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Luxembourg 2013
- 1. Laser Based DualSpinning Disk Technology Andrew Hubbard February 11, 2013 www.andor.com
- 2. Fluorescence Illumination Illumination in widefield microscopy and confocal microscopy: Petri Dish Oil Objective Wide Field Laser Scanning Spinning Disk 2 February 11, 2013 www.andor.com
- 3. The confocal principle… Point Illumination, scanned across specimen in raster format Fluorescence is detected through confocal pinhole aperture Out-of focus information is rejected by pinhole Direct optical sectioning w/o computation and assumptions Best contrast and resolution 3 February 11, 2013 www.andor.com
- 4. How to createa confocal image By moving the point of light Raster the focussed point of laser across and down the sample using one or two galvanometer driven mirrors Not the fastest method of scanning, very popular By moving the confocal pinholes Use a spinning disk of pinholes to scan the light Nipkow disk principle, very fast 4 February 11, 2013 www.andor.com
- 5. How to createa confocal image By moving the point of light Raster the focussed point of laser across and down the sample using one or two galvanometer driven mirrors Not the fastest method of scanning, very popular By moving the confocal pinholes Use a spinning disk of pinholes to scan the light Nipkow disk principle, very fast 5 February 11, 2013 www.andor.com
- 6. Galvo mirrors –laser scanners The most common method of scanning Advantages: Good spatial resolution and confocality Single laser beam Disadvantages: Slow and high level of photobleaching and phototoxicity 6 February 11, 2013 www.andor.com
- 7. Moving the pointof light “Classical Confocal” – the most common method of scanning Galvo mirror scan and photomultiplier tube (PMT) detection of fluorescence 7 February 11, 2013 www.andor.com
- 8. The Challenge ofLive Cell Imaging Key Parameters Lateral resolution Axial resolution Temporal resolution Low photobleaching Low phototoxicity Fast intra cellular trafficking events captured at high temporal resolution in a region within a fibroblast cell. 3D rendered images made from 8 Z sections with a 0.8 micron Z spacing. Each stack of images took 0.7 seconds to capture and this was repeated over 90 seconds. The endosome in the middle is 3 microns in diameter and it fuses with an endosome of 1 micron in diameter. Data courtesy of Frode Skjeldal, who works in Professor Oddmund Bakkes lab in the department of Molecular Biosciences at Oslo University. 8 February 11, 2013 www.andor.com
- 9. How to createa confocal image By moving the confocal pinholes Use a spinning disk of pinholes to scan the light Nipkow disk principle, very fast 9 February 11, 2013 www.andor.com
- 10. The Nipkow disk– Petran 1968 It’s a The first proposed Pinholes MAMMOTH method of scanning Multi-point scanner Advantages: Fast, real time confocal Disadvantages: Historically - Poor light efficiency through the disk 10 February 11, 2013 www.andor.com
- 11. Nipkow Spinning Disk 1-2% Nipkow disc with pinholes 11 February 11, 2013 www.andor.com
- 12. Dual Spinning Disk(Yokogawa) Collector disc with microlenses 70% Nipkow disc with pinholes 12 February 11, 2013 www.andor.com
- 13. Dual Spinning DiskTechnology Real-Time DichroicMovies mirror EMCCD camera 13 February 11, 2013 www.andor.com
- 14. Confocal Imaging –Conjugate focal planes Pinhole array scanning Single point scanning e.g. galvo scanners 14 April 5 2011 11, 2013 February www.andor.com
- 15. What makes adetector sensitive? Two key parameters: Quantum Efficiency Noise Camera must be designed to ensure these parameters are optimised. 15 February 11, 2013 www.andor.com
- 16. Typical Quantum Efficiency– EM and I-CCD BI CCD 100 Virtual Phase 90 FI CCD Quantum Efficiency (%) 80 70 FI CCD 60 Gen III ICCD 50 40 30 20 10 0 200 300 400 500 600 700 800 900 1000 Wavelength (nm) 16 February 11, 2013 www.andor.com
- 17. Electron Multiplication –EM Gain Low readout noise ~ 5-6 e rms EM Readout noise ~ 45 e rms Probability of Impact Ionization = p Number of Gain register stages, n Gain ~ (1+p) n e.g. p=0.01, n=500, Gain = 145 p=0.015, n=500, Gain = 1,710 17 February 11, 2013 www.andor.com
- 18. Effect of EMCCDGain on S/N EMCCD Gain Gain x1 Gain x10 Gain x100 Gain x500 18 February 11, 2013 www.andor.com
- 19. Benefits: It’s still ALIVE! • Fast, real time confocal due to multi point spinning disk excitation and multi point EMCCD detection • Good S/N due to highly sensitive EMCCD detection • Reduced photobleaching • Reduced phototoxicity 19 February 11, 2013 www.andor.com
- 20. XYZT imaging (4D) Longterm 4d imaging of Zebrafish embryo as it undergoes early cell division. 192 Z sections were taken with a step size of 0.3 micron. The stack took 20 seconds to acquire with an interval of 100 seconds between stacks. This series of maximum projection images is made up from 51840 frames that were acquired over a time period of ~9 hours. 20 February 11, 2013 www.andor.com
- 21. XYZTλ imaging (5D) Key Parameters Lateral resolution Axial resolution Temporal resolution Spectral resolution Low photobleaching Low phototoxicity Drosophila development, chromosomes in red, tubulin in green. 5 z sections, 206 time points 21 February 11, 2013 www.andor.com
- 22. Dual Spinning Diskvs. Point Scanning Point scanner vs. Dual Disk Scanner LSCM CSU No of points scanned 1 1000 Parallel detection No Yes Detector PMT CCD/EMCCD Detector QE ~30 % ~ 90% Frame rate (Hz) @512x512 0.5 to 4 10 to 30 Laser power per point 50 to 80 uW 1 uW Bleach rate Hi Low Frame time skew Significant Low Programmable scan pattern Yes No Simultaneous Programmable scan Yes No Pinhole Variable Fixed (50um) 22 April 5 2011 11, 2013 February www.andor.com
- 23. Photobleaching analysis Spinning Disk Point Scanning Data from Wang et al, Journal of Microscopy, May 2005 23 February 11, 2013 www.andor.com
- 24. Limitations of SpinningDisk Resolution Fixed pinhole of SD is matched to high mag high NA objectives 24 February 11, 2013 www.andor.com
- 25. Limitations of SpinningDisk Axial Resolution and Pinhole Crosstalk •A question of balancing pinhole size and spacing for optimal resolution, light efficiency and speed •The distance between pinholes can be increased to improve the axial resolution at the cost of signal •Depending on staining pattern and localisation thick specimens can be challenging 25 February 11, 2013 www.andor.com
- 26. Active Illumination Bleaching (e.g. Fluorescence Recovery After Photobleaching & Fluorescence Loss In Photobleaching) Photochemical destruction of a fluorophore with excessive illumination FRAP Cell Compartmentalisation & Continuity Protein dynamics and turnover 26 February 11, 2013 www.andor.com
- 27. FRAPPA Rapidly raster scans the sample, causing chemical changes to fluorescent dyes. Uses CW laser from Andor ALC. Mainly used for •FRAP – Fluorescent Recovery After Photobleaching •PA – Photoactivation/Photoconversion FRAP + PA = FRAPPA Used with the XD Spinning Disk 27 February 11, 2013 www.andor.com
- 28. CS U Laser from MPU/ALC CS U Laser from MPU/ALC 28 February 11, 2013 www.andor.com
- 29. Thanks for yourattention 29 February 11, 2013 www.andor.com
Editor's Notes
- #3 In a basic microscope with fluorescence illumination, the sample is bathed in light In confocal techniques the sample is illuminated with a focal spot of light which is required as part of the imaging technique. The focussed light is achieved by passing the light through a pinhole
- #4 * 07/16/96 * But, how does it work? Basically illumination via a laser source is passed through a pinhole. The light from the pinhole is focussed to a particular point in the sample. Light passing deeper into the sample (pale green line) causes fluorescence emission from the sample below the confocal plane. The light from this point does not pass back through the pinhole and the detector ignores it. Emitted light from the focussed point, at the confocal plane, passes in its entirety back through the pinhole and the detector uses it to form the image. Thus light from out of focus planes is discarded and does not form part of the image.
- #5 * 07/16/96 * By comparing these two methods of canning the confocal light.
- #6 * 07/16/96 * By comparing these two methods of canning the confocal light.
- #7 * 07/16/96 *
- #8 * 07/16/96 * This cartoon shows the point of light rasterring across and down the sample. Each point of emitted light passes back through the confocal pinhole and is detected on a photomultiplier tube. The PMT then converts the photons into electrons and ultimately an image formed from grey levels is displayed on a computer screen. These systems are very good in resolution, but they may bleach the sample quickly or kill living samples very quickly.
- #9 * 07/16/96 * A good example of an application where a point scanner would struggle. Endosome fusion. The larger endosome is 3 microns in diameter and fuses with the smaller one. Crucial parameters here are in red, the resolution has to be good to see such small endosomes, the system needs to be fast to acquire the stack in under a second, and the sample cannot bleach or die as this is imaged for 90 seconds. Temporal Resolution may or may not be important depending on the biology being investigated.
- #10 * 07/16/96 * By comparing these two methods of canning the confocal light.
- #11 * 07/16/96 * The principle of imaging confocally using multiple pinholes came before the now popular point scanning confocal microscopes.
- #12 * 07/16/96 * Back in Petran’s day the disk threw away 98% of the excitation light, this proved to be a major hurdle especially with insensitive detection.
- #13 * 07/16/96 * So a second disk was introduced, this one containing microlenses. Each lens focuses the light, which is now a laser source, through its pinhole. This increase the throughput of the scanning mechanics up to 70%. This increase in transmission across the pinholes means spinning disk systems can be used for imaging low fluorescent label expressing, delicate living biology.
- #14 The emitted light from the sample is detected back through the pinholes, so all is confocal, and the photons sent to the ultra sensitive iXon camera. The iXon detects the photons and converts to electrons, the image is displayed in real time on the computer screen and movies of dynamic living biology can be made at high resolution and sensitivity.
- #15 * 07/16/96 * So the Revolution spinning disk confocal, with its method of scan and superior low light detection capability means the researcher can capture biological events as they happen with minimal damage to both the fluorescent molecules and the sample itself. Because the light delivered to the sample is of a ‘low intensity, high frequency’ dose (the laser is dispersed into many micro-lasers, 1000 beams in fact, and then scanned very fast, 1000 times a second), effects of photo bleaching and photo toxicity are reduced. Further benefits in these two crucial points can be achieved as the iXon EMCCD is so sensitive the actual laser power used can be kept very low.
- #16 * 07/16/96 *
- #17 * 07/16/96 *
- #18 * 07/16/96 *
- #19 * 07/16/96 *
- #20 * 07/16/96 * With the low light levels that Spinning disk requires/utilizes, live cell imaging with good signal to noise and confocal resolution is possible.
- #22 * 07/16/96 * Now all the parameters are red (crucial) as in 5D imaging you also need good spectral resolution as you are imaging more than one colour. This is on the edge imaging and the demands on the technology are huge. If laser power is too high then cell division will be blocked, and if exposures are too long then events are missed.
- #23 * 07/16/96 * So the Revolution spinning disk confocal, with its method of scan and superior low light detection capability means the researcher can capture biological events as they happen with minimal damage to both the fluorescent molecules and the sample itself. Because the light delivered to the sample is of a ‘low intensity, high frequency’ dose (the laser is dispersed into many micro-lasers, 1000 beams in fact, and then scanned very fast, 1000 times a second), effects of photo bleaching and photo toxicity are reduced. Further benefits in these two crucial points can be achieved as the iXon is so sensitive the actual laser power used can be kept very low.
- #26 The Nipkow disk pinhole diameter in the figure (a) is assumed to be a single Airy pattern unit in diameter with reference to the focal plane (in effect, approximately 0.5 micrometers). It is also assumed that essentially all of the fluorescence emission representing the central maximum of the Airy disk represented by the point object proceeds through the pinhole and towards the objective. A view of the Nipkow disk from the side opposite the objective is presented in (b), and the pinhole diameter ( D ; 0.5 micrometers) and inter-pinhole spacing ( S ; 2.5 micrometers) are indicated on the drawing. The total light transmission through a disk having a D/S ratio of 1/5 is approximately 4 percent, consistent with typical spinning disk microscopes that are not equipped with microlens arrays. Relocating the specimen point approximately 1 micrometer beneath the focal plane (c) reduces the amount of light passing through the pinhole due to the fact that much of the light emanating from the point now strikes the bottom of the disk (d) and is reflected from the surface. Relocating the specimen point to a distance equal to S (2.5 micrometers) away from the focal plane enables some of the emission light to pass through the first ring of neighboring pinholes (e) and (f). As a result, more fluorescence emission now passes through the six peripheral pinholes than through the central pinhole, mimicking the background signal haze for a highly fluorescent point source positioned away from the focal plane in a thick specimen. Note that when the specimen point is positioned at the location in (e), emission light is now spread over a much larger diameter on the Nipkow disk and the excitation is likewise diminished. As the specimen point is lowered still farther away from the focal plane, the number of photons passing through the disk continues to diminish until some of the emission light begins to pass through the ring of secondary neighbours when the point is approximately 5 micrometers beneath the disk.
- #27 In most cases photobleaching is avoided in live cell imaging as previously discussed. However, targetted photobleaching can be used to further understand the physiology of a cell.