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Laser Based Dual Spinning Disk Technology
Andrew Hubbard
www.andor.comApril 12, 2023
www.andor.comApril 12, 20232
Fluorescence Illumination
Objective
Petri Dish
Oil
Wide Field Spinning DiskLaser Scanning
Illumination in widefield microscopy and confocal microscopy:
www.andor.comApril 12, 20233
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
www.andor.comApril 12, 20234
How to create a 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
www.andor.comApril 12, 20235
How to create a 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
www.andor.comApril 12, 20236
Single laser beam
The most common The most common method of scanningmethod of scanning
Advantages: Good spatial resolutionGood spatial resolution
and confocalityand confocality
Disadvantages:Disadvantages: Slow and high level of photobleaching Slow and high level of photobleaching and phototoxicity and phototoxicity
Galvo mirrors – laser scanners
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Galvo mirror scan Galvo mirror scan and photomultiplier and photomultiplier tube (PMT) detection tube (PMT) detection of fluorescenceof fluorescence
““Classical Confocal” – the most Classical Confocal” – the most common method of scanningcommon method of scanning
Moving the point of light
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The Challenge of Live Cell Imaging
Key ParametersLateral resolutionAxial resolutionTemporal resolutionLow photobleachingLow 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.
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How to create a confocal image
By moving the confocal pinholes Use a spinning disk of pinholes to scan the light Nipkow disk principle, very fast
www.andor.comApril 12, 202310
Multi-point scanner
PinholesThe first proposed The first proposed method of scanningmethod of scanning
Advantages:Advantages:
Fast, real time confocalFast, real time confocal
Disadvantages:Disadvantages:Historically - Poor light Historically - Poor light
efficiency through the diskefficiency through the disk
It’s a MAMMOTH
The Nipkow disk – Petran 1968
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1-2%1-2%
Nipkow Nipkow disc with disc with pinholespinholes
Nipkow Spinning Disk
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70%70%
Collector Collector disc withdisc with
microlensesmicrolenses
Nipkow Nipkow disc with disc with pinholespinholes
Dual Spinning Disk (Yokogawa)
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DichroicDichroicmirrormirror
EMCCD EMCCD cameracamera
Dual Spinning Disk Technology
Real-TimeReal-TimeMoviesMovies
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Confocal Imaging – Conjugate focal planes
Pinhole array scanningPinhole array scanning
Single point scanningSingle point scanninge.g. galvo scannerse.g. galvo scanners
April 5 2011
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Two key parameters:
Camera must be designed to ensure these parameters are optimised.
NoiseNoise
Quantum EfficiencyQuantum Efficiency
What makes a detector sensitive?
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0
10
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200 300 400 500 600 700 800 900 1000
Wavelength (nm)
BI CCD
FI CCDGen III ICCD
Qua
ntum
Effi
cienc
y (%
)
Virtual PhaseFI CCD
Typical Quantum Efficiency – EM and I-CCD
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Electron Multiplication – EM Gain
Low readout noise ~ 5-6 e rms
EM Readout noise ~ 45 e rms
Probability of Impact Ionization = pNumber of Gain register stages, n
Gain ~ (1+p) n
e.g. p=0.01, n=500, Gain = 145p=0.015, n=500, Gain = 1,710
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Gain x1 Gain x10
Gain x100 Gain x500
Effect of EMCCD Gain on S/N
EM
CC
D G
ain
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It’s still ALIVE!Benefits:• 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
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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.
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XYZT imaging (5D)
Key ParametersLateral resolutionAxial resolutionTemporal resolutionSpectral resolutionLow photobleachingLow phototoxicity
Drosophila development, chromosomes in red, tubulin in green. 5 z sections, 206 time points
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Dual Spinning Disk vs. Point Scanning
Point scanner vs. Dual Disk ScannerLSCM CSU
No of points scanned 1 1000Parallel detection No YesDetector PMT CCD/EMCCDDetector QE ~30 % ~ 90%Frame rate (Hz) @512x512 0.5 to 4 10 to 30Laser power per point 50 to 80 uW 1 uWBleach rate Hi LowFrame time skew Significant LowProgrammable scan pattern Yes NoSimultaneous Programmable scan Yes NoPinhole Variable Fixed (50um)
April 5 2011
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Photobleaching analysis
Spinning Disk
Point Scanning
Data from Wang et al, Journal of Microscopy, May 2005
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Limitations of Spinning Disk
Resolution
Fixed pinhole of SD is matched to high mag high NA objectives
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Limitations of Spinning Disk
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
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Bleaching (e.g. Fluorescence Recovery After Photobleaching & Fluorescence Loss In Photobleaching)
Photochemical destruction of a fluorophore with excessive illumination
FRAPFRAP
Cell Compartmentalisation & Continuity
Protein dynamics and turnover
Cell Compartmentalisation & Continuity
Protein dynamics and turnover
Active Illumination
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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
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Laser from MPU/ALC
CSU
Laser from MPU/ALC
CSU
www.andor.comApril 12, 202329
Thanks for your attentionThanks for your attention
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