Here we define Field of View (FOV) by detector size and microscope objective, and Field of Illumination (FOI) relative to the detector and in the image plane.

Ellipticallypolarized light

The extraordinary ray violates both Snell’s Law and the Law of Reflection.  It is not necessarily confined to the plane of incidence.  Its speed changes with direction.  The index of refraction for the extraordinary ray is a continuous function of direction.  The index of refraction for the ordinary ray is independent of direction.  When the ordinary index of refraction is plotted against wavelength, the dispersion curve for the ordinary ray is a single unique curve.  The dispersion curve for the extraordinary ray is a family of curves with different curves for different directions.  A ray normally incident on a birefringent crystalline surface will be divided into two rays at the boundary, unless it is in a special polarization state or unless the crystalline surface is perpendicular to an optic axis.  The extraordinary ray will deviate from the incident direction while the ordinary ray will not.  The ordinary ray index n0 and the most extreme extraordinary ray index ne are together known as the principal indices of refraction of the material.  The direction of the lesser index is called the fast axis because light polarized in that direction has the higher speed.

A half-wave plate δ = π can be used to rotate the plane of linearly polarized light.  The angle of rotation is 2θ, where θ is the angle between the angle of polarization and the wave plate's fast axis.

Right-circularlypolarized light

The two beams within the birefringent crystal are referred to as the ordinary and extraordinary ray, respectively.  The polarization of the extraordinary ray lies in the plane containing the direction of propagation and the optic axis, and the polarization of the ordinary ray is perpendicular to this plane.

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The electric field vector E can always be resolved into two perpendicular components.  The light is elliptically polarized, then the two components have a constant phase difference, and the tip of the electric field vector traces out an ellipse in the plane perpendicular to the direction of propagation.

Linearlypolarized light

Channelrhodopsin2 (ChRh2) is a light activated cation channel which can be expressed in neurons and used to control behavior in host organisms, including mice, c. elegans and drosophila. Stimulation with blue light (~470 nm), the power density, for photo-activation of ChRh2 is in the range 0.1-10 mW mm-2 and has a wide dynamic range.

Linear polarization

If a beam of linearly polarized monochromatic light enters a birefringent crystal along a direction not parallel to the optical axis of the crystal, the beam will may be divided into two separate beams.  Each will be polarized at right angles to the other, and they will travel in different directions.  The intensity of the original beam will be divided between the two new beams in a manner which depends on the original orientation of the electric field vector with respect to the crystal.  The ratio or the intensities of the two orthogonally polarized beams can have any value.

In other devices the changes in direction of propagation between the two rays is used to separate the incoming beam into two orthogonally polarized beams as in the Wollaston and Thompson beam-splitting prisms.

Subject to application requirements, Nyquist may or may not be necessary, Using an objective lens of 100X, 1.4 NA we see that Neo, Clara, iXon3 885 and Luca R are all capable of achieving the Nyquist criterion: 2 * Px = 22 µm. While at 60X 1.4 NA, only Neo and Clara can provide small enough pixels.

In a microscope system, the camera is coupled via a C-mount adapter and located in a primary image plane (PIP). The PIP is our reference for the definition of FOV and FOI.

A quarter-wave plate δ = π/2 can be used to convert linearly polarized light to circularly polarized light.  The incident linearly polarized light must be oriented at 45o to the wave plate's axes.  A half-wave plate δ = π can be used to rotate the plane of linearly polarized light.  The angle of rotation is 2θ, where θ is the angle between the angle of polarization and the wave plate's fast axis.

Circularpolarization

The figure below shows the trace of the field vector Ex = E0exp(i(kz - ωt)), Ey = E0exp(i(kz - ωt + φ)) in a plane perpendicular to the z-axis when looking towards the source.  (E0x = E0y = E0)

1/RXY is a good approximation of the maximum spatial frequency in the image. To capture all information in the image (e.g. with a CCD detector) we must sample at frequency F to avoid “aliasing errors”. This is known as the Nyquist criterion:

Circular polarized lightformula

This is the size of the smallest object the microscope can resolve, sometimes called the diffraction limit, and is also the diameter of the smallest spot to which a collimated beam of incoherent light can be focused. The shape of the spot is an Airy disk or optical point spread function, PSF, characteristic of the system.

The following variation on the Rayleigh criterion provides a definition for spatial resolution SXY of the illumination system:

Circular polarized lightmeaning

Power density, PD in the specimen plane is estimated from the ratio of beam power and area. Spectral transmission and chromatic errors in the microscope objective are critical to performance. To estimate specimen plane PDS, the Mosaic output beam power density is multiplied by the square of the magnification and the systems spectral transmission, T(l).

Circularpolarization example

Translating into the PIP with objective magnification of MO, we can compute the sensor pixel size required to fulfill the Nyquist criterion:

In a fluorescence microscope resolution is dominated by the objective lens which both illuminates and images the specimen. The objective numerical aperture (NA) and the wavelength of detected light (l) define Resolution, RXY by the Rayleigh criterion as follows:

As mentioned above, it is common to match FOV and FOI, but with active illumination other factors such as Power Density (PD) or Resolution may also be important considerations. Mosaic is equipped a 2X zoom laser collimator so you can trade FOI for PD.

When the sun is at a low angle in the sky, the sunlight reflecting off the surface of water is nearly 100% horizontally polarized because the angle of incidence is close to the Brewster angle.  Glare-reducing sunglasses are coated with a polarizer with a vertical transmission axis and therefore block the reflected light.

MicroPoint and FRAPPA use Gaussian laser beams and Gaussian beams remain Gaussian with a theoretical minimum focus spot diameter of l, where l is the wavelength. This can only be achieved if the collimated laser beam fills the objective aperture.

In the PIP, FOV is defined as the extent of the image sensor in X and Y dimensions. For consistency we define FOI as the extent of illumination in the PIP. This is convenient because we can easily calculate overlap between the two.

In the Glan-Taylor polarizing prism shown on the right the rejected (ordinary) ray is absorbed by black mounting material in the prism housing.

Linearly polarized light is a special case of elliptically polarized light.  If the light is linearly polarized, then the two components oscillate in phase,  for example Ex = E0xexp(i(kz - ωt)), Ey = E0yexp(i(kz - ωt)), φ = 0.  The direction of E and the direction of propagation define a plane.  The electric vector traces out a straight line.  For example, E = Ei = E0xexp(i(kz - ωt))i.