Boulevard du Temple Daguerrotype (Paris,1838) a busy street? Nyquist sampling for movement
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1 Boulevard du Temple Daguerrotype (Paris,1838) a busy street? Nyquist sampling for movement
2 CONFOCAL MICROSCOPY BioVis Uppsala, 2017 Jeremy Adler Matyas Molnar Dirk Pacholsky
3 Widefield & Confocal Microscopy Widefield Confocal Laser Scanning Microscopy (LSM)
4 MRC 500 confocal microscope
5 First Confocal Microscope Patent application 1957
6 Illumination Widefield Confocal AREA SPOT
7 Cells moving during image acquisition confocal
8 Build an image by scanning a single illumination spot Bidrectional misaligned undirectional
9 Illumination - fluorescence Lens Fluorophores inside the illumination cones are excited Focal plane Compare with Multiphoton fluorescence
10 From Wikipedia
11
12 Widefield Confocal LSM
13 Confocal Components
14 Objectives Numerical aperture (resolution) Immersion medium: air, water, oil Corrections: spherical, chromatic Working distance Coverslip thickness Transmission Magnification not very important
15 Miniature objectives
16 Same magnification - different NA 20x NA 0.8 5x NA 0.16
17 3 000 Sampling Interval nm Z axis Z-Axis XY- plane plane Variation of Resolution with the NA & NA of objective Nyquist sampling according to SVI Numerical Aperture NA
18 Confocal Light sources Lasers monochromatic coherent types: gas, solid, diode, etc Argon Ion Krypton -Argon Helium Neon Helium Cadmium Diode lasers White light lasers , 488, 514 nm 488, 568, 647 nm 543 nm, 633 nm 543 nm, 633 nm 405, 488, 635 nm etc tunable
19 Uneven illumination From Andor
20 Pinhole The maximum resolution is app µm lateral 0.40 µm axial Back Projected Pinhole size in the specimen Objective Magnification NA pinhole size (µms) 60x x x x
21 Open Pinhole Fluorescent microspheres NA 1.4 oil, pixels 45nm
22 Pinhole size and intensity % Max out badly out infocus Pinhole Airy units
23 Image Quality Photon Noise /Poisson Noise/Shot Noise Compare 2 sequential mages Difference Need More Photons
24 Poisson Noise how many photons? 160 photons 128 Single pixels in a timeseries sequential values photons mean SD sqrt
25 Improving Image Quality Doubling the Pixel Integration time: scan speed & averaging image MORE PHOTONS per pixel
26 More Photons increase laser power? Lens Fluorophore saturation Focal plane More photons from fluorophores outside the focused spot.
27 The Photomultiplier tube (PMT) 1930s Fluorescent photon hits photocathode emits photoelectron which cascades along the dynode chain each step amplifies electron numbers finally output at the anode depends on voltage Photon counting possible Best PMTs reach 30% quantum efficiency How good are camera?
28 The PMT gain (voltage) settings At each stage the number of electrons is multiplied the gain depends on the voltage
29 PMT Problem
30 Pinhole Size: nucleus LSM700 NA 1.4 oil adjusted for equal maximum intensity
31 Where do pixels come from? Camera Confocal?
32 Pixels (8bit) Best sampling rate or pixel size? Ideally : infinite small BUT each pixel generates noise The incoming signal has to be more intense than the noise (Signal:Noise ratio) Small pixels get less photons, but generate same/more noise like large pixels (who get more photons) Practically: pixel size is twice as small as smallest detail to be resolved LSM allows you to choose the pixel size
33 Imaging Nyquist theorem Nyquist found that in order to reconstruct a pure sine wave, it must be sampled at least twice during each cycle of the wave, 2x the frequency.
34 Pixels how large?
35 How many pixels? Nyquist flatbed scanner
36 Nyquist Pixel size: fluorophore and NA CLSMs have an OPTIMAL button which calculates the pixel size for Nyquist Drawbacks of small pixels : Slow Bleaching Find a compromise between Image quality required versus Sample robustness/time
37 IMAGING WITH LSM
38 Acquisition information: metadata recorded with the image RE:USE same setup measurements Pixel size: Objective s magnification Area scanned ZOOM Number of pixels selected
39 Optical slice from certain depth in sample Optical sectioning: Many slices from adjacent depths Z series reconstruction from slices
40 3D information from LSM images Orthogonal view 3D surface reconstruction * * 3D information, dashed lines in blue, red, green indicate position in ZXY and are movable Observe that light could not penetrate material on certain areas* 3 dimensional reconstruction of image
41 POSSIBILITIES WITH THE LSM
42 Photobleaching Live cells Laurdan
43 Spectral (lambda) scan with LSM (Zeiss) Emission spectra for fluorophore or autofluorescence QUASAR detector of LSM710 with 32 detectors or adjust the detection range of individual PMTs
44 Lambda Scan linear unmixing separating overlapping fluorophores Linear Unmixing determines the relative contribution from each fluorophore for every pixel of the image.
45 Live imaging (time lapse) Frame size in pixel: frames/sec 2048 x x x x x Smaller images faster Use oblong images Line scan 1 pixel wide
46 Scan a line - quickly Whole image of kidney sample Line scan & Z series
47 Fluorescent microspheres in a matrix confocal images
48 Faster Confocal Imaging many points Difficult to change pinhole Camera not a PMT Line scanning confocals Zeiss 5 Live Multi spot confocals
49 z POINT SPREAD FUNCTION A sub resolution object becomes a blob in the image 1. the NA of the objective 2. the confocal pinhole 3. wavelength of emission 4. Refractive index matching x
50 RI refractive inded Matching objective to sample 0 µm 2µm 4µm 6µm PSF: RI mismatch oil objective water (living) sample. loose resolution & intensity Live Cell water immersion objective Dipping objectives do not need a coverslip
51 Improving resolution d 2nsin(2 ) λ : shorter wavelengths better (blue light) n : high refractive index objective and specimen medium (oil: 1.5) ѳ : NA of the objective: use high as possible (1.4) Small confocal pinhole
52 Matching objective to sample z Airy disk in XZ x A) B) C) Z direction depth A) Actual situation in the sample B) Good match of embedding and preparation of sample C) mis match/ bad sample preparation
53 Techniques for the LSM and Live Cell Imaging Photoactivation /uncaging FRAP CALI Chromophore Assisted Light inactivation
54 Photoactivatable Fluorophores GFP activated by 413nm observe with 488nm excitation Pulse chase experiments Science (2002), 297,
55 Long Image acquisition times - problems
56 Temperature Control
57 Solution protect the microscope from temperature changes
58 Confocal v Widefield Point scanning Optical sectioning pinhole, Z series Z series Variable magnification zooming Higher resolution (NA of objective) Timeseries live imaging faster Motorized XY stage tiling also Spectral scanning Problems Slow faster Pinhole throws away photons all photons Poor detectors better Photobleaching reduced
59 Worth Looking At The 39 steps: a cautionary tale of quantitative 3-D microscopy Pawley Biotechniques, 2000, 884- Seeing is believing beginners guide to practical pitfalls in image acquisition Pawley J Cell Biol, 2006, 172, 9-18 Websites Microscopy: Fluorophores: Filters: Zeiss, Leica, Nikon, Olympus, BioRad Thermo Fisher Scientific Chroma, Omega, Semrock
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Why and How? Daniel Gitler Dept. of Physiology Ben-Gurion University of the Negev. Microscopy course, Michmoret Dec 2005
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