1. ECES 490/690 Cell & Tissue Image Analysis Lecture #3: Microscopy Architecture (snow day recap) Andrew R. Cohen, Ph.D. 1/26/2015

2. Dealing with Toxicity of the Fluorophore Simple Idea: –Make a cell produce proteins that are naturally fluorescent! No need to inject an extrinsic contrast agent –Inspired by the discovery of green fluorescent proteins (GFP) in a jellyfish (aequoria victoria) by Osamu Shimomura and Frank Johnson in 1961 http://www.lifesci.ucsb.edu/~biolum/organism/photo.html

3. Green Fluorescent Proteins Green fluorescent protein (GFP): –Isolated in the 60’s from a jellyfish Aequoria Victoria –When excited, it glows green –Turned out that it has a protein “aequorin” that produces blue light (470nm) which excites GFP molecule which produces green (508nm). The gene for this protein was sequenced in 1992 The detailed structure and properties of this protein are now known This gene has been mutated to produce a large number of variants of the original GFP This has revolutionized biological imaging! –Especially, the study of live cells Wikipedia

4. http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm Green fluorescent protein (GFP)

5. Reporter Gene Technology Bottom line: GFP is Produced whenever the factors triggering the gene of interest are ON Promoter for gene of interest GFP cDNAAAAA GFP Gene expression DNA Fragment Artificially inserted “Poly(A) Tail”

6. DNA Fragment Fusion Protein Technology Promoter For gene of interest GFP cDNAAAAAGene of interest GFP Protein Produces the protein of interest with a GFP unit attached! If the GFP can be verified (by other means) to not affect the behavior of the protein of interest, we have a way of fluorescently tagging the protein of interest! Gene expression Artificially inserted

7. Example: See the Microtubules! Basic Idea: –Attach a GFP to each of the  -tubulin protein molecules (“red balls” below) http://www.olympusfluoview.com/applications/gfpintro.html

8. The “classic” GFP –395nm excitation (ultraviolet)/509nm response –Works at 28 degrees (too cold for mammalian cells) EGFP: enhanced GFP –Brighter, more convenient 484nm excitation –Works at 37 degrees for mammalian use EYFP (yellow), ECFP(cyan), EBFP(blue), mOrange, DsRed,… –Many of these are derived from other creatures including reef corals, and anemones. –They have all been modified to work in warmer mammalian cells, and to make them brighter, and easier to excite, etc. –Rapidly growing field – newer variants being produced every year! Modern Palette of Fluorescent Proteins

9. Imaging Intra-Cellular Transport Vesicles are miniature “taxicabs” carrying cargo within the cells They slide over cytoskeletal fibers as they go from one place to another They have molecular equivalents of “address labels” so there is considerable specificity http://www.ohsu.edu/croet/faculty/banker/bankerlab.html Vesicle transport in a neuron

10. Considerations for Live-Cell Imaging The microscopy should not hurt/kill the cells: –Toxicity of the fluorescent label Could change the chemical function of molecule of interest Could be outright toxic to the cell(s) –Photo-toxicity (damage caused by light) Ultraviolet disrupts molecules Infrared heats up tissue Prolonged exposure to any wavelength can be bad –Configuration of microscope Inverted microscopes preferable –In vitro imaging: Need instrumentation for keeping cells alive in a dish Temperature, humidity, oxygen, Carbon dioxide, –In vivo imaging: Much harder – image a part of a living animal Surgical techniques to provide optical access the tissue of interest

11. Inverted Fluorescence Microscopes Olympus Microscopy

12. Recap: Fluorescence Molecular imaging systems –Produce spatial maps capturing distributions/locations of specific molecules Interactions of molecules with light –Intrinsic imaging –Imaging with contrast agents, especially fluorophores –Fluorescence is a hugely important phenomenon Imaging genes and gene activity –FISH: fluorescence in-situ hybridization Imaging proteins – the products of gene activity –Immunofluorescence: The use of fluorescently conjugated antibodies to “tag” specific proteins of interest –Multiplexing: Simultaneous use of multiple tags to image multiple proteins preserving relative context Absorption Non-radiative Transition / Loss Radiative Transition

13. Multi-Photon Fluorescence Basic Idea: –Hit the molecule with multiple photons, say 2, simultaneously –The molecule cannot distinguish this excitation from a single photon with twice the energy Non-radiative transition Emission 3-photon works the same way Short-lived Virtual State

14. What does it take to achieve multi- photon fluorescence? The probability of two photons hitting a molecule at nearly the same time, (within 10 -18 seconds), is extremely low! Need to achieve a super high concentration of photons –Megawatt per cubic micrometer Can’t do this on a sustained basis! –Idea #1: Use a pulsed laser, so average energy is still low enough to make specimen damage negligible 100mW –Idea #2: Concentrate the energy in space by using a lens Intense focal spot –Idea #3: Concentrate the energy in time by using ultra-short pulses from a “mode locked laser” 50-100 femtosecond pulses 1 femtosecond = 10 -15 sec Expensive stuff (  $100,000)! Lens Laser Pulse Focal Spot Laser T W P

15. What does it Buy Us? A dye that would ordinarily require ultraviolet excitation at 400nm, could now be excited using two infrared 800nm photons –Infrared is dramatically less damaging to molecular structures compared to ultraviolet No chemical disruption, just causes a little heating –Infrared is absorbed and scattered much less, so one can achieve deeper penetration into tissue –Ability to image autofluorescence without damage –It allows “super localization” We’ll explain that shortly

16. Localized Multi-photon Response Probability of simultaneous absorption falls off steeply away from the focal volume, localizing the response along the optical axis  3-D Imaging Possible! 1P 2P

17. Reduced Photobleaching Single-photon excitation happens throughput the cone of illumination Multi-photon excitation only happens near the focus  negligible photobleaching above/below the plane of focus Lens

18. Example: In-vivo Imaging of Brain Tumors Courtesy: Dr. R. Jain, MGH

19. Can we do 3-D Imaging without Multi-Photon? Yes, several methods exist: –Deconvolution Microscopy –Confocal Microscopy We need to understand the optical behavior of a lens first! –The objective lens of the microscope is key

20. Light sheet microscopy Santi, P. A. Light sheet fluorescence microscopy: A review. Journal of Histochemistry and Cytochemistry 59: 129-138 (2011). Dr. Santi introduces a nicely composed review article that covers the basic principles of light sheet microscopy that includes a historical perspective, instrumentation requirements, and details about how to process specimens for imaging.

21. LSM vs. TPM Bouchard, M. B., et al. (2015). "Swept confocally-aligned planar excitation (SCAPE) microscopy for high- speed volumetric imaging of behaving organisms." Nat Photon advance online publication. We report a three-dimensional microscopy technique—swept, confocally-aligned planar excitation (SCAPE) microscopy—that allows volumetric imaging of living samples at ultrahigh speeds. Although confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image three-dimensional volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light sheet in a single-objective, en face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless three-dimensional imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

22. Numerical Aperture (N.A.) n = refractive index of medium MediumRefractive Index Air1.0 Water1.33 Immersion oil1.55

23. Airy patterns and resolution Also known as the point-spread function

24. Obtaining 3-D Structure without the Multi-Photon Effect “Confocal Microscopy” –Name comes from “conjugate foci” of lenses: f 1 and f 2 Basic idea: –Put a tiny pinhole at the conjugate focus f 2 –Almost all of the light from the point in the specimen “squeezes through” the pinhole Point in the specimen Pinhole

25. Effect of Pinhole Better axial resolution, but Fewer photons

26. Optical Slices Move the z stage up/down At each z value, scan across the x-y field to collect an “optical slice” A “stack” of optical slices is a volumetric (3-D) image

27. How do we view a 3-D image? The computer screen is two-dimensional, so we have to project the 3-D volume I(x,y,z) onto a 2-D image P(x,y) –Two basic choices to make: Projection angle –Simplest: Aligned with axes –Best: Oblique angles Projection formula/algorithm –Sum / Average / Median –Max / Min –Surface Rendering

28. Viewing by Projections Neuron: TR054Z1 Step Size: Zoom: 1.0 Dimension: 512x480x323 The “maximum value” projection is most commonly used for fluorescence data

29. Volumetric Rendering Choose a viewing angle Ray casting: Shoot rays back into the volume from each pixel in P(x,y) –Compute an intensity value by summation / max / min / …etc. Good: Objective, since we’re not “messing with” the data Bad: Computationally expensive –We often desire to rotate the object interactively to look from different angles Could be slow on a regular computer –Expensive hardware accelerators and parallel computation algorithms available for real-time volume rendering.

30. Practical Volumetric Rendering First find all surfaces in the 3-D image –We’re messing with the data.. Use graphics routines to create artificial lighting effects to render the surfaces –Very fast since most computers have support for this type of graphics –Today’s “kiddie game cards” outperform expensive ($100K) rendering engines from 10 years ago –Pretty, but potentially misleading if the surfaces are identified inaccurately Need to pay attention to unequal axial/lateral resolution

31. Faster Confocals Spinning Disk Systems –Nipkow Disks Scans lots of points at once using a rotating disk with a spiral array of holes, and a CCD camera instead of photomultipler tubes –The basis for all modern high-throughput microscopes http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/spinningdiskfundamentals/index.html

32. Impure Channels There are basically two kinds of impurities to consider: –Case 1: Morphologically impure The fluorescent label is not specific enough We can get two or more types of “things” in a channel Solution 1 (preferred): work with the biologist to either choose different things to label, or different labels if at all possible Solution 2: develop algorithms that can handle morphologically mixed data –Case 2: Spectrally impure The fluorescent labels are specific, but their spectra overlap heavily Solution 1 (preferred): seek out alternative fluorescent labels Solution 2: computationally “unmix” the data

33. Dealing with Overlapping Spectra Nucleus: histone GFP Fusion Actin filaments: fluorescein conjugated phalloidin The peaks are separated by only 7nm ! 2 1

34. Dealing with Overlapping Spectra “Unmixing Result”: A 1 and A 2 Compute A 1 and A 2 at each pixel subject to constraint A 1 + A 2 = 1 Reference Spectra

35. Ultimate Optical Microscope of the Future Isotropic and high-resolution sampling of 3-D space (x, y, z) –Recent microscopes have broken past the Rayleigh resolution limit No wasted photons – 100% detection Complete spectrum at each pixel –Measure absorption & emission spectrum –Complete flexibility to shape the excitation spectrum –Complete flexibility to capture and analyze the emission spectrum Complete lifetime response at each pixel –Photon counting hardware at each detector –Time response at different spectral wavelengths Multiple modalities looking at the same specimen –One of the holy grails that continues to be pursued