Boulevard du Temple Daguerrotype (Paris,1838)

Nyquist sampling for movement

a busy

street ?

CONFOCAL

MICROSCOPY

BioVis

Uppsala, 2017

Jeremy Adler

Matyas Molnar

Dirk Pacholsky

Widefield & Confocal Microscopy

Widefield Confocal Laser Scanning Microscopy (LSM)

MRC 500 confocal

microscope

Patent application 1957

First Confocal Microscope

Illumination

Confocal

Widefield

AREA SPOT

Build an image by scanning a single illumination spot

Bidrectional undirectional misaligned

Cells moving during image acquisition confocal

Illumination - fluorescence

Lens

Fluorophores inside

the illumination cones are excited

Compare with Multiphoton fluorescence

Focal plane

Widefield

Confocal LSM

Confocal Components

Objectives

Numerical aperture (resolution)

Immersion medium: air, water, oil

Corrections: spherical, chromatic

Working distance

Coverslip thickness

Transmission

Magnification – not very important

Miniature objectives

Same magnification - different NA

20x NA 0.8 5x NA 0.16

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

2 200

2 400

2 600

2 800

3 000

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4

Numerical Aperture

Sampling Interval nM

Z-Axis

XY- plane

Variation of Resolution with the NA Resolution & NA of objective

Nyquist sampling – according to SVI

NA

Z axis

XY plane

Confocal Illumination Lasers

monochromatic coherent

types: gas, solid, diode

Argon Ion 353-361, 488, 514 nm

Krypton -Argon 488, 568, 647 nm

Helium Neon 543 nm, 633 nm

Helium Cadmium 543 nm, 633 nm

Diode lasers 405, 488, 635 nm etc

White light lasers tunable

Uneven illumination

From Andor

Pinhole

The maximum resolution is app. 0.15 µm lateral 0.40 µm axial

Objective Magnification NA pinhole size (µms) 60x 1.40 .40 1.90 40x 1.30 .60 3.30 25x 0.80 1.40 7.00 4x 0.20 20 100.00

Back Projected Pinhole size in the specimen

Open Pinhole

Fluorescent microspheres NA 1.4 oil, pixels 45nm

0

25

50

75

100

0 1 2 3 4 5 6 7 8 9 10

% Max

Pinhole Airy units

out

badly out

infocus

Pinhole size and intensity

Image Quality Photon Noise /Poisson Noise/Shot Noise

Compare 2 sequential mages

Difference Need More Photons

Poisson Noise – how many photons ?

0

32

64

96

128

160

0 32 64 96 128

sequential values

photons

photons 128 64 32 16

mean 126.31 64.26 31.22 15.72

SD 11.81 7.66 5.57 3.91

sqrt 11.31 8.00 5.66 4.00

Single pixels in a timeseries

Improving Image Quality Doubling the Pixel Integration time: scan speed & averaging image

MORE PHOTONS per pixel

More Photons – increase laser power ?

Lens

Fluorophore saturation More photons from fluorophores outside the focused spot.

Focal plane

Best PMTs reach 30% quantum efficiency

How good are camera ?

The Photomultiplier tube (PMT) 1930s

Fluorescent photon hits photocathode

emits photoelectron

which cascades along the dynode chain

each step amplifies electron numbers

finally output at the anode

depends on

voltage

Photon counting possible

PMT Problem

Pinhole Size: nucleus

LSM700 NA 1.4 oil adjusted for equal maximum intensity

Where do pixels come from ?

Camera

Confocal ?

Pixels

0-255

(8bit)

Best sampling rate or pixel size? Ideally : infinite small BUT each pixel generates noise The incoming signal has to be more intense than the noise (Signal:Noise ratio) Small pixels get less photons, but generate same/more noise like large pixels (who get more photons)

Practically: pixel size is twice as small as smallest detail to be resolved LSM allows you to choose the pixel size

Imaging – Nyquist theorem

Nyquist found that in order to reconstruct a pure sine wave, it must be sampled at least twice during each cycle of the wave, 2x the frequency.

Pixels – how large ?

How many pixels ? Nyquist flatbed scanner

Nyquist Pixel size: fluorophore and NA

CLSMs have an OPTIMAL button which calculates the pixel size for Nyquist

Drawbacks of small pixels :

Slow Bleaching

Find a compromise between

Image quality required versus

Sample robustness/Time

IMAGING WITH LSM

Fluorescence (excite 555nm) Transmission (555nm)

Fluorescence (488nm) & Reflection (405nm)

Single cell –GFP in a nanowire matrix

Acquisition information: metadata

recorded with the image

Reuse same setup measurements

Pixel size:

Objective’s magnification

Area scanned – ZOOM

Number of pixels

Optical sectioning: Z series Optical slice from certain depth in sample

Many slices from adjacent depths

reconstruction from slices

3D information from LSM images Orthogonal view 3D surface reconstruction

Observe that light could not´penetrate´ material on certain areas*

*

*

3 dimensional reconstruction of image

3D information , dashed lines in blue, red, green indicate position in ZXY and are movable

3D rendered Images

Spectral (lambda) scan with LSM (Zeiss)

480 490 500 510 520 530 540

550 560 570 580 590 600 610

620 630 640 650 660 670 680

QUASAR detector of LSM710 with 32 detectors or

adjust the detection range of individual PMTs

Emission spectra for fluorophore or autofluorescence

Lambda Scan– linear unmixing – separate overlapping fluorophores

Linear Unmixing determines the relative contribution from each fluorophore

for every pixel of the image.

Live imaging (time lapse)

Frame size in pixel: frames/sec 2048 x 2048 0.03 1024 x 1024 0.13 512 x 512 0.53 256 x 256 2.00 128 x 128 5.00 Smaller images – faster Use oblong images Line scan – 1 pixel wide

Fluorescent microspheres in a matrix confocal images

Faster Confocal Imaging many points

Camera not a PMT

Difficult to change pinhole

Line scanning confocals

Zeiss 5 Live

Multi spot confocals

Airyscan – replaces pinhole 32 detectors

POINT SPREAD FUNCTION A sub resolution object becomes a blob in the image

1. the NA of the objective

2. the confocal pinhole

3. wavelength of emission

4. Refractive index matching

x

z

Improving resolution

)2sin(2

nd

• λ : shorter wavelengths better (blue light)

• n : high refractive index – objective and specimen medium (oil: 1.5)

• ѳ : NA of the objective: use high as possible (1.4)

• Small confocal pinhole

Matching objective to sample

Z directio

n ”d

epth

”

A) B) C)

A) Actual situation in the sample

B) Good match of embedding and preparation of sample

C) mis match/ ´bad´sample preparation

x

z

Airy disk in XZ

Techniques for the LSM and Live Cell Imaging

http://www.cellmigration.org/resource/imaging/imaging_approaches_photomanipulation.shtml

FRAP

Photoactivation /uncaging

CALI Chromophore Assisted Light inactivation

Photoactivatable Fluorophores

• GFP activated by 413nm – observe with 488nm excitation

• Pulse chase experiments

Science (2002), 297, 1873-1877

Long Image acquisition times - problems

Temperature Control

Solution

protect the microscope

from temperature changes

Confocal v Widefield • Point scanning

• Optical sectioning – pinhole, Z series Z series

• Variable magnification – zooming

• Higher resolution (NA of objective)

• Timeseries – live imaging faster

• Motorized XY stage – tiling also

• Spectral scanning

Problems

• Slow faster

• Pinhole throws away photons all photons

• Poor detectors better

• Photobleaching reduced

Worth Looking At

The 39 steps: a cautionary tale of quantitative 3-D microscopy

Pawley Biotechniques, 2000, 884-

Seeing is believing – beginners guide to practical pitfalls in image acquisition

Pawley J Cell Biol, 2006, 172, 9-18

Websites

Microscopy: Zeiss, Leica, Nikon, Olympus, BioRad

Fluorophores: Thermo Fisher Scientific

Filters: Chroma, Omega, Semrock