introduction to confocal microscopy - crick · the calm lectures 17th oct intro to light microscopy...
TRANSCRIPT
![Page 1: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/1.jpg)
INTRODUCTION TO CONFOCAL MICROSCOPY
Matt Renshaw
October 2018
![Page 2: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/2.jpg)
The CALM lectures
■ 17th Oct Intro to Light Microscopy (Kurt Anderson)
Auditorium 1 @10:00
■ 24th Oct Intro to Confocal Microscopy (Matt Renshaw)
Auditorium 1 @10:00
■ 31st Oct Intro to Live Cell Imaging (Deborah Aubyn)
Seminar room 4 @10:00
■ 7th Nov Intro to Optical Sectioning (Donald Bell)
Seminar room 3 & 4 @11:00
■ 14th Nov Intro to Light Sheet Microscopy (Alessandro Ciccarelli)
Seminar room 4 & 5 @10:00
28th Nov Intro to Multiphoton Microscopy (Rocco D’Antuono)
Seminar room 4 & 5 at 15:00
![Page 3: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/3.jpg)
Introduction to confocal microscopy
Widefield Confocal
(yes, both are GFP z stacks)
![Page 4: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/4.jpg)
widefield confocal
0
0.2
0.4
0.6
0.8
1
0 6 11 17 22
no
rma
lise
d in
ten
sit
y
distance (microns)
wf
cf
![Page 5: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/5.jpg)
Take home message
Confocal microscopy improves the optical resolution and contrast
of your images by reducing out of focus light
![Page 6: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/6.jpg)
Talk outline…
■ Basics of fluorescence microscopy and the principle of confocal microscopy
■ Multi-dimensional image acquisition with a point scanning confocal microscope
■ Advanced applications
![Page 7: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/7.jpg)
Epifluorescence light path
www.leica-microsystems.com/science-lab/fluorescence-in-microscopy/
svi.nl/FluorescenceMicroscope
Jablonski diagram Stokes shift
![Page 8: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/8.jpg)
Components of blur…
1. Point Spread Function
How does the microscope image an infinitely small point of light?
Single point of light Airy Disk
Light waves interfere and converge on the focal point, creating a
diffraction pattern called the Airy Disk.
Sub-resolution
bead
Microscopy is:
• creating a magnified, blurred image of your object of interest.
![Page 9: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/9.jpg)
Separating objects – the Airy Disk
http://olympus.magnet.fsu.edu/
Rayleigh limit is when the central maxima of one Airy Disk is in line
with the first minima of the second Airy Disk
Size of the Airy Disk determines minimum resolvable distance of 2 objects
![Page 10: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/10.jpg)
Size of the Airy Disk determines minimum resolvable distance of 2 objects
![Page 11: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/11.jpg)
Sub-resolution bead in 3DXY YZ
YZ
Focal plane
Focal plane
![Page 12: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/12.jpg)
Point Spread Function: the 3D diffraction pattern
microscopy.fsu.edu
X
Z
![Page 13: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/13.jpg)
Sources of blur1. Point Spread Function
2. Light from out of focus areas
samplefocal
plane
excitation light
detector
pinhole
pinhole excludes out of focus
light, creating an optical section
X
Z
![Page 14: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/14.jpg)
x
y
Pinhole
Optical section at resolution limit
Pinhole blocks diffraction rings
![Page 15: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/15.jpg)
Pinhole increases contrast by excluding light from the diffraction rings
Does the pinhole increase resolution?
![Page 16: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/16.jpg)
Laser scanning confocal
Widefieldillumination
Laser scanning
![Page 17: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/17.jpg)
Image is built up pixel by pixel
Emission light is descanned = focussed onto a static position
![Page 18: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/18.jpg)
No pinhole
![Page 19: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/19.jpg)
Pinhole excludes out of focus light
10 µm
![Page 20: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/20.jpg)
Numerical Aperture
■ Depth of focus
■ PSF
■ size of the airy disk
are all related and defined by the
angle of light collected by the objective
![Page 21: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/21.jpg)
Resolution of 0.175µ Bead Pair
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
-1.1
0
-0.9
6
-0.8
1
-0.6
6
-0.5
1
-0.3
7
-0.2
2
-0.0
7
0.0
7
0.2
2
0.3
7
0.5
1
0.6
6
0.8
1
0.9
6
1.1
0
Microns
No
rm
alized
In
ten
sit
y
1.4 NA
0.7 NA
Measured Width of 0.175µ Bead
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
-1.1
8
-1.0
3
-0.8
8
-0.7
4
-0.5
9
-0.4
4
-0.2
9
-0.1
5
0.0
0
0.1
5
0.2
9
0.4
4
0.5
9
0.7
4
0.8
8
1.0
3
Microns
No
rm
alized
In
ten
sit
y
1.4 NA
0.7 NA
210 nm
570 nm
• Numerical Aperture determines:
• how much light from the sample is collected by the objective
• resolution
• the thickness of sample that appears in focus
![Page 22: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/22.jpg)
■ Numerical aperture
■ Resolution
■ Depth of focusn•
NA2D
d =1.22 •
2• NA
NA = n sin α
![Page 23: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/23.jpg)
Optical Sectioning: Confocal Microscopy
Other optical sectioning
techniques are available.
See:
Intro to Optical Sectioning
talk
![Page 24: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/24.jpg)
Talk outline…
■ Basics of fluorescence microscopy and the principle of confocal microscopy
■ Multi-dimensional image acquisition with a point scanning confocal microscope
■ Advanced applications
![Page 25: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/25.jpg)
The Tetrahedron of Frustration
![Page 26: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/26.jpg)
Standard applications
■ Although there are subtle differences, all of our confocals have these basic functions
■ Software varies, but settings are there somewhere…
![Page 27: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/27.jpg)
Excitation lasers
Excitation and Emission
![Page 28: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/28.jpg)
Detection
■ Adaptable wavelength selection
■ Simultaneous or sequential detection
![Page 29: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/29.jpg)
Brief comparison of detectors
www.leica-microsystems.com/science-lab/
![Page 30: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/30.jpg)
Optimising acquisition settings
■ Laser power
■ Gain
■ Number of pixels
■ Scanning speed
■ Size of scanning area
■ Averaging
![Page 31: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/31.jpg)
Increase laser power = increase signal
Increase laser power = increase photo damage
![Page 32: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/32.jpg)
Increase laser power = increase signal
Increase laser power = increase photo damage
![Page 33: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/33.jpg)
Increase gain = increase signal
Increase gain = increase noise
![Page 34: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/34.jpg)
Increase gain = increase signal
Increase gain = increase noise
![Page 35: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/35.jpg)
Finding a compromise
![Page 36: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/36.jpg)
Finding a compromise
Noise can be negative, addition of offset prevents false zeros
![Page 37: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/37.jpg)
Finding a compromise
Noise can be negative, addition of offset prevents false zeros
![Page 38: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/38.jpg)
Achieving resolution: number of pixels
More pixels, more time
512 x 512 ~500µm per pixel 1024 x 1024 ~250µm per pixel
![Page 39: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/39.jpg)
2048 x 2048 ~125µm per pixel
Achieving resolution: number of pixels
More pixels, more time
512 x 512 ~500µm per pixel
![Page 40: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/40.jpg)
Achieving resolution: optical zoom
Zooming reduces size of scanned area
1024 x 1024 ~250µm per pixel 1024 x 1024 ~125µm per pixel
![Page 41: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/41.jpg)
Line averaging reduces noise, but takes longer
Averaging 1
![Page 42: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/42.jpg)
Line averaging reduces noise, but takes longer
Averaging 4
![Page 43: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/43.jpg)
Averaging 8
Line averaging reduces noise, but takes longer
![Page 44: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/44.jpg)
Reduce scanning speed, reduce noise
![Page 45: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/45.jpg)
Reduce scanning speed, reduce noise
![Page 46: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/46.jpg)
Imaging 3D volumes
www.thorlabs.com
![Page 47: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/47.jpg)
Imaging deep, increases light scattering
Laser power and gain can be adjusted through z stack to compensate
![Page 48: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/48.jpg)
Talk outline…
■ Basics of fluorescence microscopy and the principle of confocal microscopy
■ Multi-dimensional image acquisition with a point scanning confocal microscope
■ Advanced applications
![Page 49: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/49.jpg)
Nikon W1 spinning disk confocal
![Page 50: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/50.jpg)
■ Speed
■ Reduced photo-toxicity
■ Photo manipulation
![Page 51: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/51.jpg)
Resonant scanning on the Olympus FV3000
■ rapidly oscillating resonant mirror
scanners
■ Very fast frame speeds (~400 per
second)
■ Permits very low laser power
■ Combined with rolling averaging
(post processing) can detect high
quality images of dynamic
processes
![Page 52: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/52.jpg)
Zeiss LSM 880 with Airyscan
![Page 53: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/53.jpg)
0.5 1.0 1.5 2.0 2.5 3.0 4.0 4.5
0
5.0
20
40
60
100
80
0
Pin
ho
le t
ran
sm
issio
n %
Pinhole diameter (AU)
Pinhole diameter, resolution and transmission
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
5.
0
1.1
1.0
0.9
0.8
0.7
Pinhole diameter (AU)
Re
so
luti
on
5.0
0.2 AU = 1.4 x improvement
![Page 54: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/54.jpg)
Joseph Huff Nature Methods volume12, page1205 (2015)
0.2 AU
Airyscan detector is 32x 0.2 AU mini GaAsP detectors
![Page 55: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/55.jpg)
Alex van Vliet, Tooze Lab
Conventional
Airyscan
![Page 56: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/56.jpg)
Spectral imaging and unmixing
■ separating fluorophores with close emission spectra
e.g. Alexa fluor 555 and Mito Tracker orange
Wavelength (nm)
Re
lati
ve in
ten
sit
y (%
)
![Page 57: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/57.jpg)
http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html
Whole spectrum of emitted light collected by spectral detector (ChS)
![Page 58: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/58.jpg)
415 nm
686 nm
8.7 nm intensity bins
![Page 59: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/59.jpg)
Alexa 555 labelled microtubules and Mito tracker orange
mitochondria
unmixing
![Page 60: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/60.jpg)
Multiphoton
■ Multiphoton laser allows very deep tissue imaging
■ See Introduction to Multiphoton Imaging talk (Rocco, 28th Nov)
■ MP lasers can also be used for cutting experiments
![Page 61: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/61.jpg)
MP – ablation experiments
Sophie Herszterg, Vincent lab
![Page 62: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/62.jpg)
Fluorescent Recovery After Photobleaching
■ Bleach region of interest
■ Measure recovery of fluorescence
![Page 63: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/63.jpg)
0 30 60 90 1200
10
20
30
40
50
60
70
80
90
100
110
Time (s)
Inte
nsit
y
(a.u
. p
erc
en
tag
e r
ela
tive t
o p
re-b
leach
)
180815 CAP cKO +CAP 14
1
2
3
4
5
6
7
8 0 30 60 90 1200
10
20
30
40
50
60
70
80
90
100
110
Time (s)
Inte
nsit
y
(a.u
. p
erc
en
tag
e r
ela
tive t
o p
re-b
leach
)
180815 CAP cKO 7
1
2
3
4
5
6
7
8
Alex Hunt, Treeck lab
Using FRAP to investigate cell-cell interactions
![Page 64: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/64.jpg)
New Leica SP8 FALCON
Pulsed, white-light laser
460 700
Time-resolved,
single-photon
detection
![Page 65: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/65.jpg)
GFP mCherry
2658 ps
Mean tau
= 2320 ps
Roman Fedoryshchak, Treisman lab
Fluorescence Lifetime Imaging (FLIM)
■ Quantify FRET
■ Detect protein interactions
![Page 66: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/66.jpg)
Thank you for your attention!
Good sources of info… (Google is always useful too)
■ https://www.leica-microsystems.com/science-lab/
■ https://www.microscopyu.com/
■ http://olympus.magnet.fsu.edu/index.html
■ http://zeiss-campus.magnet.fsu.edu/
■ http://micro.magnet.fsu.edu/
■ https://svi.nl/
■ Imaging Helpdesk (twice monthly in cafe)
![Page 67: Introduction to confocal microscopy - Crick · The CALM lectures 17th Oct Intro to Light Microscopy (Kurt Anderson) Auditorium 1 @10:00 24th Oct Intro to Confocal Microscopy (Matt](https://reader030.vdocuments.us/reader030/viewer/2022040814/5e5ab02288bd643c1c0a118f/html5/thumbnails/67.jpg)