3D light microscopy techniques
The image of a point is a 3D feature In-focus image Out-of-focus image
The image of a point is not a point Point Spread Function (PSF) 1D imaging 1 1 2! NA = 0.5! NA 2D imaging 3.83 2"! NA =! 0.61 NA! 2 NA n2 3D imaging
Resolution is now an arbitrary measure of how close two point images can come such that they are perceived as separate Lord Rayleigh s criterion: " R = 0.61 # NA " z R = 2 #n NA 2 λ = 488 nm (NA = 1.4) δ = 212 nm; δ z = 780 nm (NA = 0.4) δ = 744 nm; δ z = 9.56 micron
Point image in optical sections
Two dimensional Image formation In real space, image formation is described by a convolution of the point spread function (PSF) with the emitted light from the object. In reciprocal the image spectrum is formed by multiplication of the object spectrum with the object transfer function (OTF)
3D Information transfer In analogy to the twodimensional image formation, we can determine a 3D Point spread function (PSF) and a 3D Optical Transfer function (OTF). PSF z OTF kz z=0 z=2µm
In a 3D object we have cross-talk between in- and outof-focus parts In-focus part Out-of-focus part
Result is a blurred image with substantial background intensity
Reduce out-of-focus information by inserting a pinhole Illumination / exitation pinhole emission pinhole confocal planes
Result: much sharper pictures non-confocal = wide-field confocal
Another example using high resolution imaging 1 µm Nuclear pore complexes
In practice, confocal microscopes are point scanners PMT replaces the CCD camera Laser replaces the arc lamp
Thick sample imaging
Confocal vs widefield microscope J sharp optical sectioning L point-scanning method (slow) L majority of returned photons not detected wait for a long time to get robust signal even slower Photodetector noise gets critical (weak SNR) Photodamage on sample J nice additional features: use programmability of laser scans for bleaching experiments for selective point measurements in small volumes (spectroscopy, fluorescence correlation spectroscopy)
Image formation in the confocal microscope Again the image is formed by a convolution, but the confocal PSF is smaller and has no butterfly wings. z kz Widefield PSF confocal The optical transfer function has an ellipsoidal shape and has no discontinuity in the middle- optical sectioning confocal OTF
Optical sectioning with the confocal microscope z object The confocal microscope can t resolve fine features along the z direction, but can create sharp images in the focal plane optical sectioning z confocal image x z y
Fibrinogen on Ti/Ti surface Control using photobleaching 50 µm 50 µm
Addressing the speed issue by parallel scanning methods
Practical problems and a solution Low light efficiency (1% of light energy used) Unequal illumination (not all pinholes equally bright) Reflection of unused light in emission path Micro-lenses laser spot pinholes collector disk Nipkow disk After Perkin Elmer http://lifesciences.perkinelmer.com
Light efficiency 60% Max 350 images / s (longer exposures by integration) The micro-lens array solution
Summary: spinning disk confocal J Ideal for confocal live cell imaging (and other realtime processes) L Cross-talk between pinholes (less optical sectioning power than LSM; in particular with the plane mirror test) K Synchronization between camera and disk K Non-modular (one disk for one objective lens setup)
Multi-photon microscopy
Fluorescence fundamentals
2 photon microscopy 10ns 100fs lens Pulsed lasers (typically Ti:Saph ) and tight focusing increase the photon flux. Linescan using confocal and 2 photon microscopy Because of the extremly high photon density at the focal point, it is possible that two photons interact simultaneously with a fluorophore. No bleaching in the out-offocus planes, but increased photo-bleaching in the focal plane (~10faster)!
The multi-photon microscope (in comparison to conventional and confocal microscopy)
The major advantage is the ability to reduce the influence of light scattering in the sample Scattering of emitted rays Capture of scattered, emitted rays Scattering of excitation rays Less scattering of excitation rays (long wavelength)
Demonstration of 2-photon performance on a pollen grain 20 µm
Major advantages (and usefulness) Imaging of scattering samples Deep sections and whole tissue imaging Maximal use of light Shorter exposure times and levels Low photobleaching outside the focal volume Long observation possible Low photo-toxicity
Summary: multiphoton microscopy J Thick section imaging J Long duration live cell microscopy L Lower resolution compared to confocal Long wavelength excitation L Thermal damage from chromophores that absorb in the IR spectrum L Dependent on fluorescence L Expensive (requires a pulsed laser setup)