Cmb551 1a: Microscopy And Image Analysis In Cell Biology Sam Johnson

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CMB551 1A: Microscopy and Image Analysis in Cell Biology Sam Johnson http://microscopy.duke.edu/learn/CMB551.html [email protected] August 2015 Fluorescence High-contrast, multi-channel imaging. Relies on intrinsic contrast, little specificity Allows many modes of imaging – confocal, TIRF, 2-photon, singlemolecule, fluorescent proteins= live cell imaging, FRAP, photoconversion, FRET, FCS, super resolution. . . Fluorescence can be . . . 488 2˚ 1˚ Basis of all fluorescence scopes Photo-bleaching Chemical reaction causing irreversible loss of fluorescence Reactive Reactive and long-lived Photo-toxicity Same principle as photo-bleaching but this time it’s bad for the cell not just the fluorophore Oxygen Radicals Generally UV light is more toxic than far-red wavelength, with or without any exogenous fluorophore around Modalities | Photon budget The insides of a microscope: Fluorescence Adapted from http://www.microscopyu.com/articles/formulas/formulasconjugate.html Microscope configurations The objectives point in different directions but the optical principles are the same The objective Collects light from the sample and forms an image up the microscope near the eyepieces Intermediate image SAMPLE Objectives are the most important parts Three important concepts in microscopy • Magnification • Resolution • Contrast Magnification “How many times bigger the image is than the object” Magnification is not a very useful concept on its own Resolution “The smallest distance between two objects that can be observed as two objects” It doesn’t mean anything else (e.g. how nice the image looks) Does the resolution also limit the smallest object we can see? Imaging a tiny fluorescent object Imaging a tiny fluorescent object In 3D this is a point-spread function XZ XY Resolution in fluorescence terms Object Airy disk http://www.olympusmicro.com/primer/java/imageformation/rayleighdisks/index.html Resolution limit of Light Microscopy About 200 nm • Naked eye = 100 mm • Electron microscopy = <1 nm http://learn.genetics.utah.edu/content/begin/cells/scale/ Reference sizes • • • • • • • Diameter of an adherent fibroblast 100 mm Mammalian cell nucleus 10 mm Red blood cell 7 mm Bacteria 1 mm Virus 50 nm Ribosome 20 nm Globular protein 2 nm Resolution in fluorescence terms Imaging green fluorescent beads of different sizes 1000 nm Objects Images 750 nm 500 nm 250 nm 125 nm Resolution in transmitted light terms With brightfield TL you don’t see things below the resolution Ok Small objects diffract light at a greater angle, not captured by the lens Brightfield illumination Contrast Contrast is needed in the image to be able to resolve anything Contrast = DI I Signal to noise is a related concept Numerical Aperture NA = n sin (m) Numerical Aperture determines. . . Brightness = NA4 / Mag2 (epi) = NA2 / Mag2 (trans) Resolution = 0.61 l / NA Example set objectives for fluorescence Brightness Magnification NA = NA4 / Mag2 = NA2 / Mag2 Immersion (epi) (trans) Resolution (nm) Relative brightness 5x 0.15 DRY 2033 1.0 10x 0.30 DRY 1017 4.0 20x 0.50 DRY 610 7.7 40x 0.75 DRY 407 10 63x 1.40 OIL 218 48 100x 1.40 OIL 218 19 This formula ignores transmission efficiency Numerical Aperture and working distance Low NA High NA Working distance NA and depth of . . . • Depth of field – Z- range of image that is sharp • (Depth of focus – Range focus can be moved and image still sharp, more a photography term but sometimes used interchangeably) Optical cost comparison $3.29 $11,822 Aberrations Simple lenses Problems . . . chip Field curvature curved image plane Objectives correct for some aberrations Good optics are relatively free of aberrations. Microscopes are expensive because high magnification requires the best optics. Many types of objective Achromats – Fluorite Apochromats – Plan Apochromats – Limited color correction Good all round objectives, good transmission, NA up to 1.3 Highly color corrected Additional correction for field curvature $ $$$ http://www.olympusmicro.com/primer/anatomy/objectives.html Many types of objective immersion Dry Dipping Oil Silicon n=1.4 Water Glycerol Multi Why use oil or water objectives? Higher NA = more resolution and brightness NA = n sin (m) (sin (90) = 1) n = refractive index Effective limit of NA Air = 1.0003 ~0.95 Water = 1.33 ~1.2 Glass = 1.515 ~1.4 Why use oil or water objectives? Higher NA = more resolution and brightness NA = n sin (m) (sin (90) = 1) n = refractive index Effective limit of NA Air = 1.0003 ~0.95 Water = 1.33 ~1.2 Glass = 1.515 ~1.4 Why use oil or water objectives? Virtual fish Fish Matching the refractive index of the sample and the objective immersion helps keep aberrations and degradation to a minimum Quiz 5 – (sorry about the numbering) Coverslips Number Ideal thickness Range #0 100 μm 80-130 μm #1 150 μm 130-170 μm #1.5 170 μm 160-190 μm #2.0 220 μm 190-250 μm http://microscopy.duke.edu/coverslip.html Coverslip thickness is very important Worst for high NA dry objectives Adjustable correction collar to minimize spherical aberration and light loss http://www.microscopyu.com/articles/formulas/formulascoverslipcorrection.html Infinity optics A missing bit from a few slides ago Infinity advantages - Easier to add extra components (eg filters) Can focus by moving the objective http://www.olympusmicro.com/primer/anatomy/infinityintro.html /The objective Collects light from the sample and forms an image up the microscope near the eyepieces Intermediate image SAMPLE The eyepiece (s) Intermediate image Magnifies the intermediate image and allows us to see the image SAMPLE Total magnification = Mobjective x Meyepeice Attaching a camera to a scope 1.4 Mpixels 12 Mpixels ~$12 000 from Photometrics ~$80 from BestBuy The details required for microscopy cameras • Quantum Efficiency – Chance of a photon being recorded • Dynamic range – Full well capacity (how many electrons each pixel holds)/noise • Cooling – Low light levels, noise is significant and cooling helps. • Noise – Cooling reduces dark noise a lot. Electronics optimized for low read noise. • Frame rate – 100 fps can be useful sometimes. • Grade – No (or few dead pixels) CCDs, charge-coupled devices Photons  Charge  Pixels http://www.microscopyu.com/articles/digitalimaging/ccdintro.html CCD sensors Photons  Charge  Pixels http://www.microscopyu.com/articles/digitalimaging/ccdintro.html Binning Combining pixels on the CCD chip to make superpixels Bin 8: Bin 4: Bin 2: 672x512 Bin 1: 1344x1024 Binning More photons collected in each output pixel Electron Multiplying-CCD cameras Also tend to be built for highest QE – peak ~95% (cf ~70% normal) Good for very fast, dim or photosensitive samples http://learn.hamamatsu.com/articles/emccds.html CMOS CCD Each column in a CMOS chip has own readout structure Possible to read the image faster (and use a larger chip = larger fov or more resolution) 2048 x 2048 px resolution at 100 fps ~50 GB/min! Fluorescence in more detail http://www.olympusmicro.com/primer/java/jablonski/jabintro/ Excitation and emission Stokes shift Reducing energy Notice the “tails” http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html Emission is excitation-independent Always back to here before fluorescing Illumination sources Arc lamp Metal Halide http://zeiss-campus.magnet.fsu.edu/articles/lightsources/mercuryarc.html Regulation of exposure to excitation Arc lamps tend to bright, unregulatable and slow to turn on/off (30 min minimum to extend life) •Fast electromechanical shutter •About 10 mSec cycle • • • Neutral Density Usually expressed as a transmission ND20 = 20% of light goes through Illumination sources: Arc lamps and similar Mercury Arc lamp Spectra uneven and much in UV Xenon Spectra more even, weak in UV Arc lamp Visible Illumination sources: LEDs and similar Last ~forever (50 000 hr) Fast switching and power regulation Taking off for the future - cheaper, brighter, more l . . . Filters and dichroics T Filter terminology Bandpass (BP) – 480/40 CWL/bandwidth Full-Width at Half Maximal (FWHM) Central wavelength Longpass – LP600 (Defined around center of cut-on l) (guess what, say, Short Pass 670 means) http://micro.magnet.fsu.edu/primer/techniques/fluorescence/filters.html Filters and dichroics Even better filter set T Filter choice and efficiency Choose filter to match your fluorophore (tools to help) More photons = better Does this help? Filter choice and efficiency A fluor’ with a fairly small stokes shift Best excitation Better emission Multi-channel imaging More than one color; more than one fluorescent protein, probe, antibody . . . AF488 AF568 Multi-channel imaging Filter cubes are an easy way of switching excitation filter, dichroic and emission filter Spectraviewer quiz http://www.invitrogen.com/site/us/en/home/support/Research-Tools/FluorescenceSpectraViewer.html Use a spectra viewer to choose which of these filter sets you would prefer to use for a sample labelled with AF488 and AF568 Set A: Cube1 - x480/25 dichroic 495 m520/40 Cube2 - x570/20 dichroic 585 mLP590 Set B: Cube1 - x500/25 dichroic 515 m540/30 Cube2 - x595/10 dichroic 610 mLP615 Bleedthrough DAPI AF488 • Down the spectrum (1st law of thermodynamics) • Worse when intensities are unbalanced Other multi-channel methods DAPI Can be faster Better registration Some disadvanatges GFP AF568 Exciters Dichroic Emitters The insides of a microscope: transmitted light Adapted from http://www.microscopyu.com/articles/formulas/formulasconjugate.html Light source: Halogen bulb The condenser The condenser collects the light and concentrates it onto to the specimen May also have some special parts in for phase contrast or DIC etc Kohler illumination Bright and even illumination with good contrast and resolution Optimal alignment of the condenser How do we do this alignment in practice? http://www.microscopyu.com/tutorials/java/kohler/ Kohler illumination in practice Open the apertures and focus on your specimen as best you can in brightfield. Don’t change the focus through out. You can adjust the lamp brightness at any stage • Close down the field diaphragm/aperture so it becomes visible - you should see an octagon shaped aperture appear but it may be very blurry • Focus the condenser with the knobs that raise/lower entire condenser - the octagon shaped field aperture should be made as sharp as possible • Then center the condenser using the two centering pins • Open the field diaphragm until it is just out of view – now the whole area is evenly illuminated • Adjust the condenser aperture so the contrast of the image is good – you can do this empirically or remove the eyepiece and adjust so 2/3 to 5/6 of the pupil is filled Components on an inverted scope Components on an upright scope Back focal plane of the objective Image Diffraction Object Conjugate planes Image, object or field planes Illumination, aperture, or diffraction planes D = Retina of the eye Eye C = Intermediate image 4 = Pupil of the eye Eyepiece 3 = Objective pupil B = Specimen plane Objective Condenser 2 = Condenser aperture A = Field diaphragm Lamp 1 = Lamp filament If that sounds useful to you please find a simple scope and try the alignment process (or use this virtual scope) http://www.microscopyu.com/tutorials/java/kohler/ Contrast in transmitted light images Cells don’t absorb much light, there isn’t much contrast based only on that Staining isn’t ideal Many methods and variations to increase the contrast over brightfield. . . Phase contrast Brightfield Phase Simple to setup, good depth of field, copes with plastic (ideal for TC cells) . . . but not much contrast is produced Surround wave Diffracted waveWhy is it smaller amplitude? Combined wave Much more contrast in this condition . . . Surround wave Diffracted waveNote the similar amplitude? Combined wave Phase contrast Interference at BFP of objective Objective Scattered light has its phase shifted by 1/4l Condenser Phase contrast alignment Kohler alignment with the condenser aperture fully open DIC (Normarski) Brightfield DIC More complex and $$ Pseudo-3D Two beams of different polarization pass through the sample and are recombined where they interfere and produce an image with high contrast Differential Interference Contrast microscopy 0˚ 90˚ http://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy Differential Interference Contrast microscopy http://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy The problem with widefield microscopes and biology Sectioning Vibratome Microtome Cryostat Non-physical sectioning – optical sectioning by . . . Confocal | Multiphoton | Spinning disk | TIRF | SPIM The confocal principle How a laser scanning confocal microscope works http://www.olympusfluoview.com/theory/index.html The confocal advantage Optical sectioning of thick samples 3D reconstruction What a confocal looks like Scanhead A microscope (inverted or upright) Lasers and electronics Probably on an air table Computer LASERs are used for excitation Ideal for point scanning: •Narrow collimated beam, low divergence •Powerful http://www.olympusfluoview.com/theory/laserintro.html Many lasers available . . . Gas lasers Diode lasers (Kr/Ar 488 568 647) 405 Ar/Ar 458 488 488 HeNe 543 594 633 514 561 635 (Color coding refers to the color of the fluorophore for which the laser line is most commonly used) Adjusting the laser power Relatively fixed output Rapid control 0-100% T Laser AOTF Laser AOTF l selection and intensity control Fluorophore saturation Bleaching is proportionately worse here Emission intensity Image will suffer loss of contrast and quantification issues Excitation intensity Widefield is normally in the linear range, the concentrated laser spot in confocal may not be. Start low and increase to the minimum necessary Scanning mirrors The relative position of the two mirrors can point the spot anywhere in the field http://www.olympusconfocal.com/theory/confocalscanningsystems.html Scan speed Fast scans • Fast processes • Useful for focusing and adjustments – eg 1 fps Slower scans • More light gathered • Better images • More damage Averaging Scanning each pixel multiple times and averaging improves the noisy signal . . . Averaging: how much do you need? 1 2 8 4 16 Decreasing rate of improvement, empirically determine a good balance between final SNR and time/damage in acquisition Pinhole adjustment Axial (z) or lateral (xy) resolution Is the pinhole . . . A. A big waste of signal? B. Only good as we didn’t want that light? C. A bit of both Signal : Noise Signal : background Photomultiplier tubes (PMTs) are used as detectors • • • • • Fast (good for scanning) Large collection area Good SNR Very large dynamic range (with gain&offset adjustment) Adequate dynamic range at a single gain&offset position • QE <30% (not as good as CCD) Gain and offset adjustments 100 msec 200 msec 400 msec 600 msec 800 msec 1000 msec 2000 msec Camera: Confocal: Increasing gain (voltage on PMT) Offset to set the background to black Optimal gain and offset Confocals have special display modes to highlight saturated and 0 intensity pixels Should you have no, a few or many saturated and zero intensity pixels? Multi-channel confocals Most confocals have several laser lines and PMTs 2˚ dichroic 1˚ dichroic Why don’t we in general do this with widefield systems? Simultaneous or sequential acquisition Blue Green Red Faster Blue then Green then Red Less bleedthrough Line vs frame switching Blue line1 then Green line1 then Red line1 Blue line2 then Green line2 then Red line2 The AOTFs switch the laser light on and off very rapidly Good to see all the channels appearing together Blue image then Green image then Red image Good when something physical happens between colours (eg change dichroic) Use this when you have more channels than PMTs Spectral imaging: serial Leica spectral detector Select l Prism PMT Lamda-scan over time Spectral imaging: parallel Zeiss META/QUASAR detector Array of 32 PMTs Unmixing overlapping signals Reference spectra (or ACE) Overlapping signals Separate signals Ideally, avoid having to do this. But maybe . . . • • • You’ve only GFP-YFP mice You have a strong autofluorescence (eg chloroplasts) You need many colours Scan area: zoom The area swept by the galvo mirrors can be adjusted . . . Is this meaningful zoom or just digital zoom? . . . Scan area and number of pixels Any particular frame can have different numbers of pixels. . . Zoom and number of pixels are obviously related How many pixels do I need in my image? Same area: 225 mm across 225 mm/512 = 440 nm 225 mm/1024 = 220 nm The most possible, right? The Leica SP5 goes up to 8K by 8K, shall we have 64 Mpx for every scan? How many pixels do I need in my image for the best resolution? Nyquist sampling theorem: Sample at twice the resolution Resolution = 0.61 l / NA Increasing number of pixels per area Signal under-sampled Not capturing all the resolution of the system Signal Well sampled Just right, The Nyquist rate Signal Over-sampled Not gaining any more resolution, more bleaching waste of time and disk space Sampling rate Aliasing Over-sampled How do frequencies relate to resolution? But I don’t image wavy green lines or brick walls Our object has a spatial frequency in the distribution of the contrast we are imaging Nyquist calculator for microscopy http://support.svi.nl/wiki/NyquistCalculator Q Consider this principle and a few more subtle factors A Do I really need to listen to Nyquist? • You might not always be seeking the best resolution • You might need to under-sample for speed, phototoxicity . . . • Over-sampling is effectively averaging But optimal sampling is important and beneficial in many cases 3D acquisition Confocals are good for acquiring stacks of images because of the optical sectioning ability Z-stack acquisition control The point-spread function Calibration of the microscope, the 3D image of a point source XY XZ Point spread function 3D 3D 2D Widefield Confocal FWHM for 63x/1.4 NA is about 200 * 500 nm Blur degradation in 3D wf images Too many PSFs to draw Image: Sharp image +dimmer blur Overwhelmed by blur Lateral and axial resolution XY rxy ~ 0.61 l / NA YZ rz  l / NA2 Resolution is always worse in Z than XY Optical section thickness 10x/0.3 20x/0.45 Optical section (micron) Smaller with higher NA 63x/1.2 40x/1.3 63x/1.4 100x/1.4 Larger with more open PH Pinhole size (AU) Sampling in the z-axis The same principle as in XY Some regions not imaged Covered Covered and well sampled Transmitted image For transmitted light For fluorescent light It is NOT confocal (why?), beware of the overlay Setting up a transmitted image 1. Setup Kohler TL 2. Confocal forms a TL image You can use whichever lasers you are using for fluorescence, but it tends to be best with far-red (Gain and offset controls are physical dials on a Leica) http://www.olympusfluoview.com/java/confocalsimulator/index.html Other ways of doing something like this Without these problems . . . Point based consequences: • Speed (eg 260,000 fold for 512 image) • SNR (if quicker than 7 hr per image) • Photodamage (bright spot to compensate) Low detector QE, few photons Without these problems . . . Point based consequences: • Speed (eg 260,000 fold for 512 image) • SNR (if quicker than 7 hr per image) • Photodamage (bright spot to compensate) Low detector QE, few photons Fixed Live What can we do about this? Improve confocals GaAsP detectors improve QE Drive confocals wisely • • • • Keep laser power to a minimum Open the pinhole a bit Under sample Get ok-ish images • Use the best reagents • Keep live samples otherwise as happy as possible Resonant scanners Smaller max amplitude so zoom is >1 8000 Hz resonant scanner vs say 400 Hz standard scanner Leica SP5 and SP8 have this feature Everything else is the same except the scanner Emission intensity Consequences of scanning faster: reduced photobleaching Excitation intensity Faster scanning = lower fluorophore saturation Phototoxicity and photobleaching are reduced Consequences of scanning faster: reduced photobleaching t ~3 ns t ~30 ms Example pixel dwell times: ~2 mSec standard, ~100 nSec resonant The longer you illuminate, the greater the % of GFP accumulates in T Triplet state is effectively permanent within the scale of pixel dwell time But the time between scans of the same spot is >tT http://www.leica-microsystems.com/science-lab/brighter-fluorescence-by-resonant-scanning/ The spinning disk principle 1. Pinholes are in an image plane so project many spots onto the sample Defocused laser 4. Image captured on a CCD 3. Disk descans the light – motion is trivial relative to speed of light 2. Spots scan the sample as the disk spins The spinning disk principle 1. Pinholes are in an image plane so project many spots onto the sample Defocused laser 4. Image captured on a CCD 3. Disk decans the light – motion is trivial relative to speed of light 2. Spots scan the sample as the disk spins Detector comparison CCD PMT EMCCD Photodetectors hv QE Something Results dark noise PMT 20% CCD 70% EMCCD 95% An electron Amplification (perhaps) multiplicative noise Something bigger A2D Read noise Gain regulated cascade More electrons Charge, e in well No Same Charge, e in well em gain register wf 1,000-20,000 PSC 20-100 Disk 10-3,000 Pixels More electrons S:B over SNR Shot Noise Statistical noise in photon arrival, not from the detector How many photons emitted in 1 second? kf kf 100,000 photons/sec ~100,000 (normal distribution) 2 photons/sec Probably not 2, Poisson distribution SNR = N N = N NSR: 5% error 400 with 1% error with 10,000 photons Photobleaching advantages t ~3 ns Emission intensity t ~30 ms Excitation intensity Each spot is less intense than in a point scanner The stroboscopic avoidance of triplet How does a spinning disk work? 99% disk ~40% T No zoom Synch required above ~20 fps Sectioning in spinning disks Ideal pinhole diameter = 0.5 l M/NA • 100x/1.4 = 20 mm • 20x/0.5 = 11 mm Trade-off for excitation intensity and emission confocality: 50-70 microns and fixed The pinhole is > AU1 (often much more at low power lenses) so the sectioning and z-axis resolution will be less good than a point scanner Sectioning in spinning disks Pinhole cross talk (Both these factors would be much worse without the microlens disk= increased transmission without having bigger pinholes) What works well on a spinning disk Living things that need sectioning . . . • Things which match the high magnification, high NA optimizations (eg subcellular imaging) • Photosensitive samples • Fast imaging Not the dimmest samples Not thick homogenous samples Things quite like a Yokogawa spinning disk . . . Slits not spots Changeable Swept field Visitech Infinity Prairie/Nikon Similar to the microlens spinning disk in terms of strengths and weaknesses Sectioning by excitation Excite a defined region of the z-axis and image pretty much all the fluorescence available • TIRF • Multi-photon excitation • SPIM Total internal reflection Some reflected, some refracted Evanescent wave Evanescent wave Exponential decay of intensity Iz = I0e-bz Evanescent wave Evanescent wave Plane of excitation ~100 nm thick This is much thinner than a confocal slice Widefield vs TIRF Myosin Actin Two ways of generating and imaging TIRF Prism-based A bit more to align Better SNR, lower background Slight constraint on imaging objective Sample access is difficult in some setups Have to build your own Objective-based More convenient Needs >1.45 NA objective SNR still very good Components of a TIRF system Inverted microscope with a special TIRF objective Opaque incubator TIRF angle autoalignment optics round the back Fiber-coupled lasers EMCCD What is TIRF good for? Anything at the edge of the cell/tissue • Exocytosis/endocytosis • Vesicle dynamics • Cytoskeletal activity at the membrane – Focal adhesions • Signalling in the membrane – translocation Relatively distinct subset of samples gain from TIRF imaging Single molecule imaging Single molecule sensitivity allows single molecule biochemistry TIRF imaging is the highest SNR fluorescence imaging modality so good for single molecule studies, for which you need . . . • A sensitive way of imaging • A way of only having a few molecules in your imaging volume • (also the basis of some super-resolution techniques . . .) Two photon excitation Single photon Two-photon Two photon excitation Single photon Two-photon Excitation is limited to a small focal volume where photons are most concentrated Differences to a single photon confocal Can use all the light, no pinhole needed Non-descanned detector Since we don’t need to go through a pinhole, we don’t even need to descan and the PMT can be close to the objective and efficient Is it a confocal? (this arrangement makes it very sensitive to room light) 2-photon advantages Main advantage: Imaging thicker specimens The longer wavelength excitation penetrates further into the sample. The scattered excitation light doesn’t cause background fluorescence. The excitation is also not attenuated by fluorophore absorption above the plane of focus Tissue absorption of light of l . . . Tissue H20 700-1200 nm is a good window between absorption/scatter by tissue and absorption by water Emission advantage Because we are able to image all the light (no pinhole) this is less affected by scatter With pinhole No pinhole The NDD is closer and more efficient for a scattered beam path (which is hard to move efficiently through several lenses) 1P 2P Pretty pictures from the Olympus FV1000 MPE brochure A Pulsed laser is required for MPE For efficient MPE we need photon concentration in space and time . . . http://www.olympusmicro.com/primer/techniques/fluorescence/multiphoton/multiphotonintro.html A pulsed laser Chameleon Ultra II Femtosecond pulsed laser 3-4 W of power at peak Tunable 680-1080 nm @40 nm/sec This gives flexibility, since these laser cost about $200,000 each its not normally practical to have several per machine like with 1 photon Excitation spectra Most good 1P fluor’s are ok for 2P (weak ones are normally even worse in 2P) Not exactly double 1photon Tends to be broader and blue shifted This makes multichannel imaging easier in some ways and more difficult in others Photobleaching and toxicity Good luck! Extremely toxic l Won’t go through glass Much less phototoxicity associated with these l Multi-photon excitation provides a means of exciting UV and blue fluorophores with less phototoxity Photobleaching and toxicity Photodamage in 2P is confined to the thin layer being imaged Power (photons/mm2 Wide-field X 1 Photon 105X 2 Photon (average) 106X 2 Photon (peak) 1011X But the damage in that area can be worse In general, for thick samples 2P has an advantage over 1P For thin samples, 2P is often worse than 1P There may be strange forms of damage due to very high field strength – ROS, DNA breaks, tweezing 2-photon vs. 1-photon • Improved SNR with thick samples • The IR 2PE is less phototoxic in many cases, especially for UV dyes. • Photobleaching/damage restricted to plane being imaged. Photobleaching or uncaging is possible with fine z-axis resolution • Resolution is slightly less good • Multi-channel acquisition is harder (excitation cross-sections are normally much broader) and limited in excitation l • No control of optical section thickness • Lasers are expensive and require exact alignment and can produce heating and other damage Don’t use a 2-photon system unless you need the advantages Final sectioning by excitation technique for today . . . A brief mention Selective Plane Illumination Microscopy (SPIM) Light sheet fluorescence microscopy Widefield CCD detection Sheet illumination from the side Components of an example SPIM system Often a low-ish mag low NA dipping objective For TL Laser(s) for excitation shaped into a sheet Plane illumination photodamage advantage Reduced region of illumination and photodamage n times better for a z-stack of n slices The excitation intensities are overall much lower than confocal Selective plane illumination microscopy techniques in developmental biology Jan Huisken and Didier Y. R. Stainier Development 2009 Samples good for SPIM etc The sectioning, speed, efficiency and low photodamage make it ideal for study of living samples up to a few mm across Thin-sheet laser imaging microscopy for optical sectioning of thick tissues, Santi et al 2009 Reconstruction of zebrafish early embryonic development by Scanned Light Sheet Microscopy Keller et al 2009 SPIM systems are good for a range of samples imaged poorly by other techniques Optical clearing: SCALE reagent • • • • 4 M urea 10% (wt/vol) Glycerol 0.1% (wt/vol) Triton X-100 pH of 7.7 Refractive index of 1.387 and 486 nm Soak your sample in it for a couple of weeks . . . http://www.nature.com/neuro/journal/v14/n11/full/nn.2928.html It clears fixed samples by removing refractive index changes Doesn’t destroy fluorescence Preserves tissue structure And allows . . . SCALE reagent 24 tiled stacks =9000 images Movies of data Tools available for you Light Microscopy Core Facility DUKE UNIVERSITY AND DUKE UNIVERSITY MEDICAL CENTER Choose wisely Modality comparison Modality Sensitivity Speed Photodamage Sectioning Samples WF Fluorescence Good Camera fast Not bad No Cells, thin sections Not bad “100 nm” Things close to the membrane, single molecules A concern Adjustable >500 nm Nearly anything, only helps with thick samples/sectioning Better than normal confocal Adjustable >500 nm Living samples that need high-speed/ low photodamage Good Fixed by disk, depends on objective Live samples, cells to embryos It depends By excitation Things too thick for 1P TIRF Good Camera fast Confocal point scanning Poor Slow as point based Resonant PS confocal Poor Point-based still Spinning Disk Good (but goes through a disk) 2 Photon Poor Camera fast Slow as point based But testing a few is often a good idea Commercial choices: the big four The best microscopes are definitely from (Redacted by the LMCF legal department) Super resolution How to improve the resolution of fluorescence microscopy Resolution = 0.61 l / NA It should be said that it’s really quite good already ~200 nm 1. STED: STimulated Emission Depletion More strange things about the quantum mechanics of fluorophores A long wavelength photon can deplete the fluorescence How STED can get us better resolution Normal 1P excitation spot Doughnut of depletion Bigger doughnut, smaller spot STED system Strengths and weaknesses of STED  Point scanning confocal with improved XY resolution ~3X  Fast – pretty much as a standard confocal • Fluorophore limitations, multiple fluors difficult • Requires precise alignment • The power required for depletion is not ideal for living cells • Corruption with depth • Z-resolution not improved 2. Structured illumination extends the passband We can image the moire fringes and with knowledge of the illumination structure we can capture the object in finer detail than ordinarily possible 3D Structured illumination Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy SIM in practice • • • • 100 nm XY by 200-300 nm Z resolution Essentially a widefield technique Normal dyes Two fold resolution improvement still pretty useful 3. Core principle of the next approach We can’t separate two objects beyond our diffraction limited resolution but . . . . . . If you only have one object, you can position the centroid with very high accuracy, say 1 to a few nm Dark sample with PAfluorophore Weak photoactivation to produce a few isolated fluorphores Image and bleach individual fluorophores and map their position Many iterations to produce super-resolution map/image TIRF image PALM Super-resolution Resolution can be improved more than 10 fold Imaging Intracellular Fluorescent Proteins at Nanometer Resolution Processing the images • 10,000 images, ~10 min, few GB • Fiducials help correct drift • Individual molecules must be separated by >resolution • But sequential so >105 molecules per mm2 possible • Accuracy of centroid fitting is l/2NA √#photons about 50 photons per point (STORM more than FPs) Astigmatism: 3D STORM iPALM Which super resolution approach is best? Well they do pretty different things . . . STED– Confocal like Limited fluorophores, damage, no z-axis improvement Structured illumination Smallest improvement in resolution, but 3D improvement Most versatile and like “normal” microscopy Probably live cell compatible PALM/STORM family Best resolution Temporal constraint How easy are the 3D versions? Unlike “normal” imaging http://www.nature.com/nmeth/collections/superresmicroscopy/index.html http://www.annualreviews.org/doi/full/10.1146/annurev-cellbio-100109-104048 What should I understand? • How fluorescence works • How all those spectra, filters, lamps, objectives add up to a photon efficient imaging combination. How to choose a filter for a particular fluorophore. • Resolution (in fluorescence terms) – what it means, what it doesn’t • What factors into a wise choice of an objective • NA and its consequences • How CCD cameras work and are used in microscopy. When to use an EMCCD. • What all the components in the TL path do and HOW TO KOHLER A SCOPE (maybe the conjugate planes explanation) • Contrast – principles of brightfield, Phase contrast, DIC • The confocal principle, advantages and disadvantages • How a confocal works, what the components do and how to adjust them • Resolution and sampling in 2D and 3D • Advantages and disadvantages of Spinning disk • TIRF and the type of samples it works for • Advantages and disadvantages of multiphoton More information This is a fairly comprehensive collection of review articles and interactive tutorials about the optics involved in microscopes http://www.olympusmicro.com/primer/anatomy/anatomy.html This book has a very good for transmitted light and optical basics: Fundamentals of Light Microscopy and Electronic Imaging – Douglas B Murphy (Duke has an eBook) Optical Microscopy by Davidson and Abramowitz is a 40 page review article you can download here http://www.olympusmicro.com/primer/opticalmicroscopy.html Review articles about fluorescence microscopy http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html Spinning disks TIRF http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html SPIM http://dev.biologists.org/content/136/12/1963 Multiphoton articles/tutorials http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphotonhome.html Reviews on multiphoton imaging http://zeiss-campus.magnet.fsu.edu/referencelibrary/multiphoton.html