INTRODUCTION TO CONFOCAL MICROSCOPY

Matt Renshaw October 2018 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 Introduction to confocal microscopy

Widefield Confocal

(yes, both are GFP z stacks) widefield confocal

1

0.8 wf 0.6 cf

0.4 normalised intensity 0.2

0 0 6 11 17 22 distance (microns) Take home message

Confocal microscopy improves the optical resolution and contrast of your images by reducing out of focus light Talk outline…

■ Basics of fluorescence microscopy and the principle of confocal microscopy ■ Multi-dimensional image acquisition with a point scanning confocal microscope ■ Advanced applications Jablonski diagram Stokes shift Epifluorescence light path

www.leica-microsystems.com/science-lab/fluorescence-in-microscopy/ svi.nl/FluorescenceMicroscope Microscopy is: • creating a magnified, blurred image of your object of interest. Components of blur… 1. Point Spread Function

How does the microscope image an infinitely small point of light? Sub-resolution bead

Single point of light Airy Disk

Light waves interfere and converge on the focal point, creating a diffraction pattern called the Airy Disk. Separating objects – the Airy Disk

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 http://olympus.magnet.fsu.edu/ Size of the Airy Disk determines minimum resolvable distance of 2 objects Sub-resolution bead in 3D XY YZ

Focal plane

YZ Focal plane Point Spread Function: the 3D diffraction pattern

X

Z

microscopy.fsu.edu detector Sources of blur pinhole excludes out of focus light, creating an optical section 1. Point Spread Function 2. Light from out of focus areas pinhole

X

Z excitation light

focal sample plane Pinhole blocks diffraction rings

Pinhole

y

x

Optical section at resolution limit Pinhole increases contrast by excluding light from the diffraction rings

Does the pinhole increase resolution? Laser scanning confocal

Widefield Laser illumination scanning Image is built up pixel by pixel

Emission light is descanned = focussed onto a static position No pinhole Pinhole excludes out of focus light

10 µm 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 • Numerical Aperture determines: • how much light from the sample is collected by the objective • resolution • the thickness of sample that appears in focus

Measured Width of 0.175µ Bead Resolution of 0.175µ Bead Pair

1.1 1.1 1.0 1.0 0.9 0.9

0.8 1.4 NA 0.8 1.4 NA 0.7 0.7 210 nm 0.6 0.6 0.5 0.5 0.4 0.4 0.7 NA 0.7 NA 0.3 0.3

Normalized Intensity Normalized 570 nm Intensity Normalized 0.2 0.2 0.1 0.1 0.0 0.0

0.00 0.15 0.29 0.44 0.59 0.74 0.88 1.03 0.07 0.22 0.37 0.51 0.66 0.81 0.96 1.10

-1.18 -1.03 -0.88 -0.74 -0.59 -0.44 -0.29 -0.15 -1.10 -0.96 -0.81 -0.66 -0.51 -0.37 -0.22 -0.07 Microns Microns ■ Numerical aperture NA = n sin α

1.22 •  ■ Resolution d = 2• NA n• ■ Depth of focus D  NA2 Optical Sectioning: Confocal Microscopy

Other optical sectioning techniques are available.

See: Intro to Optical Sectioning talk Talk outline…

■ Basics of fluorescence microscopy and the principle of confocal microscopy ■ Multi-dimensional image acquisition with a point scanning confocal microscope ■ Advanced applications The Tetrahedron of Frustration Standard applications

■ Although there are subtle differences, all of our confocals have these basic functions ■ Software varies, but settings are there somewhere… Excitation and Emission Excitation lasers Detection

■ Adaptable wavelength selection ■ Simultaneous or sequential detection Brief comparison of detectors

www.leica-microsystems.com/science-lab/ Optimising acquisition settings

■ Laser power ■ Gain ■ Number of pixels ■ Scanning speed ■ Size of scanning area ■ Averaging Increase laser power = increase signal

Increase laser power = increase photo damage Increase laser power = increase signal

Increase laser power = increase photo damage Increase gain = increase signal

Increase gain = increase noise Increase gain = increase signal

Increase gain = increase noise Finding a compromise Finding a compromise

Noise can be negative, addition of offset prevents false zeros Finding a compromise

Noise can be negative, addition of offset prevents false zeros Achieving resolution: number of pixels 512 x 512 ~500µm per pixel 1024 x 1024 ~250µm per pixel

More pixels, more time Achieving resolution: number of pixels 512 x 512 ~500µm per pixel 2048 x 2048 ~125µm per pixel

More pixels, more time Achieving resolution: optical zoom 1024 x 1024 ~250µm per pixel 1024 x 1024 ~125µm per pixel

Zooming reduces size of scanned area Line averaging reduces noise, but takes longer

Averaging 1 Line averaging reduces noise, but takes longer

Averaging 4 Line averaging reduces noise, but takes longer

Averaging 8 Reduce scanning speed, reduce noise Reduce scanning speed, reduce noise Imaging 3D volumes

www.thorlabs.com Imaging deep, increases light scattering

Laser power and gain can be adjusted through z stack to compensate Talk outline…

■ Basics of fluorescence microscopy and the principle of confocal microscopy ■ Multi-dimensional image acquisition with a point scanning confocal microscope ■ Advanced applications Nikon W1 spinning disk confocal ■ Speed ■ Reduced photo-toxicity ■ Photo manipulation 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 Zeiss LSM 880 with Airyscan Pinhole diameter, resolution and transmission

0.2 AU = 1.4 x improvement 1.1 100

1.0 80

0.9 60

0.8 40 Resolution 20 0.7 5.

Pinhole transmission % Pinhole transmission 0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 0.5 1.0 1.5 2.0 2.5 3.0 4.0 4.5 5.0 Pinhole diameter (AU) Pinhole diameter (AU) Airyscan detector is 32x 0.2 AU mini GaAsP detectors

0.2 AU

Joseph Huff Nature Methods volume12, page1205 (2015) Conventional

Airyscan

Alex van Vliet, Tooze Lab Spectral imaging and unmixing

■ separating fluorophores with close emission spectra

e.g. Alexa fluor 555 and Mito Tracker orange Relative intensity (%) intensity Relative

Wavelength (nm) Whole spectrum of emitted light collected by spectral detector (ChS)

http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html 8.7 nm intensity bins

415 nm

686 nm Alexa 555 labelled microtubules and Mito tracker orange mitochondria

unmixing 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 MP – ablation experiments

Sophie Herszterg, Vincent lab Fluorescent Recovery After Photobleaching

■ Bleach region of interest ■ Measure recovery of fluorescence Using FRAP to investigate cell-cell interactions

180815 CAP cKO +CAP 14 180815 CAP cKO 7

)

)

h

h

c

c

a

a

e

e

l l 110

110 b

b

- - 1

1 e e 100 100

r

r

p

p

90 90 2 2

o

o

t t 80

80

e

e

y y 3 70 3

t 70 v

t

v

i

i

i

i

t

t

s s 60

60 a

a

l

n

l n 4 4

e

e

e e 50 50

t

r

t

r

n

n

e

I

e I 40 5 40 5

g

g

a a 30 30

t t 6 6

n n 20 20

e

e

c c 10 7

10 7 r

r

e e 0 0

p

p

8 8 . . 0 30 60 90 120 0 30 60 90 120

u

u

.

.

a

a

( ( Time (s) Time (s)

Alex Hunt, Treeck lab New Leica SP8 FALCON Time-resolved, single-photon detection

460 700

Pulsed, white-light laser Fluorescence Lifetime Imaging (FLIM)

GFP mCherry

■ Quantify FRET ■ Detect protein interactions

Mean tau = 2320 ps

2658 ps

Roman Fedoryshchak, Treisman lab 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)