked by the conventional equilibrium binding technique. In FCS the small laser 

volume element (0.2 fl) allows the detection of single molecules as well as the 

measurement of molecular properties at specific coordinates in the cell membrane (Rigler 

et al. (1999) PNAS 96:13318-13323). FCS allows detection of the interaction of ligands 

with binding sites of receptors on the molecular level in their native environment on cell 

surfaces with single molecule detection sensitivity (Rigler et al. (1999) PNAS 96:13318- 

13323); Pramanik et al. (1999) Biomed. Chromatogr. 13:119-120; Boonen et al. (2000) 

Planta Med. 66:7-10). This technique permits the identification of receptors or target 

molecules which were not possible before to detect by isotope labeling. With FCS one 

can analyze a mixture of multiple ligand-receptor complexes possessing different 

molecular weights (M 1 

, M 2 

, M 3 

) and different diffusion times ( τ D1 

, τ D2 

, τ D3 

), respectively. 

The beauty of FCS technique is that there is no need for separating unbound ligand from 

bound one to calculate the receptor bound and free ligand fractions y 

1, 

y 2 

and y 3 

corresponding to M 1 

/τ D1 

, M 2 

/τ D2 

and M 3 

/τ D3 

, respectively or to calculate other parameters 

describing binding of the fluorescent labeled ligand. Thus, it is possible to perform largescale 

drug and/or active compound screening in cell cultures using FCS technique. 

To demonstrate receptor binding in the membranes of living cells we will report our 

studies conducted on interactions between the ligands galanin (GAL), a neuropeptide 

that displays a variety of important biological actions and is thought to be implicated in 

several human disorders such as Alzheimer’s disease, Depression and Feeding Disorders 

(Bartfai et al. (1993) Crit. Rev. Neurobiol. 7:229-274) and the epidermal growth factor 

(EGF) that plays a crucial role in the molecular network communication of physiological 

processes (Boonstra et al. (1995) Cell Biol. Int. 19:413-430; Zwick et al. (1999) Trends 

Pharmacol. Sci. 20:408-412). Both GAL and EGF have been labeled by a fluorophore 

tetramethyl rhodamine isothiocyanate (Rh) which provides the fluorescence signal in the 

interaction of the ligands with their respective receptors. The results demonstrate the 

presence of specific binding of GAL and EGF to their respective membrane-bound 

receptors in cultured cells. The specificity of the binding is attested by the consistent 

displacement of bound Rh-GAL and Rh-EGF following addition of 1,000 -fold molar 

excess of unlabeled GAL and EGF, respectively. 


- 43 -



The binding specificity of GAL is further confirmed when its binding was displaced by the 

GAL antagonist M40 whereas vascular EGF was unable to displace Rh-EGF binding, 

demonstrating no cross-reaction.Evidence for these specific interactions was verified by 

an equilibrium saturation binding experiment. Rh-GAL and EGF binding to the cell 

membranes are saturated at nanomolar concentration. The Scatchard plots show a 

binding process with K ass 

of 0.8·10 9 M -1 

for GAL and K ass 

of 1.5·10 9 M -1 for EGF. The 

dissociation kinetics follow a single exponential function characteristic for a relatively slow 

dissociation process with k diss 

= 3.7·10 -4 s -1 for GAL and k diss 

= 2.9·10 -4 s -1 for EGF. The 

appearance of two binding complexes through distribution of diffusion times may 

suggest that these are representations of two different forms or subtypes of GAL or EGF 

receptors. The fact that exposure of the cells to pertussis toxin interferes with the binding 

of GAL and EGF to their respective receptors considers an allosteric system (Monod et al. 

(1965) J. Mol. Biol. 12:88-118), involving at least two or possibly several conformational 

states of both the receptor and the G-protein. Finally, we have demonstrated the 

possibility to determine directly association rate constants for ligand-receptor interactions 

on the cell surface i.e. in the natural situation. This study is of pharmaceutical significance 

since it will provide insights that FCS can be used as a rapid technique for studying 

ligand-receptor interactions in cell cultures, which is one step forward for large-scale 

drug screening in cell cultures. 

1.Tjernberg, L., Pramanik, A., Björling, S., Thyberg, P, Thyberg, J., Nordstedt, C., Terenius, L., and 

Rigler,R., (1999) “Amyloid β-peptide polymerization studied by fluorescence correlation spectroscopy”. 

Chem. Biol. 6, 53-62. 

2. Pramanik, A., Juréus, A., Langel, Ü., Bartfai, T. and Rigler, R. (1999) “Galanin receptor binding in 

the membranes of cultured cells measured by Fluorescence Correlation Spectroscopy”. Biomed. 

Chromatogr. 13, 119-120. 

3. Rigler, R., Pramanik, A., Jonasson, P., Kratz, G., Jansson, O. T., Nygren, P.-Å., Ståhl, S., Ekberg, K., 

Johansson, B.-L., Uhlén, S., Uhlén, M., Jörnvall, H. and Wahren, J. (1999) “Specific Binding of Proinsulin 

C-Peptide to Human Cell Membranes”. Proc. Natl. Acad. Sci. U.S.A., 96, 13318-13323. 

4.Pramanik, A., Thyberg, P. and Rigler, R. (2000) “Molecular interactions of peptides with phospholipid 

vesicle membranes as studied by Fluorescence Correlation Spectroscopy”. Chem. Phys. Lipids 104, 35-47. 

5.Boonen, G., Pramanik, A., Rigler, R. and Häberlein, H. (2000) “Evidence for specific interactions between 

a kavain derivative and human cortical neurons measured by Fluorescence Correlation Spectroscopy”. 

Planta Med. 66, 7-10. 

6. Reznikov, K., Kolesnikova, L., Pramanik, A.,T., Tan-no, K., Gileva, I., Yakovleva., T., Rigler, R., Terenius, 

L., and Bakalkin, G. (2000). Clustering of apoptotic cells via bystander killing by peroxides. Faseb J., 000, 

000-000. 

7. Wahren, J., Ekberg, K., Johansson, J., Henriksson, M. Pramanik, A., Johansson, B.-L., Rigler, R., and 

Jörnvall, H. (2000) “Role of C-peptide in human Physiology”. A. J. Physiol., 278, 000-000. 

8. Pramanik, A. and Rigler, R. (2000) “FCS-assay of ligand-receptor interactions in living cells”. In: 

Fluorescence Correlation Spectroscopy. Theory and Applications (Elson, E. L. and Rigler, R., eds.). Springer- 

Verlag, in press. 

- 44 -



Interactions of mutant EGF receptors analysed by Fluorescence Correlation Spectroscopy 

(FCS) 

Jagdish Rao, Alastair Nicol, Peter Jordan, and Daniel Zicha 

NR6 cells (kindly provided by Alan Wells) and fluorescently labelled EGF were used in an 

FCS analysis of the mobility of mutant receptors. FCS can distinguish interactions 

between molecules of different sizes by measuring their diffusion times. Non-specific 

interactions required absence of free dye. Albumin also needed to be eliminated since it 

was introducing an unacceptable background signal. Measurements in live cells 

confirmed increased mobility of receptors possessing truncated cytosolic domains and 

indicated additional interactions after internalisation. 

- 45 -



Influence of solvent conditions and chaperones on Prion Protein aggregation 

Jens Schell, Oliver Schäfer, Jörg Tatzelt and Detlev Riesner 

Institut für Physikalische Biologie, Heinrich Heine Universität Düsseldorf; Department of Cellular 

Biochemistry, Max-Planck Institut für Biochemie, Martinsried; both Germany 

Prions are the causative agents of Creutzfeldt-Jakob disease in human, scrapie in sheep 

and bovine spongiform encephalopathy. They are largely composed of the pathogenic 

isoform, designated PrP Sc , of the cellular prion protein (PrP C ). In contrast to PrP C , PrP Sc and 

the N-terminal truncated form PrP 27-30, reveal a high content of β-sheet structure, are 

not soluble in mild detergents, partially resistant to proteolytic digestion and infectious. 

Recombinant (r)PrP and solubilized (s)PrP27-30 adjusted to 0.2 % SDS aquire properties 

reminiscent to that of PrP C , and after removing the SDS by dilution at neutral pH they 

assume structural features of PrP Sc . After a fast structural transition oligomeric multimers 

are formed within minutes to hours. In a slow process up to days large amorphous 

aggregates composed of these multimers are formed. 

These aggregation processes were analyzed by fluorescence correlation spectroscopy and 

differential ultracentrifugation experiments. The influence of solvent conditions and of 

the molecular chaperone GroEL were analyzed. 

The experiments revealed that molecular chaperones can inhibit the aggregation of PrP 

but were unable to productively fold PrP into a soluble form at neutral pH after release. 

If PrP is released from GroEL or GroEL/ES by addition of ATP, aggregation of PrP can be 

observed. The details of this aggregation process were different as compared to that in 

the absence of GroEL. Significant differences in respect to the proteolytic stability of 

these aggregates were not found so far. Ultrastructural alterations are under 

investigation. 

Chemical chaperones as glycerol, DMSO or TMAO did not inhibit the PrP aggregation, 

but they affected the intermediate states of the aggregation process. 

- 46 -



Investigation of aptamer/moenomycin interaction by FCS 

Heike Schürer 1 , Andrej Buchynskyy 2 , Kerstin Korn 1 , Peter Welzel 2 and Ulrich Hahn 1 

1 

Institut für Biochemie, Fakultät für Biowissenschaften, 

2 

Institut für Organische Chemie, Fakultät für Chemie und Mineralogie, Universität Leipzig, Germany 

The antibiotic moenomycin is the only known inhibitor of the enzyme which catalyzes 

transglycosylation, one of the last steps in peptidoglycon biosynthesis of the bacterial cell 

wall [1]. The transglycosylation reaction is a highly promising target for the design of new 

antibiotics. The moenomycins inhibit this reaction due to binding to the enzyme. They 

represent interesting lead compounds for novel therapeutics. In vitro selection technology 

(SELEX: systematic evolution of ligands by exponential enrichment) is a promising tool 

for identifying oligonucleotide sequences from large random sequence libraries that bind 

to a variety of molecular targets with high affinity and specificity [2]. Using the SELEXtechnology 

we selected 2’-NH 2 

-modified RNA molecules which bind to moenomycin with 

high affinity. 

Binding studies of aptamers with moenomycin using fluorecsence correlation 

spectroscopy (FCS) have shown, that FCS represents a new and highly sensitive method 

for the investigation of aptamer/target interactions. For the FCS measurements a 

tetramethylrhodamine-labelled analogue of moenomycin was used [3]. Binding of 

aptamers to moenomycin* by formation of RNA-moenomycin* complexes was identified 

by a change of the autocorrelation signal. Due to the differences of diffusion times of 

free moenomycin* (75µs) versus moenomycin*/aptamer complex (400µs) we were able 

to calculate the percentage of the formed complex. Detailed analysis of these 

aptamer/moenomycin interactions are still in progress. 

In summary, FCS technology will be a powerful tool for the fast screening of 

aptamer/target interactions and the use of FCS will eliminate the handling of 

radioactively labelled aptamers. 

[1] For leading references, see El-Abadla, N., Lampilas, M., Hennig, L., Findeisen, M., Welzel, P., 

Müller, D., Markus, A. and Heijenoort, J. (1999) Tetrahedron 55, 699-722. 

[2] For reviews on in vitro selection, see Tuerk, C. and Gold, L. (1990) Science 249, 505-510; Ellington, 

A. D. and Szostak, J. W. (1990) Nature 346, 818-822; Famulok, M. (1999) Curr. Opin. Struct. Biol. 

9, 324-329. 

[3] Unpublished results. 

- 47 -



Molecular dynamics in cells and on membranes: new challenges for fluorescence 

correlation spectroscopy 

Petra Schwille 

AG Experimentelle Biophysik, MPI für biophysikalische Chemie, Göttingen 

Intracellular applications of fluorescence correlation spectroscopy (FCS) promise direct 

access to a large variety of molecular parameters like concentrations, particle mobility 

and internal dynamics, rates of association, dissociation, recognition or enzyme activity in 

the cytosol, membranes, and organelles on the single molecule level. However, most 

applications to date have been performed in aqueous buffer solution, while accessing the 

cellular environment bears several difficulties in experimental performance, like 

photobleaching and long-term depletion of dye resources, as well as enhanced 

background, e.g. by scattering and cellular autofluorescence. Beyond that, exposure to 

intense light as required for high signal-to noise ratios in FCS may induce severe 

photodamage resulting in malfunction of the cellular machinery. We show that these 

difficulties can be overcome by proper instrumentation and design of the experiments, 

e.g. employing multiphoton excitation. Applying both one- and two-photon excitation 

(1PE and 2PE), several examples are given for intracellular diffusion and active transport 

in tubular structures, as well as internal dynamics of fluorescent particles such as GFP 

(green fluorescent protein) in mammalian and plant cells through diffraction limited focal 

spots with a spatial resolution of ca. 0.4 µm 3 . Mobility parameters can be measured over 

a wide range of characteristic molecular residence times from about 10 -3 to 10 3 ms with 

data collection times of 10-100 s. Deviations from normal diffusion of fluorescent probes 

that may be explained by interaction with cellular structures are found in various cell 

compartments. Translational membrane diffusion in live RBL (rat basophilic leukemia) cells 

was studied on single lipid analogs and IgE receptor proteins and also showed significant 

deviations from Brownian motion possibly due to spatial restrictions. Giant unilamellar 

vesicles were employed as model membrane systems in order to characterize diffusion of 

fluorescent lipid analogs in different lipid phases, their coexistence clearly distinguished 

by multiphasic curve shapes of the autocorrelation function, with diffusion coefficients 

ranging from 10 -7 to 10 -11 cm 2 /s. These results on model membrane systems may 

implicate phase heterogeneity as an explanation for the anomalous subdiffusion 

phenomenon on cell surfaces. From our experience with different applications of 

intracellular FCS we conclude that although both excitation alternatives work well in thin 

preparations like single cells, 2PE can substantially improve signal quality in turbid 

preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE 

furthermore minimizes long-term photobleaching of spatially restricted dye resources 

which is an essential advantage if single molecule experiments or measurements at ultralow 

concentrations are considered. In the cell lines observed, background 

autofluorescence did not seriously inhibit intracellular FCS with both 1PE and 2PE. 

Schwille P., Haupts U., Maiti S., and Webb W.W. (1999) 

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and twophoton 

excitation, Biophys. J. 77(4):2251-2265 

- 48 -



A statistical analysis of fluorescence fluctuation data with rare events 

E. Van Craenenbroeck, G. Matthys, J. Beirlant and Y. Engelborghs. 

Laboratory of Biomolecular Dynamics, University of Leuven, Celestijnenlaan 200 D, B3001 Leuven, Belgium. 

A new statistical method is proposed to analyse fluorescence correlation spectroscopy 

(FCS) measurements that cannot be evaluated with a classical autocorrelation 

function [1]. It applies to binding studies where one of the interacting particles has a 

much brighter fluorescence intensity with respect to the other, which causes high 

fluorescence bursts whenever these molecules are detected. This biases the 

autocorrelation function, making it in most cases impossible to use this function as a 

fitting equation. 

Here, a statistical approach is used to quantify the amount of fluorescence found in 

bursts, thereby enabling to perform binding studies in cases where the fluorescence 

per molecule of both interacting species differs greatly. The method is demonstrated 

on a system of known composition, making it a promising tool for future FCS 

measurements. 

The analysis presented here can be considered complementary to the photon 

counting histogram (PCH) method introduced by Chen and coworkers [2] or FIDA as 

presented by Kask et al. [3]. Unlike PCH, the method can be applied in conditions 

where the fluorescence bursts are rare events. PCH on the other hand is more 

convenient to analyze systems in which the interacting particles show a small 

difference in fluorescence intensity. 

E. Van Craenenbroeck, G. Matthys, J. Beirlant and Y. Engelborghs, ´A statistical analysis of fluorescence 

correlation data´ J Fluoresc 9 (1999) 325. 

Y. Chen, J.D. Muller, P.T.C. So and E. Gratton, ´The photon counting histogram in fluorescence fluctuation 

spectroscopy´ Biophys J 77 (1999) 553. 

P. Kask, K. Palo, D. Ullmann, and K. Gall . Fluorescence-intensity distribution analysis and its application in 

biomolecular detection technology . Proc. Natl. Acad. Sci. (USA) (1999) 96, 13756-13761. 

- 49 -



Fluorescence Correlation Spectroscopy of Flavins and Flavoproteins 

Antonie Visser, Petra van den Berg, Mark Hink, Valentin Petushkov 

MicroSpectroscopy Centre, Department of Agrotechnology and Food Sciences, Laboratory of 

Biochemistry, Wageningen University Dreijenlaan 3, 6703 HA Wageningen, The Netherlands 

Fluorescence correlation spectroscopy has been applied to the free flavin cofactors 

FMN and FAD and riboflavin (vitamin B2) and to some highly fluorescent 

flavoproteins functioning as antenna proteins in bacterial bioluminescence. The 

experimental conditions have been optimized to obtain autocorrelation traces, which 

can be interpreted on the basis of photophysical and structural characteristics. Both 

FMN and riboflavin undergo instanteneous photobleaching, which is manifested by a 

distinctly reduced particle number. From the ratio of FAD- and FMNautocorrellograms 

a sub-microsecond dynamic effect can be observed, which is 

originating most likely from the stacking and unstacking of the two aromatic moieties 

in FAD. The autocorrelation traces of antenna proteins reflect translation diffusion 

only at relatively low laser intensity. Higher excitation intensity induced irreversible 

photodissociation of the flavin ligand. 

- 50 -



The Characterization of a Transmembrane Receptor Protein by Fluorescence Correlation 

Spectrosocpy 

Thorsten Wohland, Ruud Hovius, and Horst Vogel 

Many receptor proteins play an essential role in cell functioning and are important targets 

for therapeutic agents. Fluorescence Correlation Spectroscopy (FCS) is a suitable 

technique to investigate molecular interactions with and between these receptors and 

thus to elucidate receptor function. The advantage of FCS lies in its wide dynamic range 

in the time and concentration domain, its sensitivity, and thus in the small amounts of 

material needed. The characteristic time of the processes that can be measured spans 

more than eleven orders of magnitude, between several nanoseconds up to hours; the 

measurable concentration range extends from 0.1 nM to several µM. 

Here we report on experiments to determine concentrations and the translational 

diffusion coefficients by FCS to investigate the properties of the 5-hydroxytryptamine 

(=serotonin) receptor of type 3 (5HT 3 

-R) in vitro and in vivo. The 5HT 3 

-R is a 

homopentamer, consisting of subunits of a relative molecular mass of 54 kDa. This 

neuroreceptor functions as a ligand-gated ion channel, which influences among other 

things anxiety and depression in human beings. It is therefore a good example for a 

pharmacologically important membrane receptor. 

First, ligand binding to detergent-solubilized 5HT 3 

-R was measured using fluorescent 

antagonists. These experiments yielded directly values for the equilibrium constants and 

the stoichiometry of the binding reactions as well as on the molecular mass of the 

purified receptor protein. Interestingly, the homopentameric receptor binds only one 

antagonist; this is an important result which cannot be obtained easily and directly by 

other methods. 

We then proceeded to characterize the diffusion of the 5HT 3 

-R in transiently transfected 

HEK293 cells. FCS measurements over the whole cell body could distinguish specifically 

between labeled receptors in different cellular compartments. The receptors show a wide 

range of diffusion constants probably reflecting the inhomogeneous composition of the 

plasma membrane, but their diffusion is in all cases much slower than diffusion of 

hydrophobic membrane probes. At membrane areas of high receptor concentrations 

strong photobleaching can be seen indicating that the receptor is immobile or very slowly 

diffusing, possibly arranged in clusters with reduced mobility. 

This work shows that FCS delivers important information of molecular interactions on a 

cellular level. The measurable concentrations as well as the time and spatial resolution lie 

well inside the range of biological interest. 

- 51 -

Analyse 

Molecular Interactions 

Efficiently: 

A Nanoliter 

is Enough 

Ideal for solutions 

Miniaturization, as everywhere, is on the 

advance in biochemistry. ConfoCor 2, the 

fluorescence correlation microscope, now 

provides a direct measurement method for 

characterizing molecular interactions in nanoliter 

assays. It yields the ratio of free and 

bound molecules in a homogeneous, freesolution 

assay comprising a single step. 

Results are obtained within seconds. 

Ideal inside cells 

The LSM 510, the confocal laser microscope, 

will show you the location of labeled 

molecules. The ConfoCor 2 will help you to 

understand how these molecules interact. 

The measurement volume, not bigger than 

an E.coil bacterium, easily fits into a cell. So 

biochemical processes can be investigated 

in their natural environment. Interested 

Contact us for details! 

<strong>Carl</strong> <strong>Zeiss</strong> · Microscopy · D-07740 Jena 

Phone ++49-36 41-64-16 16 · Fax -64-3144 

USA (800)2 33-23 43 · Japan (03)33 55-03 41 

micro@zeiss.de · www.zeiss.de/micro

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