Confocal Microscopy and Living Cell Studies Soňa Leqartová Institute of Biophysics of the Czech Academy of Sciences BJ9393 Table 7.1 Different Types of Llight Microscopy: A Comparison Type of Microscopy Brightfield {unstained specimen) Passes light directly through specimen; unless cell is naturally pigmented or artificialJy stained, image has little contrast. Brightfield (stained specimen). Staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved). Light Micrographs of Human Cheek Epithelial Cells Type of Microscopy Phase-contrast. Enhances contrast in unstained cells by amplifying variations in density within specimen; especially useful for examining living, un pigmented cells. D ifferen [ n I'm i u- rfrren ce-oo tit rast (Nomarski). Like phase-contrast microscopy, it uses optical modifications to exaggerate differences in density Fluorescence. Shows the locations of specific molecules in the cell. Fluorescent substances absorb short-wavelength, ultraviolet radiation and emit longer-wavelength, visible light. The fluorescing molecules may occur naturally in the specimen but more often are made by tagging the molecules of interest with fluorescent molecules. Confbcal. Uses lasers and special optics for "optical sectioning." Only those regions within a narrow depth of focus are imaged. Regions above and below the selected plane of view appeal" black rather than blurry. This microscope is typically used with fJuorescently stained specimens, as in the example here. Copyright© Pearson Education, Inc., rxjMiahing as Benjamin Cummingg https://inhabitatxom/5-bioluminescent-species-that-light-up-the-world/bioluminescent-fungus-2/ Introduction to Fluorescence http://rstl.royalsocietypublishing.org/content/142/463.full.pdf+html Sir George Gabriel Stokes (1819-1903) a British physicist and mathematician YELLOW-GLASS OF WINE EMISSION FILTER TRANSMITS * 400nm 6 G STOKES BLUE -6LASS IN CHURCH WINDOW EXCITATION FILTER t 400nm If! Stokes shift H absorption emission wavelength Lakowiczetal., 2006 Ishikawa-Ankerhold et al., 2012 Introduction to Fluorescence Perrin-Jablonski diagram (1935) Aleksander Jabtohski (1898-1980) https://vwwv.researchgate.net/Perrin-Jablonski-diagram-The-vibrational-manifold-associated-with-elect • ground state (singlet S0) • vibrational relaxation • internal conversion (IC) -> the lowest singlet state (S:) • intersystem crossing (ISC) -> triplet state (T:) Introduction to Fluorescence Aequorea victoria GFP-derived mRFP1 -derived Evolved by SHM -*-, ,-^-j^Srv Ext 3SO 433/451 488 516 4a7/so4 540 548 554 568 574 587 595 596 605 590 rim Em 440 475/505 509 529 537*562 553 562 581 585 596 610 620 625 636 648 nm f111*1111111111 TI Ti TI ^ a =J ^ 3 3 Di to wüj = 3. Q. fU « O 3, S (6 10 3 2 2 =1 q •The Nobel Prize in Chemistry 200S Osamu Shimomura, Martin Chalfie, Roger Y. Tsien Share this: The Nobel Prize in Chemistry 2008 Photo: U. Montan Photo: U. Montan Photo: U. Montan Osamu Shimomura Martin Chalfie Roger Y. Tsien Prize share: 1/3 Prize share: 1/3 Prize share:'.O The Nobel Prize in Chemistry 2008 was awarded jointly to Osamu Shimomura, Martin Chalfie and Roger Y. Tsien "for the discovery and development of the green fluorescent protein, GFP". Photos: Copyright ©The Nobel Foundation https://www.nobelprize.org/nobeljDrizes/chemistry/laureates/2008/ http://photobiology.info/Zimmer.html Introduction to Fluorescence Fluorophores • chemical compounds: re-emit light upon light excitation absorb light (a particular wavelength) —► transiently excited —► return to ground state Ishikawa-Ankerhold et al., 2012 contain several combined aromatic groups, or plane or cyclic molecules with several tt groups not all energy is emitted as fluorescence, some is dissipated as heat or vibrational energy M.inncfir. : *M rnt ii-ti: SlBl I- ■■ (VFP I \" ,-'— Hh^jJTi-1#Li M I Hilt LnS7S Carl Zeiss Microjmaging GmbH http://www.atdbio.com/content/34/Alexa-dyes Photoconversion Dendra2: improved green to red photoswitchable fluorescent protein • derived from octocoral Dendronephthya sp. (Gurskaya et al., 2006) • low phototoxicity monitoring selective cell fate real-time tracking protein dynamics (movement, degradation, etc.) H4-Dendra2 3 • • • • * 9* 9 0 0 * * Cvackova etal., 2009 Resolving power of microscopes 6 Copyright. 2012, university of warkato, All Rights Reserve«. www-sciencel earn. org. nz History of Microscopy: Fiŕít git of term "microscope" 1850-1873 Abbe equation f M f"i|:liUi ="■-■■ II! 1956, 1957- 1978, 1986 - 1990, 1991, 1993, 1994 > 2002 - 2006, 2008 - 2014 I'll I I I I / I I 1 : : Oplkdl microst ope for use. by physicians during eye surgery Contocal principle Čariaca I laser scanning NůbelPriZtr Multipficton rniicnoíCúpv 5TED/SIM Super-resolution microscopy 50 OCT PALM FPALM 5TORN Cherntitry Prite CFP OfT h CŕrpjriJítry Hobe) Prite I Robert Hoofce microscope (1665) - 7«.. iiyj .•is í ť ÍL Marvin L. Minsky (1927-2016) The Nobel Prize in Chemistry 2014 Eric Betzig, Stefan W. Hell, William E. Moerner Share this: The Nobel Prize in Chemistry 2014 Photo: A. Mahmoud Eric Betzig Prize share: 1/3 Photo: A. Mahmoud Stefan W. Hell Prize share: 1 (I Photo: A. Mahmoud William E. Moerner Prize share: 1 /I The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy". Numerical Aperture (NA) ability to gather light and resolve fine specimen detail at a fixed object distance http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html • most oil immersion objectives —> a maximum numerical aperture of 1.4 • the most common numerical apertures ranging from 1.0 to 1.35 Numerical Aperture (NA) Application Magnification Special Design Properties Lens Image Distance/ Coverslip Thickness (mm) Numerical Aperture/ Immersion Medium Working Distance Color-Coded Ring for Magnification Magnification IX 11 3X 4X 10X 20X 40X 60X 100X Color Code Black Gray Red Yellow Green Light Blue Light Blue Dark Blue White https://www.edmundoptics.com/resources/application-notes/microscopy/understandin Numerical Aperture (NA) The Abbe diffraction limit http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html 2n sin a The Abbe diffraction limit Resolved Rayleigh Limit Nat Resolved https://wwwJeica-microsystems.com/science-lab/microscope-resolution-concepts-factors-and-calculation/ https://phys.org/news/2016-09-quantum-mechanics-technique-rayleigh-curse.html ant hair ABBE'S DIFFRACTION LIMIT (0.2 urn] mammalian cell bacterium mitochondrion virus protein small molecule 1 mm 100um lOum 1 urn 100 nm 10 nm http://www.kurzweilai.net/the-nobel-prize-in-chemistry-2014-beyond-the-diffraction-limit-in-microscopy 1 nm The Abbe diffraction limit Confocal Microscopy Dec 19, 1961 Marvin L. Minsky (1927-2016) • basic concept of confocal microscopy (1950s) • advances in computer technology M. MINSKY MICROSCOPY APPARATUS Filed Nov. 7. 1957 3,013,46 FIG. 3. INVENTOR. /MAR.VIN MIN$KV Light A mplification S timulated E mission R adiation IIP https://escooptics.com/blogs/news/what-is-the-international-day-of-light • coherent monochromatic liqht (stimulated emission of photons from excited atoms or molecules) COHERENT HI ^ ' ft V NON-COHERENT ,. ji ff / 1 mm ^ it Detector Pinhole Aperture iFluorescenee-Barrier Filter In-Focus Light Rays Dichromatic-Mirror Out-of-Focus Light Rays Objective Photomultiplier Detector , m 0 Laser Scanning Confocal Microscope| Optical Configuration Laser Excitation Source I Light Source "Pinhole Aperture ~I Focal —I-Planes Figure 2 Specimen' FIG. 3. INVENTOR. MARVIN MINSKV ATTof}Me.i-S^ I http://fluoview.magnet.fsu.edu/theory/confocalintro.html 1. Laser Excitation Source 2. Reflected through dichroic mirror 3. Into lens (Objective) 4. Focussed to the point in specimen 5. Emitted light (from specimen) 6. Into same lens 7. Beam splitter 8. Detector (Photomultiplier) Confocal Microscope Confocal Microscope Scanning System Nipkow disk Galvanometer Scan Mirror J Galvano meter Gal va no mete r | i ^^^1 ib) } Single Galvanometer single Mirror Laser Beam Scan Mirror Dual Galvanometer Single Mirror lojmir Peträn lilan Hadravsky Nipkow Disk Nipkow/ Petrah Disk Architect Li re 10* Rotation m « Archimedean Spiral Figure 2 KAISERLICHES PATENTAMT PATENTSCHRIFT -- Jft 30105 - Kl-ASfiG Öl: Elf*™™ Ahmhi PACX NIPKQ^r in BERLIN. E-lL-hLriftclie* Tsk&top. PilrrHiil Irti EX /u der P*r#nct()brifi.l 30105. Nipkow Disk lojmir Peträn lilan Hadravsky https://www.juliantrubin.com/bigten/baird_nipkow_television.html John Baird mechanical television patent RE19169 Il9=an arc-lamp in the infra-red spectrum for not blinding photographed people 20=lens that intensify the light (by 19) reflected from the transmitted object 21=the transmitted object light reflection (cross) passing a framing mask 22=spiral lenses mounted on a rotating disc fo r scanning the object 14=other possible scanning disk arrangements for different radiations or needs 23=photoelectric cell (selenium) for infrared light detection 24=line amplifier transmitting amplified electrical signals from the cell to the receiver 25=gas-discharge lamp (neon), converts the arriving varying electrical signals into light 26=a rotaing disc for the detection of the arriving image 27=projection screen Confocal Microscope Nipkow Disk June 30, 1970 m. petran etal 3,517,980 UBIttOD AND ARRANGEMENT FOR IMPROVING THE RSSOLVIHO rOKER MID CONtBJLST Filed Oac. 4, 19B7 5 Sheet3-Shi»t s Rotation „ Direction Nipkow Dish lojmir Petran lilan Hadravsky Nipkow J Petran Disk Architecture no. 2 INVENTORS Vetr&n, "iff/an Confocal Microscope Confocal Microscope Scanning System Nipkow disk Galvanometer Scan Mirror - ^ Galvanometer Galvanometer! ,1 Figure 6 Laser Benin Scan Mirror Dual Galvanometer Single Mirror lojmir Peträn lilan Hadravsky Nipkow Disk Nipkow / Petrah Disk Architecture M 2 Archimedean Spiral Figure 2 Disk Speed = D Light Sheet Fluorescence Microscopy (LSFM) • splits fluorescence excitation and detection —► two separate light paths • camera-based detector —► collect images faster —► less excitation light • 3D imaging extremely fast —► imaging samples = millimeter scale (developing organisms or large cleared tissue samples) Light Sheet Fluorescence Microscopy (LSFM) Video I © Sample courtesy of E.Diel, D. Richardson, Harvard University, Cambridge, USA Light Sheet Fluorescence Microscopy (LSFM) https://www.photonicsxom/Articles/Light_Sheet_Microscopy_Transforming_3D/a65704 CLEM SMLM SIM T-REX RESOLFT STORM Super-Resolution Microscopy FPALM dSTORM DyMIN STED REDCue STED SOFI SIM (Structured Illumination Microscopy) Constructive vs. destructive interference; Coherent vs. incoherent interference Waves that combine in phase add up to relatively high irradiance. wv Constructive interference (coherent) Waves that combine 180° out of phase cancel out and yield zero irradiance. WV Destructive interference (coherent) Waves that combine with nearly cancel out and yield very low irradiance. wv wv wv addition Source: Tribino, Georgia Tech SIM (Structured Illumination Microscopy) 541 7 631 SIM (Structured Illumination Microscopy) Advantages • 2x increase in spatial resolution over wide-field microscopy -> lateral (in xy) -100 nm • 3D imaging at fast frame rate • labelling using conventional fluorophores • up to 3 simultaneous colour imaging (other super-resolution microscopy modalities are often limited to 2) Disadvantages • artefacts generated during image reconstruction • sensitive to out-of-focus light and so difficult on thick or too densely labelled samples. Stimulated emission depletion (STED) ■s*™ microscopy super-resolution microscopy J 1M INSTRUMENTS overcomes the diffraction limit of light microscopy The Nobel Prize in Chemistry 2014 Eric Betzig, Stefan W. Hell, William E. Moerner Share this: The Nobel Prize in Chemistry 2014 Photo: A. Mahmoud Eric Betzig Prize share: 1/3 Photo: A. Mahmoud Stefan W. Hell Prize share: 1/3 Photo: A. Mahmoud William E. Moerner Prize share: 1/3 The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy". https://www.nobelprize.org/nobeljDrizes/chemistry/laureates/2014/ Stimulated emission depletion (STED) microscopy • switching off the fluorescence by intense laser light —► in outer regions of diffraction limited excitation focus • detected fluorescence in center excitation focus —► high resolution images Stimulated emission depletion (STED) microscopy Applications ❖ Structural analysis -> instead of Electron Microscopy (EM) ❖ Correlative methods -> combining AFM + STED ♦> Multicolor ♦> Live-cell (ONLY plasma membrane with organic dyes) —► RECENTLY: multicolor live-cell STED (pulsed far-red laser) http://www.spiegel.de/fotostrecke/sted-mikroskopie-scharfer-blick-in-die-nanowelt-fotostrecke-51431-13.html Single-Molecule Localization Microscopy (SMLM) Conventional fluorescence microscopy; fluorophores loo clow to resolve Multiple rouradsof stochastic activation and localisation of individual molecules single motecule i:--.^,c + • 1- • 1- —► • +, # + • Ccntroid Computer localisation rendered super- resolution image Thorley et al., 2014 w Original Image ■ - Pjrlitls Detertion Reconstruction RE50LFT/STED fl? cc-r. ■_ 00 D Structured illumination microscopy (SIM] 3 £>aiT.erfis*3 modulations - & images *" o ^ i Ac=fj..irod Images' Single molecule local izati on d c: O a "j j ■r % : :) 3 o E CC Deconvolution Acquisition (Convolution Noise) ^^^^ ^^^^^^^^1 Deconvolution (PSF + model) Convolution = Distortion http://bigwww.epfl.ch/deconvolution/ point spread function (PSF) —► response of an imaging system to a point source or point object the degree of spreading (blurring) of the point object -> the quality of an imaging system https://svi.nl/Deconvolution Deconvolution Point Spread Function (PSF) Experimental Theoretical quantum dots or fluorescent beads resolution size limit isolated one, direct injection to sample use same setting all tthe time average PSF Both approaches are advisable Deconvolution Fourier transformation rtime" domain frequency domain Impulse, or "delta" function Boxcar AA/W Sin wave comb Sync Function 1/T 1/T Deconvolution methods No neighbors Nearest neighbors Linear methods Wiener filter, inverse filtering Linear least squares (LLS) Constrained iterative Jansson van Cittert Nonlinear least squares Statistical image restoration Maximum likelihood Maximum a posteriori Maximum penalized likelihood Blind deconvolution Deconvolution Huygens Deconvolution Software Lightning CLSM https://svi.nl/Deconvolution © Sofia Legartova Deconvolution Electron Microscopes I Light Microscopes Maximum resolution is 0.5nm Maximum resolution is 200nm Useful magnification is up to 250,000xinTEM, 100,000x in SEM Useful magnification is around 1000x (1500x at best) Wavelength is 1.0nm. Wavelength is between 400-700nm. Highly detailed images, and even 3D surface imaging. See reasonable detail, with true colours. Can see organelles of cells, bacteria and even viruses. Good for small organisms, invertebrates and whole cells. sample detector I TEM 5,5 nm ^ I I w TEM SEM Electron beam passes through thin sample. Electron beam scans over surface of sample. Specially prepared thin samples are supported on TEM grids. Sample can be any thickness and is mounted on an aluminum stub. Specimen stage halfway down column. Specimen stage in the chamber at the bottom of the column. Image shown on fluorescent screen. Image shown on TV monitor. Image is a two dimensional projection of the sample. ! Image is of the stirface of the sample^ I SEM https://www.majordifferencesxom Laboratory of Cellular Biophysics (2009) • cultivation chamber (5% C02 and • cultivation chamber (5% C02 and temperature control, temperature control, Live cell experiments) Live cell experiments) • WLL (470-670 nm, Image acquisition) • WLL (470-670 nm, Image acquisition, FLIM-FRET) • Argon laser (Fluorescence Recovery After • Argon laser (Fluorescence Recovery After Photobleaching, FRAP) Photobleaching, FRAP) • UV-lasers (355 nm and 405 nm, DNA repair • UV-laser (405 nm, FLIM-FRET) studies) • FLIM-FRET Methods DNA repair studies ^^^^^^^^ DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. 1. an irreversible state of dormancy, known as senescence 2. cell suicide, also known as apoptosis (programmed cell death) 3. unregulated cell division, which can lead to the formation of a tumor that is cancerous Methods DNA repair studies Base- excision 1 1 Nucleotide excision 1 H Mismatch repair (BER) II 1 repair (NER) I Brepair (MMR) Single-strand damage Base Excision Repair (BER) • repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or deamination Nucleotide Excision Repair (NER) • recognizes bulky, helix-distorting lesions such as pyrimidine dimers and 6,4 photoproducts Mismatch Repair (MMR) • corrects errors of DNA replication and recombination that result in mispaired (but undamaged) nucleotides Hoeijmakers et al., 2001 Methods DNA repair studies Hoeijmakers et al., 2001 Double-strand breaks Non-Homologous End Joining (NHEJ) Homologous Recombination (HR) Microhomology-Mediated End Joining (MMEJ) Methods DNA repair studies • activation of DNA damage response (DDR) system Leica TCS SP-5 X • Nucleotide excision repair • cyclobutane pyrimidine dimers (CPDs) UV-laser 355 nm IGFP-I UV-laser 405 nm Hoechst sensitization 405 nm without Hoechst sensitization GFP-H2B UVA-irradiation 0 min > 5 h ■-• W 20 urn ^ ^ 6 h 4 • 20 urn f <• 23 h Stixova et al., Folia Biologica, 2014 Legartova and Suchankova et al., JoVE, 2017 ivncroirraaiai Methods rois Single cell DNA repair studies UV-lasers Transfection (\%nnm)or " • transfer of non-viral genetic material into eucarytic cells _| — Time-laps Goal: to express a particular gene in the host cell Jr?™™„ - 1 1 u microscopy Used: to study gene expression regulation, protein function, gene silencing or gene therapy • Stable Transfection (H2B-GFP) • Transient Transfection (PCNA or 53BP1-RFP) imunostainingl H2B-GFP i ■ H2B-GFP ■ I Suchánkova et al., 2015 Methods DNA repair studies Hel_a-GFP-H2B cells DNA lesions GFP-H2B ROl DAPI GFP-H2B https://ibidi.eom/griddecl-clishes-slicles/178-dish-35-mm-high-g rid-500-g lass-bottom, html B Tunc imcjiu-.il of protein accumuI.nian c „ D CD ■il 11 II. ■ y ✓ ✓ Fixed calls ✓ ^ ^ Living colls - Prcttaln .lccuiiiulatiori ■ Maximum accumulation b 1 ■a 100 E s .1 tr.-.i of protein accumulation sit DMA lesions on , K O i- 1 ^/vv^ //i^/y Selected proteins Suchankova et al., 2015 Methods Fluorescence Recovery After Photobleaching (FRAP) Movement (exchange (un)bleached) of molecules • Diffusion • Active transport Fluorescence Recovery After Photobleaching (FRAP) (Im)mobile fraction td diffusion time Fj fluorescence before bleaching F0 fluorescence just after bleaching F«, fluorescence in bleached region after full recovery Mobility = diffusion coeff. D -> related to td diffusion time = (F. - F0)/ (F, - F0) Reits and Neefjes, 2001 Methods Förster Resonance Energy Transfer (FRET) Ishikawa-Ankerhold et al., 2012 Methods Förster Resonance Energy Transfer (FRET) • a distance-dependent physical process by which energy is transferred nonradiativelv from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole-dipole coupling (Förster, 1965). http://research.chem.psu.edu/txlgroup/RESEARCH.html Methods Förster Resonance Energy Transfer (FRET) Fluorophore properties A good fluorophore • Large extinction coefficient (~ 105 cnr1M-1) • High fluorescence quantum yield ( > 0.8) • Large shift of the fluorescence vs. absorption (Stokes shift > 40 nm) • Low quantum yield of photobleaching ( < 106) https://images.naturexom/full/nature-assets/nprot/journal/v8/n2/images/n .jpg Methods Förster Resonance Energy Transfer (FRET) Leica TCS SP5 X • protein-protein interactions FRET Acceptor Bleaching • donor "de-quenching" in presence of an acceptor • comparing donor fluorescence intensity in the same sample before and after destroying the acceptor by photobleaching FRETeff = (Dpost - Dpre)/ D post LAP2a/ lamin A n Mm 1 5 ..-HI Methods Förster Resonance Energy Transfer (FRET) Disadvantages of FRET • fluorescent probes + molecule of interest —► creation of fusion proteins = mutation and/or chemical modification of the molecules under study • speciment movement (during the bleaching procedure) • photo-bleaching once in sample • donor fluorophore emission bleed through —► acceptor emission channel Methods Fluorescence Lifetime Imaging (FLIM) - Förster Resonance Energy Transfer (FRET) Fluorescence Lifetime (x) • average time a fluorophore remains 1. Start the clock laser pulse (picosecond frequency) in excited state before returning to 2. Stop the clock 1st photon that arrives at the detector the ground state by emitting photon 3. Reset the clock -> wait for start next signal arrivJI t mf> Yiang et al., 2015 Fluorescence lifetime histogram Fit a exponencial decay -> get the fluorescence lifetime (in ns) Methods Fluorescence Lifetime Imaging (FLIM) - Förster Resonance Energy Transfer (FRET) Donor (D) Acceptor (A) excitation state energy to the acceptor molecules Methods Fluorescence Lifetime Imaging (FLIM) Instrumental limitations - Förster Resonance Energy Transfer (FRET) 1 Liu et al., 2019 ■ light intensity = high —► loss of arriving photons/ pulse s keep probability of detecting —► one photon/ laser pulse Counting Loss i- i X i X J-► Dead Time ■ time a photon processed —► no other photon recorded Methods Fluorescence Lifetime Imaging (FLIM) - Förster Resonance Energy Transfer (FRET) Enrico Gratton Professor of Biomedical Engineering and Physics Laboratory for Fluorescence Dynamics K^O£iJi University of California, Irvine Experimental point Quenching trajectory 1 1 A B Simple Rules for FRET: 1) If the experimental point lies on a straight line then it is FRET 2) FRET efficiencies follow a "quenching trajectory" 3) Quantitative FRET efficiencies can be obtained from the position on the quenching trajectory https://biocenterat-my.sharepoint.eom/:p:/r/personal/lijuan_zhang_vbcf_ac_at/_layouts/15/Doc.aspx?sourcedoc=%7Bd0e7c7c8-a72e-42c6-8ff7-599b2235447f%7D&action=edit The "F" words FRET FFS FLIM FCS ru:> FIGS FRAP FLAM ■ FACS FCCS F L U 0 R 8 S E I C £ &9 Advanced hoton ounting K.ilany 'J* Ihody IniriuinmhJl iolog1cal confo Microscopy Molecule-; 2(112, 17, 4047-4132; doi: 10.3390/moleculesl 7044047 OFFM ACCESS molecules ISSN 1420-J049 w w w. mdp i. convj on nia 1/mol ecu les Review Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM Hellen C, Ishikawa-Ankci-holü Richard Ankcrhold 2 and Gregor P, C, Drummed J'i''* SCIENCE STUDENT How society sees me How religious people see me How it really is