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