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Laser Based Dual Spinning Disk Technology

Andrew Hubbard

www.andor.comApril 12, 2023


Fluorescence Illumination

Objective

Petri Dish

Oil

Wide Field Spinning DiskLaser Scanning

Illumination in widefield microscopy and confocal microscopy:


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


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



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



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


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


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.


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


1-2%1-2%

Nipkow Nipkow disc with disc with pinholespinholes

Nipkow Spinning Disk


70%70%

Collector Collector disc withdisc with

microlensesmicrolenses

Nipkow Nipkow disc with disc with pinholespinholes

Dual Spinning Disk (Yokogawa)


DichroicDichroicmirrormirror

EMCCD EMCCD cameracamera

Dual Spinning Disk Technology

Real-TimeReal-TimeMoviesMovies


Confocal Imaging – Conjugate focal planes

Pinhole array scanningPinhole array scanning

Single point scanningSingle point scanninge.g. galvo scannerse.g. galvo scanners

April 5 2011


Two key parameters:

Camera must be designed to ensure these parameters are optimised.

NoiseNoise

Quantum EfficiencyQuantum Efficiency

What makes a detector sensitive?


0

10

20

30

40

50

60

70

80

90

100

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


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


Gain x1 Gain x10

Gain x100 Gain x500

Effect of EMCCD Gain on S/N

EM

CC

D G

ain


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


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.


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


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


Photobleaching analysis

Spinning Disk

Point Scanning

Data from Wang et al, Journal of Microscopy, May 2005


Limitations of Spinning Disk

Resolution

Fixed pinhole of SD is matched to high mag high NA objectives


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


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


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


Laser from MPU/ALC

CSU

Laser from MPU/ALC

CSU


Thanks for your attentionThanks for your attention

luxembourg 2013

Documents

spinning disk of pinholes

confocal pinholes

confocal image

point scanning point

confocal microscopy

light nipkow disk principle

point of light raster

nipkow disk petran