Slide 1 t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Week 5 Live Cell.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Week 5 Live Cell Imaging in Confocal Microscopy Multiphoton Microscopy Spectral Imaging BME 695Y / BMS 634 Confocal Microscopy: Techniques and Application Module Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine & Department of Biomedical Engineering, Schools of Engineering These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose. A useful text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures in these lecture notes are taken from this text or of the WEB.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Lecture Summary 1. Live cell confocal microscopy 2. Live cell applications and examples 3. Multiphoton microscopy 4. Spectral Imaging

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Applications Organelle Structure Probe ratioing Conjugated antibodies DNA/RNA Cytochemical Identification Oxidative Metabolism Exotic Applications Organelle Structure & Function –Mitochondria (Rhodamine 123) –Golgi(C6-NBD-Ceramide) –Actin (NBD-Phaloidin) –Lipid (DPH)

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Specific Organelle Probes BODIPY Golgi505511 NBD Golgi488525 DPH Lipid350420 TMA-DPH Lipid350420 Rhodamine 123 Mitochondria 488525 DiOLipid488500 diI-Cn-(5)Lipid550565 diO-Cn-(3)Lipid488500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Organelle Function MitochondriaRhodamine 123 EndosomesCeramides GolgiBODIPY-Ceramide Endoplasmic ReticulumDiOC 6 (3) Carbocyanine

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Calcium Related Applications Probe Ratioing –Calcium Flux (Indo-1) –pH indicators (BCECF, SNARF) Molecule-probeExcitationEmission Calcium - Indo-1351 nm405, >460 nm Calcium- Fluo-3488 nm525 nm Calcium - Fura-2363 nm>500 nm Calcium - Calcium Green488 nm515 nm Magnesium - Mag-Indo-1351 nm405, >460 nm Phospholipase A- Acyl Pyrene351 nm405, >460 nm

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Probes for Ions INDO-1 E x 350E m 405/480 QUIN-2E x 350E m 490 Fluo-3 E x 488E m 525 Fura -2E x 330/360E m 510

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Ionic Flux Determinations CalciumIndo-1 Intracellular pH BCECF How the assay works: Fluorescent probes such as Indo-1 are able to bind to calcium in a ratiometric manner The emission wavelength decreases as the probe binds available calcium Flow CytometryImage Analysis

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Oxidative Reactions SuperoxideHydroethidine Hydrogen PeroxideDichlorofluorescein Glutathione levelsMonobromobimane Nitric Oxide DAF-FM (4-amino-5- methylamino-2',7'-difluorofluorescein)

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University DCF DCFH-DA DCFH DCF COOH H Cl O O-C-CH3 O CH3-C-O Cl O COOH H Cl OH HO Cl O COOH H Cl O HO Cl O Fluorescent Hydrolysis Oxidation 2’,7’-dichlorofluorescin 2’,7’-dichlorofluorescin diacetate 2’,7’-dichlorofluorescein Cellular Esterases H2O2H2O2 DCFH-DADCFH-DA DCFH DCF H O 2 2 2 2 Lymphocytes Monocytes Neutrophils log FITC Fluorescence.1.1 100 0 100 10 1 0 20 40 60 counts PMA-stimulated PMN Control 8080

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Hydroethidine HE EB N CH 2 CH 3 NH 2 H2NH2N H Br - N CH 2 CH 3 NH 2 H2NH2N + O2-O2- Phagocytic Vacuole SOD H2O2H2O2 NADPH NADP O2O2 NADPH Oxidase OH - O2-O2- DCF HE O2-O2-O2-O2- H2O2H2O2H2O2H2O2 DCF Example: Neutrophil Oxidative Burst

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Hydrogen peroxide measurements with DCFH-DA % change (DCF fluorescence) 525 nm 12 3 4 5 Step 6B: Export data from measured regions to Microsoft Excel Step 7B: Export data from Excel data base to Delta Graph Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Superoxide measured with hydroethidine Export data from Excel data base to Delta Graph Export data from measured regions to Microsoft Excel cell 1 cell 2 cell 3 cell 4 cell 5 Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis. %change (DCF fluorescence)

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University H 2 O 2 stimulation and DCF & EB loading in Rat Pulmonary Artery Endothelial Cells ENDO HBSS ENDO HBSS TNFa ENDO L-arg ENDO/ L-arg TNFa ENDO/ D-arg ENDO/ D-arg TNFa Endo + 200uM H2O2 Endo / TNFa + 200uM H2O2 Endo / L-arg + 200uM H2O2 Endo / L-arg TNFa + 200uM H2O2 Endo / D-arg + 200uM H2O2 Endo / D-arg TNFa + 200uM H2O2 0 20 40 60 80 100 120 140 160 180 200 020406080100120140 Time (minutes) Mean EB Fluorescence. 200uM H 2 O 2 added Time (seconds) DCF Fluorescence Confocal System - Fluorescence Measurements 200uM H 2 O 2 added 24 treatments - 5000 cells each

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University pH Sensitive Indicators SNARF-1488575 BCECF488525/620 440/488525 [2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein] ProbeExcitationEmission

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Exotic Applications of Confocal Microscopy FRAP (Fluorescence Recovery After Photobleaching) Release of “Caged” compounds Lipid Peroxidation (Parinaric Acid) Membrane Fluidity (DPH)

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University “Caged” Photoactivatable Probes Ca ++ : Nitr-5 Ca ++ - buffering: Diazo-2 IP 3 cAMP cGMP ATP ATP-  -S Available Probes Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at 340-350 nm

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Time (seconds) after UV FLASH Release of Caged Nitric Oxide in Attached PMN 0 50 100 150 200 250 020406080100120140160 Fluorescence Emission at 515 nm Release of Caged Compounds C D UV excited Control Region Time (seconds) CONTROL 0 50 100 150 200 250 CONTROL STUDY Fluorescence Emission at 515 nm 0100200300400

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Membrane Polarization Polarization/fluidityDiphenylhexatriene How the assay works: The DPH partitions into liphophilic portions of the cell and is excited by a polarized UV light source. Polarized emissions are collected and changes can be observed kinetically as cells are activated. An image showing DPH fluorescence in cultured endothelial cells.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University 1 2 3 3 2 1 405/35 nm 460 nm Calcium ratios with Indo-1 Changes in the fluorescence were measured using the Bio-Rad calcium ratioing software. The same region in each wave length was measured and the relative change in each region was recorded and exported to a spread sheet for analysis.. Export data from measured regions to Microsoft Excel Export data from Excel data base to Delta Graph 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 cell 1 cell 2 cell 3 Ratio: intensity1 (460nm) / intensity2 (405/35nm)

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University FRAP Intense laser Beam Bleaches Fluorescence Recovery of fluorescence 10 seconds 30 seconds Zero time Time %F

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Imaging 3D ECM structures Mainly collagen based materials Usually 40-120 microns thick Require both transmitted and fluorescent signals Often require significant image processing to extract information

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Thick Tissue - Bone and Cartilage Very difficult to image thick specimens Can use live specimens if appropriately stained Special preparation techniques

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University History Developed in 1961 by Kaiser and Garret A process unknown in Nature except in stars Can be reproduced in a laser beam whereby more than one photon can be absorbed by a molecule in a short time The energy of both photons is summed in a way similar to that of a photon of shorter wavelength, but the emission is almost identical to that of a single photon

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Energy states in 2-photon Note that the end result is essentially the same for 1 photon and 2 photon. The emission is the same in both cases.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Advantages of 2 Photon  Longer observation times for live cell studies  Increased fluorescence emission detection  Reduced volume of photobleaching and phototoxicity. Only the focal-plane being imaged is excited, compared to the whole sample in the case of confocal or wide-field imaging.  Reduced autofluorescence of samples  Optical sections may be obtained from deeper within a tissue that can be achieved by confocal or wide-field imaging. There are three main reasons for this: the excitation source is not attenuated by absorption by fluorochrome above the plane of focus; the longer excitation wavelengths used suffer less Raleigh scattering; and the fluorescence signal is not degraded by scattering from within the sample as it is not imaged.  All the emitted photons from multi-photon excitation can be used for imaging (in principle) therefore no confocal blocking apertures have to be used.  It is possible to excite UV flourophores using a lens that is not corrected for UV as these wavelengths never have to pass through the lens.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University 2-Photon Excitation The sample is illuminated with a wavelength of twice the wavelength of the absorption peak of the fluorochrome being used. For example, in the case of fluorescein which has an absorption peak around 500 nm, 1000 nm excitation could be used. Essentially no excitation of the fluorochrome will occur at this wavelength and hence no bleaching will occur in the bulk of the sample. A high-powered pulsed laser is required with has a peak power of >2Kw Power should be in pulses shorter than a picosecond (so that the mean power levels are moderate and do not damage the specimen) Two-photon events will occur at the point of focus give above conditions The photon density is sufficiently high that two photons can be absorbed by the fluorochrome essentially simultaneously. This is equivalent to a single photon with an energy equal to the sum of the two that are absorbed. Thus, fluorochrome excitation will only occur at the point of focus This eliminates unnecessary phototoxicity as there is little excitation out of the plane of focus Image quality is excellent as there is practically no out-of-focus interference.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University 3-Photon Microscopy Advantages  UV fluorophore excitation without UV irradiation  Similar resolution to 2 photon excitation of UV fluorophores Three-photon excitation can also be used in certain circumstances. In this case three photons are absorbed simultaneously, effectively tripling the excitation energy. Using this technique, UV excited fluorophores may be imaged with IR excitation. Because excitation levels are dependent on the cube of the excitation power, resolution is improved compared to two photon excitation where there is a quadratic power dependence. It is possible to select fluorophores such that multiple labeled samples by can be imaged by combination of 2- and 3 photon excitation, using a single IR excitation source.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Limitations of 2-Photon  Slightly lower resolution with a given fluorochrome when compared to confocal imaging. This loss in resolution can be eliminated by the use of a confocal aperture at the expense of a loss in signal.  Thermal damage can occur in a specimen if it contains chromophores that absorb the excitation wavelengths, such as the pigment melanin.  Only works with fluorescence imaging.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Why 2-photon is very specific Fluorescence from the two-photon effect depends on the square of the incident light intensity, which in turn decreases approximately as the square of the distance from the focus. Because of this highly nonlinear (~fourth power) behavior, only those dye molecules very near the focus of the beam are excited. The tissue above and below the plane of focus is merely subjected to infrared light that causes neither photobleaching nor phototoxicity. Although the peak amplitude of the IR pulses is large, the mean power of the beam is only a few tens of milliwatts, not enough to cause substantial heating of the specimen.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Multi-Photon Fluorescence Microscopy The experimental benefits of multi-photon excitation: Localized excitation provides high spatial resolutionLocalized excitation Inherent z-axis resolution improves sensitivity and three-dimensional optical sectioningInherent z-axis resolution Reduced photodamage/ photobleaching Increased penetration depth in specimen Provides selective excitation of fluorophores by two and three photonsProvides selective excitation of fluorophores Increased detection sensitivity of fluorophores by reducing autofluorescence or background Elimination of confocal aperture Applications for multi-photon microscopy are: In-vivo and in-vitro imaging Fluorescent Lifetime Imaging Optical Tomography Imaging Semiconductor Imaging http://www.microcosm.com/tutorial/tutorial.html

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Instrumentation Typical Instrumentation for Multi-Photon Time- Resolved Microscopy: Femtosecond, Picosecond or CW Lasers Near Infra-Red Optics coated for high peak power lasers Special Dichroics for Multiphoton Excitation Laser Scanning Microscope optimized for Infra-Red high peak power lasers Time-Resolved Instrumentation for Imaging Dichroic For Two-Photon Excitation Dichroic For Three-Photon Excitation Wavelength (nm) http://www.microcosm.com/tutorial/tutorial.html

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Comparison Between Confocal and Two-Photon Detection Confocal one-photon excitation imaging compared with two-photon imaging in scattering tissue. Due to the longer wavelength, less excitation light is lost to scattering when using two-photon excitation. Ballistic and diffusing fluorescence photons can be used in the two-photon case, but only ballistic photons can be used in the confocal case. In multi-photon excitation more fluorescence photons are detected from a focal point than from a confocal method. Ref. W. Denk, J. Biomedical Optics (1996) 1(3), 296-304. Ballistic photons are non-scattering photons. http://www.microcosm.com/tutorial/tutorial.html

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University 2-photon Vs single photon (confocal) From Current Protocols in Cytometry Online Copyright © 1999 John Wiley & Sons, Inc. All rights reserved. Photo from Brad Amos The cuvette is filled with a solution of a dye, safranin O, which normally requires green light for excitation. Green light (543 nm) from a continuous-wave helium-neon laser is focused into the cuvette by the lens at upper right. It shows the expected pattern of a continuous cone, brightest near the focus and attenuated to the left. The lens at the lower left focuses an invisible 1046-nm infrared beam from a mode-locked Nd- doped yttrium lanthanum fluoride laser into the cuvette. Because of the two-photon absorption, excitation is confined to a tiny bright spot in the middle of the cuvette.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Comparison of One-Photon Excitation vs. Two-Photon Excitation One- Photon Excitation One-Photon and Two-Photon Excitation images were obtained by CW 5 mW Laser at 442 nm. (Recent findings indicate that 2-photon can be obtained with high power CW lasers) and Ti:sapphire laser at 800 nm respectively. Two-photon excitation exhibits localized excitation, the inherent advantage which accounts for the improved resolution available with this method. Two- Photon Excitation http://www.microcosm.com/tutorial/tutorial.html

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Lasers and Probes Pulsed laser source of 1047 nm which can excite most blue and red, and some green emitting fluorophores. BLUE EMITTING: AMCA, Hoechst 33342, Hoechst 33258, DAPI GREEN EMITTING: Oregon Green 514, red-shifted GFP, JC-1, FITC, Ca Green ORANGE EMITTING: Calium Orange, Mitotracker Rosamine, Rhodamine 123, FM4-64 RED EMITTING: Nile Red, Calcium Crimson, TRITC, Texas Red, DiI, PPI, CY-3

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University http://www.microcosm.com/tutorial/tutorial.html Z-axis Resolution in 3-Photon and 2-Photon Excitation Comparing the signals obtained when moving the focus from the cover glass into (a) BBO/ toluene and (b) Rhodamine 6G / immersion oil layer. This compares the axial resolution of a three-photon and two-photon microscope, respectively. The excitation wavelength is 900 nm. No confocal spatial filtering is used. The steeper signal in (a) shows the higher axial resolution of three-photon excitation microscopy. The z-axis represents the focal point in the experiment. Ref. Stefan Hell........ (1995)

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Ref. J. R. Lakowicz and I. Gryczynski, "Topics in Fluorescence Spectroscopy", volume V, Plenum Press, 1997 Calcium dependent emission spectra of Indo-1 for one-, two- and three-photon excitation at 295, 590 and 885 nm, respectively. The results suggest that the relative cross-section for three-photon excitation of Indo-1 is less for the Ca 2+ - bound form, as compared to relative cross-section for two- photon. Hence the calcium bound or free form of Indo- 1 can be selectively sense by two- or three-photon excitation respectively. Selective Detection of Fluorophores in Multi-Photon Excitation http://www.microcosm.com/tutorial/tutorial.html

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Examples DNA tagged fluorescence image without using an UV source. The DAPI stained nuclei were excited with the Nd:YLF pulsed laser (1047nm) via three-photon excitation. (349 nm) Images from: Multi-Photon Excitation Fluorescence Microscope Coordinator, Madison, WI 3-photon image of a DAPI stained Caenorhabitis elegans worm

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Sequence of images showing a comparison between confocal imaging (488nm excitation) and 2-photon imaging (1047nm excitation). The sample is a zebra fish that is heavily stained with safranine (the sample was prepared by B. Amos). As can clearly be seen, 2-photon imaging is able to give much better images deep into the specimen. Comparison of confocal and 2-photon imaging (JPEG-100K) Comparison of confocal and 2-photon imaging (JPEG-100K) Images from: Multi-Photon Excitation Fluorescence Microscope Coordinator, Madison, WI 1047 nm 488nm

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Comparison of XZ images taken by confocal and 2-photon imaging. The images were obtained by sequentially scanning a single horizontal XY line while stepping the focus into the specimen. The sample is a safranine stained zebra fish (prepared by B. Amos). The 2-photon system (left) is able to reveal structural information in regions where nothing can be seen in the confocal system (right). Comparison of XZ images of confocal and 2-photon imaging (JPEG-25K) Comparison of XZ images of confocal and 2-photon imaging (JPEG-25K) Images from: Multi-Photon Excitation Fluorescence Microscope Coordinator, Madison, WI 2-photonconfocal

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Double labeled 3t3 cell in anaphase showing microtubules (Green FITC) and actin staining (red rhodamine phalloidin). This is a fixed specimen and is included to demonstrate that double labeling is possible with the 1047 nm excitation wavelength used in the 2-photon imaging system. Double labeled 3t3 cell in anaphase showing "green" microtubules and "red" actin staining (JPEG-15K) Double labeled 3t3 cell in anaphase showing "green" microtubules and "red" actin staining (JPEG-15K) Images from: Multi-Photon Excitation Fluorescence Microscope Coordinator, Madison, WI

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University 2-photon -Bacterial Studies An other example of the use of two-photon excitation microscopy is the imaging of Dental Biofilm. It consists of various aerobic and anaerobic bacteria embedded in a matrix of polysaccharides and proteins and can reach thicknesses of several hundred micrometers. The pH is an important property of the biofilm with respect to the effect on dental enamel. Using a carboxy-fluoresceine staining the pH of the biofilm was monitored after the addition of sucrose. The lifetime of the probe is sensitive to the local pH. Calibration of the fluorescence lifetime in biofilm at several pH values allows a determination of the local pH in the measured images. http://www.phys.uu.nl/~wwwmbf/ResJV.htm In the image (right) a fluorescence intensity image of biofilm is shown. Several types of bacteria can be distinguished.

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University In the above image a fluorescence intensity image of biofilm is shown. Several types of bacteria can be distinguished. Below (left) another intensity image of biofilm is shown. After supplying the biofilm with a sucrose solution the bacterial metabolic activity increases which results in the production of H+. The fluorescence lifetime images before (middle) and 70 minutes after the addition of sucrose (right) show a clear drop in pH. Here, the lifetime range in the images runs from pH 6.5 (black) to pH 2 (white). http://www.phys.uu.nl/~wwwmbf/ResJV.htm

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Spectral Imaging Increasing the number of spectral channels collected Allows more advanced classification systems Takes more time to image Much more complex analysis

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Multispectral microscopy – Not more colors!!! Color image Multispectral image Greyscale image Expansion/rebirth of the Landsat Concept from the 1970s

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Lyot filter (static)Single bandpass LCTF (randomly tunable) 400 450 500 550 600 650 700 750 400450500550600650700750 Measured center wavelength (nm) Wavelength “dialed-in” High precision and accuracy Enabling Technology: Liquid tunable filters Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University High-resolution cytology segmentation Conventional RGB Image Spectrally segmented Image Wavelength (nm) Characteristic Spectra High spectral resolution increases utility of spectrally responsive indicator dyes Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Multispectral Imaging – Zeiss Meta Ability to select a range of wavelengths As desired by the user

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Nuance-Micro Slide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com

t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Lecture Summary Live cell applications are relatively common using confocal microscopy Correct use of fluorescent probes necessary Temperature and atmosphere control may be required Thick specimens often require advanced image processing Exotic applications are potentially useful A limited window of time is available to image live cells before cells deteriorate 2-photon microscopy can penetrate greater tissue depth 2-3 photon has advantages for excitation of lower wavelengths (UV) 2-photon is very complex technology 2-photon is very expensive Possibly the future replacing confocal imaging Spectral Imaging will be next major change in biological imaging

Slide 1 t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Week 5 Live Cell.

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Slide 1 t:/classes/BMS 602B/lecture 5 602_B.ppt Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson, Purdue University Week 5 Live Cell.

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