Laser scanning or spinning disk? Which confocal microscope to use, and why?

 
Laser Scanning confocal microscopy
Spinning disk confocal microscopy
Preferred application Typically used on fixed specimens 4D (time-lapse) imaging of living cells.
How it works
The excitation laser source is scanned across the specimen in a point-by-point raster pattern, so that, over time, a complete image of the focal plane is collected. The emitted light is then collected by the objective, passed through a pinhole aperture and detected by a photomultiplier tube. The resulting image is reconstructed and displayed by a computer. By compiling multiple optical sections of the specimen sequentially, the entire specimen can be reconstructed in three dimensions for analysis.
Our systems consist of a "sandwich" of two coaxial spinning disks. First, an array of microlenses efficiently guides the laser beams into pinholes of a second disk, the Nipkow disk. A merge module allows micro-second switch of excitation wavelengths. The Nipkow spinning disk consists of a thin wafer with hundreds of pinholes arranged in a spiral pattern. When placed in the internal light path of the confocal microscope, the spinning disk diffuses the light over the entire field. The pinholes of the disk permit only perpendicularly oriented rays of light to penetrate. Emitted light is reflected back through the microscope objective and captured by a full-frame CCD camera.
Strengths
-Offers the highest level of confocality and the ability to do extremely thin optical sections of specimens.
-Near-total control of where and how a very bright illumination source strikes the specimen.
-Optimized for use where transmission, or speed, is a higher priority than ultra-thin sectioning.
-Since this method applies low dose of multiple excitation beams to the specimen, fluorescence bleaching is very slow and the damage to sample (phototoxicity) is very low.
Weaknesses
-Emission from the specimen must be very bright, and the objective used of the highest numerical aperture, or light gathering ability.
-The intensity of the laser light causes photobleaching in fluorescent probes, and phototoxicity in the specimen itself.
-The point-by-point image acquisition is time consuming, making the system ineffective for recording short-time-period events.
-Specimen movement during the raster scan results in jagged edges in the image and poor definition of intracellular details.
-With their larger pinhole, these systems cannot deliver the same thinness of optical sectioning as their laser-based cousins, and offer thus less confocality.
The head reduces the light throughput and weak signals are hard to detect.
Caveats
- Sample preparation, protection of the sample from epifluorescence
- Manipulation of microscope to minimize exposure of sample to light
- Keep the room as dark as possible

Long Distance 20X and 40X objectives (LSM1 only): for thick samples

DIC Optics Using differential interference contrast (DIC) one can obtain a very distinct image that appears three-dimensional. Polarized source light is separated into two beams before passing through the specimen. In any part of the specimen in which adjacent regions differ in refractive index the two beams are delayed or refracted differently. A prism in the objective lens recombines beams, resulting in differences in brightness due to differences in refractive index or thickness in the specimen.

Meta Spectral Imaging: Using the concept of Emission Fingerprinting, the LSM 510 META detector is able to separate even widely overlapping emission spectra, allowing for more precision in fluorescent imaging. Fluorochromes that resemble other emission spectra peaks can be used simultaneously through the technique of linear un-mixing.

Live cell imaging: Spinning disk confocal microscopy is suited for live cell imaging. SD1 is designed for applications where high speed and high sensitivity are required. SD2 is a versatile system designed for high-resolution live cell imaging.

Multi-dimensional Imaging: z-stack (3D), time-lapse, and multi-color allow up to 6-dimension imaging.

FRET Imaging: Fluorescence Resonance Energy Transfer (FRET) occurs when the fluorescence emission energy from an excited donor (typically a CFP) excites an acceptor (typically YFP) resulting in the acceptor fluorescence. FRET only occurs if the molecules are in very close proximity so it can be used to map out interactions between molecules. So one would excite CFP but if there is FRET the fluorescence emission would be yellow. This is not a trivial experiment and requires a great deal of time to be invested in setting up good controls and good probes for analysis. A very good FRET pair will give at most 20% FRET efficiency.

FRAP (LSM1, LSM2): Fluorescence Recovery After Photobleaching (FRAP) is used to bleach out fluorophore and follow the recovery of fluorescence that will reflect the dynamics of the fluorescently labeled molecule (i.e. protein, lipid, RNA etc.).

Acquisition softwares:
  LSM1, LSM2: Zeiss LSM5
  SD1, SD2: Improvision Volocity 3DM
  Deco1: Improvision Volocity 3DM or Openlab