Research in Microfluorimetry

By Martin vandeVen

INTRODUCTION:

The microfluorimetry section of the physiology group of the Biomedical Institute of the University Hasselt is situated in Building D on the University Campus, http://www.uhasselt.be/english/algemeen/how_to_reach.asp . Facilities adjacent to cell growth and electro-physiology laboratories comprise 40 m2 of dedicated, air-conditioned spaces (3 in total) with controlled access.  They are used by a host of European university and government researchers as well as by commercial enterprises.  The latter ones on a contract basis channeled through the External Relations Office (UHasselt, interface-dienst, phone: +32-(0)11-268014, fax: +32-(0)11-268019, email: an.debacker@uhasselt.be http://www.uhasselt.be/onderzoek/interfacedienst/default.asp). 

AVAILABLE EQUIPMENT:

 Three (3) air-conditioned rooms with ozone removal equipment and controllable light levels:  

Lamps:

• Line spectrum Hg lamps

• Broad band Xe and Tungsten lamps

Lasers:

• Water cooled Argon ion laser, Spectra Physics model Beamlock 2020-03, 458, 488, 514 nm, 1.5 W all lines.

• Small frame forced air cooled 25 mW Argon ion laser

• Red Diode laser (on loan from the Biology Dept.) Coherent Fab laser, 650 nm.

• Gre-Ne laser , 543 nm, 1 mW

• He-Ne laser 633 nm, 5 mW

Microscopes:

• Nikon inverted microscope model TMD-35 with Xe-lamp, Sutter 10 position excitation filterwheel and 30 f/s CCD camera, suitable for 37 degr. cell physiology experiments.

• Zeiss Axiovert 100 inverted microscope with Xe-lamp, Sutter 10 position excitation filterwheel and 30 f/s CCD camera, suitable for 37 degr. C. cell physiology experiments

• Zeiss inverted Laser Scanning Confocal Microscope 510 META with spectral acquisition on an automated Axiovert 200M stage.  Equipped with Sutter 10 position excitation filterwheel and temperature regulated perfusion box.  Suitable for 37 degr. C. cell physiology experiments.

 

Nikon TMD-35 inverted microscope	Fura-2 intracellular Ca concentration imaging in confluent MDCK cells. Courtesy of Dr. I. Smets
Zeiss Axiovert 100 inverted microscope	Human OligodendroGlioma cells. Courtesy of Dra. E. Gielen
Zeiss inverted Laser Scanning Confocal Microscope LSM 510 META on Axiovert 200M frame	LSM 510 META confocal microscope with cell physiology peripheral equipment

Stage Scanning Phase Fluorimeter:

• Multi-frequency Phase Fluorimeter model K2 ISS Inc. for frequency domain fluorescence intensity decay measurements with two Hamamatsu R928 photomultiplier detectors.

Raman setup:

• Jobin-Yvon (Spex) H250 monochromator with DataScan software (on loan from Dr. M. Nesladek, IMO/IMOMEC)

RECENT PUBLICATIONS:

1. Wenmackers, S.; Haenen, K; Nesládek, M.; Wagner, P.; Michiels, L.; vandeVen, M. and Ameloot, M.  Covalent immobilization of DNA on CVD diamond films.  Phys. Stat. Sol. (a) 199, 1, 44-48, 2003.

2. Zamai, M; vandeVen, M.; Farao, M.; Gratton, E.; Ghiglieri, A.; Castelli, M. G.; Fontana, E.; d’Argy, R.; Fiorino, A.; Pesenti, E.; Suarato, A. and Caiolfa, V. R.  Camptothecin Poly[N-(2-Hydroxypropyl) Methacrylamide] Copolymers in Antitopoisomerase-I Tumor Therapy: Intratumor Release and Antitumor Efficacy.   Molecular Cancer Therapeutics Vol. 2, 29-40, 2003

3. Smets, I.; Caplanusi, A.; Despa, S.; Molnar, Sz.; Radu, M.; vandeVen, M.; Ameloot, M.; and Steels, P.  Ca2+  uptake in depolarized mitochondria is mediated via the reversed action of the mitochondrial Na+/Ca2+ exchanger in metabolically inhibited MDCK cells.  Am J Physiol Renal Physiol 286: F784–F794, 2004

RESEARCH:

Using natural intrinsic and man-made fluorophores the microfluorimetry section specializes in carrying out basic research on the changes in pH, membrane potential, intracellular ion concentrations.  Living and fixed cells and tissues are studied as well as  mitochondria under normal and stress conditions like hypoxia and apoptotic processes.  The research takes place within the LUC campus-wide framework of research on autoimmune diseases like Multiple Sclerosis and Rheumatic Arthritis and it complements immuno-histological and MRI research efforts. This methodology is also important to reveal the interactions of heavy metals with vesicular trafficking, an important focus of our eco- and human toxicological division. Cells studied include A6, COS7, HeLa, MDCK, and HOG cells.  Impaired transport of vesicles and rafts in normal and diseased Human Oligodendrocyte cells is visualized with a range of fluorescence methodologies. Theoretical models are developed to describe the observations.  

LSCM, Myelin and Multiple Sclerosis Research

The white matter of the brain, central nervous system and spine (myelin) encloses the nerves. This myelin sheet is formed and maintained by cellular extensions socalled processes of a special type of cel: the oligodendrocyt, Figure 3a.  When the myelin is damaged electrical impulses are no longer properly conducted along the nerves.

People with Multiple Sclerosis have locally damaged myelin.  These damage zones are called plaques.  Since they occur at  various spots and moments the disease carries the name Multiple Sclerosis. Myelin destruction is caused by a local inflamatory reaction and is accompanied by the dead of the oligodendrocyte. For more effective treatment a better insight in the origin of this process is required. 

Because confocal microscopy allows optical sectioning the localisation and dynamical processes of a range of interesting functional and structural proteins labeled with fluorophores can be monitored in living oligodendrocytes under various circumstances. To this end brain and central nervous system cells are grown at BioMed and thick tissue coupes from plaques are studied to understand the dead of oligodendrocytes under controlled and reproducible conditions e.g. the addition of cytokines.

Figure 3b,c  show LSCM images of glial cells in primary culture made visible via fluorophore labeled antibodies against the protein skeleton of the cells.   

Figure 3a

Figure 3b

Figure 3c

Figure 3a Schematic Representation of an Oligodendrocyte

Figure 3b displays in false color an astrocyte (rat primary culture) labeled with an antibody against Glial Fibrillary Acid Protein (GFAP).  Courtesy Dr. F. Vandenabeele, Image size 230x230 µm  

Figure 3c shows a group of spindle-like oligodendrocyte precursor cells (O-2A) marked with a labeled antibody against the protein Nestin. O-2A cells are precursors for astrocytes and oligodendrocytes. When stress is applied to these cells the expression of certain genes is followed by tracking the amount and localization of Green Fluorescent Protein (GFP). Image size 140 x 140 µm.  

The influence of heavy metals like Cadmium on the cellular metabolism is monitored by confocal microscopy in collaboration with the Center for Environmental Studies, CMK, UHasselt, http://www.uhasselt.be/cmk/    

Biosensor Development and Evaluation

Concurrently microfluorimetry is used to complement and optically characterize sensor and biosensor designs and components based on thin and thick layer polymers, Figure 4b, and diamond substrate, Figure 4a, with modified surface properties.  These designs being bio – and haemo compatible allow the direct electronic readout of the presence of extreme low concentration marker molecules in body fluids, http://www.imo.uhasselt.be/ .   

Figure 4a

Figure 4b

Figure 4c

Figure 4a Diamond grain boundary and bulk fluorescence, image size 920x920 µm. Courtesy of Dra. S. Wenmackers, IMO/IMOMEC. 

Figure 4b thick PPV conjugated polymer film morphology. Courtesy of Drs. R. Dams, Chemistry Dept.  Figure 4c spincoated thin film MDMO-PPV fluorescence, courtesy of Drs. P. Cooreman IMO/IMOMEC.  

Environmental Biotic and A-Biotic stress research

For Biology the influence of gene expression patterns due to biotic and a-biotic stress in plant tissue is followed via confocal and macro imaging as well as the spread of GFP-labeled plant bacteria specially detrimental for the locally important fruit growing industry and apple and pear tree varieties, their leaves and flowers.   

Figure 5a

Figure 5b

Figure 5c

Figure 5a  Starting Erwinia Amylovora infection in leaf veins.  Courtesy of Dra. K. Heyens, CMK. 

Figure 5b  Chlorophyll emission of the same leaf. 

Figure 5c  Infected flower parts. Courtesy of Dra. M. Thoelen, CMK.  Image sizes 920 x 920 µm.

Development of a Compact Space Microscope

Within the framework of a Belgian consortium consulting on and testing of a compact breadboard design microscope for the space environment.  AMME: Advanced Microscopy MEthods – General Support Technological Program, ESA.

Planned Expansion of Instrumentation Capabilities

In addition the development and implementation of new prioritized fluorescence techniques in the following areas:

• Total internal reflection microscopy to study the behavior and properties of thin, ~ 100 nm thick, interface layers of substrate attached immobilized cells and biosensor surfaces.  

• Fluorescence and image correlation spectroscopy for elucidating diffusional and transport phenomena in living cells  

• Implementation of polarization measurement capabilities for rotational dynamics studies of fluorophore labeled proteins and enzymes  

• The conversion of the stationary single-point multi-frequency phase and modulation fluorimeter to the confocal microscope  

• Raman confocal spectrometry to complement the META design for gaining a better insight in the bulk properties of diamond and the influence of grain boundaries and impurities  

• Implementation of an exchangeable CCD camera system on the confocal microscope  

• Implementation of laser light excitation on the Zeiss Axiovert 100 inverted microscope setup

Development of robust automatic image and data processing routines in collaboration with:

• University of Genoa, Physics Department, Italia  

• Laboratory of Fluorescence Dynamics, University of Illinois at Urbana-Champaign, USA. 

• Computer and Biology Department of Turku University, Finland.  

BACKGROUND INFORMATION:

Fluorescence can be observed as longer wavelength, red-shifted optical emission light.  The phenomenon occurs  when a fluorochrome is illuminated with shorter wavelength monochromatic lamp or laser light matching its absorption properties.  On an atomic or molecular level this means that electrons of the fluorochrome ground state gain energy upon absorption of the proper light color and reach an excited state.  This excited state electron returns to the ground state under the emission of longer ( = redder) wavelength photon.  The difference between excitation and emission peaks is called the Stokes shift.  As shown in Figure 6 the excited state can be reached with one blue photon and is then called single-photon excitation (1PE) or with 2 red excitation photons (two photon excitation, 2PE) or even more photons (multi-photon excitation).    

Simple Jablonski eergy dagram for one-photo (blue) and two-photon (red) excitation

STOKES SHIFT: Absorption efficiency and fluorescence emission intensities are dependent on the excitation wavelength

Figure 6a,b Origin of Fluorescence 

http://www.zeiss.de/C12567BE0045ACF1/Inhalt-Frame/252C0056834A910B41256A7100455639

Selection of just the fluorescence emission, Figure 6a,b, occurs with optical filters optimized for each fluorophore.  With the advent of sensitive CCD cameras and slow scanning XY-stages or fast scanning light beams, http://www.celanphy.sci.kun.nl/Bruce%20web/scanning%20microscopy.htm images can be collected indicating not only the temporal but also the equally important spatial and bulk variation in fluorescence signal.  With the addition of a bright Xenon lamp and a computer controlled excitation filter wheel a range of excitation wavelengths can be selected to match the fluorophore absorption properties.  A thermo-electrically Peltier cooled CCD camera allows the collection of very weak fluorescence emission.  Rotation of the excitation filter wheel allows the collection of ratiometric images for determination of for example intracellular Calcium concentrations, Smets, I.; Caplanusi, A.; Despa, S.; Molnar, Zs.; Radu, M.; vandeVen, M.; Ameloot, M.; and Steels, P. Am J Physiol Renal Physiol 286: F784–F794, 2004

Figure 7 a

Figure 7b

Figure 7c

Figure 7a  Madin-Darby Canine Kidney (MDCK)cells at 37°C with mitochondria visualized via mitotracker green dye  

Figure 7b MDCK cells labeled with Calcium concentration indicator dye Rhodamine-2  

Figure 7c MDCK cells similar to 2b after treatment with pore-forming ionomycin. Image size 70 x 70 µm.

Apart from the observation of slowly varying temporal and spatial spectral fluorescence intensities, also molecular orientational dynamics is monitored by studying the linear polarization properties of the fluorescence emission.  Faster msec and µsec kinetics and transport phenomena are followed via Fluorescence and Image Correlation and Cross-Correlation Spectroscopy.  Fluorescence emission properties on a psec and nsec time scale are monitored with fsec and psec optical pulse or modulated light excitation and provide an insight in the influence of the local environment on the decay of fluorescence intensity and changes in rotational dynamics.  

Confocal Laser Scanning Microscopy (CLSM).  Cells and tissues can be studied when immobilized and fixed, but observing their functioning under normal physiological conditions for example 37 degrees C and proper pH necessitates the use of small containers with an optically transparent bottom filled with buffer solution placed on a temperature controlled stage of an inverted microscope.  With one-photon laser excitation an hour-glass volume is illuminated.  This volume emits fluorescence in all directions.  This emission consists of the usually bright fluorophore superimposed on a autofluorescence background caused by cellular components.  A small pinhole (the confocal aperture) placed in front of the detector allows to limit the observed femto liter volume ( e.g. 0.5 x 1 µm high) to the focus spot which means complete elimination of fluorescence contribution from layers above and below the focus plane resulting in a crisp image but at the same time at the expense of a sharp reduction in intensity thereby justifying the use of laser excitation lightsources.  A scanning mirror assembly, Figure 8, makes the typical µm size diffraction-limited laser illumination spot scan a square area. Pixel dwell time are typically in the > µsec range.  This means that a 512x512 Horizontal x Vertical pixel image ( = one (1)  image frame) scanned with a 4 µsec pixel dwell time is collected in 1 sec.  By raising or lowering the objective a stack of regularly spaced images can be obtained. This is the main advance of a laser scanning confocal microscope: non-destructive imaging of live cells and tissues.    

Layout of a LSCM with infinity corrected optics

Optical sectioning on living cells, 3D mitochondrial distribution in MDCK cells.  Stack of 40 images, image size 140 x 140 µm

Figure 8 LSCM with Infinity Corrected Optics http://www.zeiss.com/C12567BE0045ACF1/Inhalt-Frame/E107868046B1D34841256A73003F785A

LIST OF ABBREVIATIONS: (as used in the text hereafter)

TUTORIAL SITES, MATERIALS AND METHODS

Colocalization

http://www.ph.tn.tudelft.nl/~lucas/education/EMBO/2002/delft-tanke_%20day5_handouts.pdf   slide 15

Confocal Microscopy

http://www.bmi2.bmt.tue.nl/vital-imaging/Extensions/viu.htm  Vital Imaging Dr. M. van Zandvoort, UM

Cytometry

http://www.ph.tn.tudelft.nl/~lucas/education/EMBO/2002/delft-tanke_%20day5_handouts.pdf

Diffusion, Rafts

http://www.biophys.leidenuniv.nl/Research/FvL/   TSLesHouches2001_3.pdf  T Schmidt Leiden 

FCS, FCCS

ICS, ICCS

Scanning FCS

http://kentlink.kent.edu/record=b2835736    Overview

http://www.drbio.cornell.edu/FCS_FPR/FCS/FCS_principles.html

http://www.ph.tn.tudelft.nl/~lucas/education/EMBO/2002/delft-tanke_%20day5_handouts.pdf  slide 30,31

http://www.biophysics.org/btol/img/petra-schwille.pdf

http://www.pci.uni-heidelberg.de/pci/fpraktikum/ws00/ws000112.pdf  FCS Deutsch

http://www.lfd.uiuc.edu/staff/chen/yanthesis.pdf

http://lfd.uiuc.edu/staff/ruan/thesis.pdf

FLIM

http://www.ph.tn.tudelft.nl/~lucas/education/EMBO/2002/delft-tanke_%20day5_handouts.pdf  slide 25-26

FRAP  /  FLIP  /  FLAP

http://www.eur.nl/fgg/pathol/research/mcb/frap.htm  Houtsmuller, EUR

http://www.eur.nl/fgg/pathol/research/mcb/flip.htm

http://www.drbio.cornell.edu/Infrastructure/FPR.html

http://www.ph.tn.tudelft.nl/~lucas/education/EMBO/2002/delft-tanke_%20day5_handouts.pdf  slides 12-13, FLAP 14

FRET

http://www.kcci.virginia.edu/FRET-FLIM/index.php

http://www-cellbio.med.unc.edu/facilities/fret.htm  Principle

http://www.kcci.virginia.edu/FRET-FLIM/process/index.php  FRET data analysis

http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/cf_tut_part2-6d.htm

http://www.ph.tn.tudelft.nl/~lucas/education/EMBO/2002/delft-tanke_%20day5_handouts.pdf  slide 17-21

http://www.m-boersch.org/PDF/single_molecule_spectroscopy-05.pdf  SMD FRET

http://lfd.uiuc.edu/staff/sophie/Sophie_thesis.pdf 

FRET-FLIM

http://www.ph.tn.tudelft.nl/~lucas/education/EMBO/2002/delft-tanke_%20day5_handouts.pdf  slide 22-24, 29

Single Particle Tracking

http://www-cellbio.med.unc.edu/dept/facilities/sptm.htm

http://www.focusonmicroscopy.org/2003/abstracts/115-Ritchie.pdf

http://people.ccmr.cornell.edu/~uli/Pages/nanobiotech3.html

Polarization

http://www.biophysj.org/cgi/content/full/83/3/1631

http://www.biophysj.org/cgi/content/full/79/1/536

Optical Tweezers

http://www.atsweb.neu.edu/mark/APL/Optical%20Tweezers.pdf

Optical Traps

http://members.yline.com/~tweezers/opticalstretcher.pdf  

Rotational Dynamics

http://www.m-boersch.org/PDF/single_molecule_spectroscopy-04.pdf

SHG

http://www.drbio.cornell.edu/Infrastructure/NonlinearMicroscopies_WWW/SHG.htm

http://www.physio.espci.fr/shg.pdf

http://www.biophysj.org/cgi/content/full/82/1/493  

SMD

http://www.bphys.uni-linz.ac.at/bioph/res/sdt/publications/mmb00.html

http://www.biophysics.org/btol/single.html

http://www.biophysik-dresden.de/research.html  Schwille, Dresden

http://www.biophys.leidenuniv.nl/Research/FvL/  Schmidt, Leiden

http://www.m-boersch.org/  tutorial

TIRF

http://micro.magnet.fsu.edu/primer/java/tirf/pandsintensities/index.html

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/tirfhome.html

http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfhome.html

http://www.olympusmicro.com/primer/java/tirf/reflect/

http://www.olympusmicro.com/primer/java/tirf/prismmorph/

http://micro.magnet.fsu.edu/primer/java/tirf/highnaobjective/index.html

http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html

http://www.embl-heidelberg.de/ExternalInfo/stelzer/pdf/rohrbach00.pdf

TIR(F) - FCS  N. Thompson

http://www.biophysj.org/cgi/content/abstract/43/1/103  several recent references

Two Photon Excitation

http://www1.phys.uu.nl/wwwmbf/ with an example of a 2-photon setup.

Multiphoton Excitation

http://www.drbio.cornell.edu/MPE/mpe.html

http://www.drbio.cornell.edu/Infrastructure/Infrastructure%20Index.html Multiphoton excitation.  Prof. Dr. Watt Webb, Cornell University

http://www.microscopy.fsu.edu/primer/resources/multiphotonweb.html  

FLUORESCENCE RESOURCES:

Young Investigators Awards

http://lfd.uiuc.edu/

http://www.iss.com/Resources/weber.html

Confocal Microscopy Tutorials

http://micro.magnet.fsu.edu/primer/virtual/confocal/index.html

http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/cf_tut_part2-6c.htm very extensive

http://www.cyto.purdue.edu/flowcyt/websites/cytsites/sitescon.htm  general

Fluorescence Centers USA [what about the center in Baltimore – lakowicz]

http://www.ncrr.nih.gov/ncrrprog/btdir/Laser.asp

http://lfd.uiuc.edu/

Fluorescence Courses

http://fluorescence-foundation.org/  Genoa

http://www.picoquant.com/trfcourse.htm  SMD  Berlin

http://www.cci.virginia.edu/workshop/workshop2004/index.php  FRET

http://www.probes.com/resources/courses.html  not kept up ?

Fluorescence Optical Filters

http://www.fluorescence.com/tutorial/fm-optic.htm

http://www.chroma.com/

http://www.omegafilters.com/

http://www.microscopyu.com/tutorials/java/noiseeater/  cubes

http://www.olympusmicro.com/primer/virtual/fluorescence/ cubes

Fluorescence Organizations

http://tango01.cit.nih.gov/sig/home.taf?_function=main&SIGInfo_SIGID=40

Fluorescence Probe Data

http://www.synthegen.com/products/fluorescent/table.lasso

http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/laser-line.pdf

http://microscopy.bio-rad.com/fluorescence/fluorophoradata.htm

Fluorescence Products

http://www.fluorescence-resource.com/  general

http://www.probes.com/resources/sites/  general

http://fluoreszenzanalytik.de/flu.htm  general

Fluorescence Protocols

http://www.probes.com/resources/sites/protocols.html

Fluorescence Image Deconvolution Software

http://powermicroscope.fisica.unige.it/  Site often not up

Fluorescence Image Analysis

http://rsb.info.nih.gov/ij/  freeware

http://bij.isi.uu.nl/vr.htm  freeware

Fluorescence Image Processing

Image Registration

http://rsb.info.nih.gov/ij/plugins/  freeware

http://bigwww.epfl.ch/thevenaz/turboreg/

Fluorescence Data Analysis Sites

http://lfd.uiuc.edu/  Globals  http://fms.physics.uiuc.edu/Lfd/Globals/lead.html

http://www.kcci.virginia.edu/FRET-FLIM/process/index.php  FRET

Fluorescence Tutorials

http://micro.magnet.fsu.edu/primer/java/scienceopticsu/jablonski/index.html

http://fmrc.pulmcc.washington.edu/DOCUMENTS/FMRC299.pdf

http://isb.epfl.ch/LCPPM/BiophysicsI/TIPS_Fluoresc.pdf 

Image Processing Tutorials

http://www.ph.tn.tudelft.nl/Courses/FIP/noframes/fip.html  Young

Microscopy Tutorials

http://micro.magnet.fsu.edu/primer/virtual/virtual.html

Spectroscopy Tutorials

http://www.nuigalway.ie/chem/AlanR/ARyderP9.html

http://www.jobinyvon.com/jy/oos/oos1.htm  Lerner and Thevenon

Upcoming Conferences

http://lfd.uiuc.edu/