Basic LM, Optics & Resolution

What is empty magnification?

• Magnification beyond 500-1000x the numerical aperture of the objective

• Increases image size but adds no detail → can lead to image degradation

What is numerical aperture (NA)?

• It is the light gathering ability of an objective

• The higher the NA, the more powerful the system, and the better the resolution

• NA = n x sin(θ)

  • n = refractive index of the immersion media,

  • θ = the half-angle of the maximum cone of light the objective can collect

What is contrast?

The visibility of structures against a background

What is resolution? What two parameters determines reolsution?

• The shortest distance between two point sources where they’re observed as two individual parts.

• The smaller the resolution, the more powerful the system.

• Determined by NA and wavelength

• d = 0.61λ/NA

What is an Airy disk and how does it impact resolution?

• It is the diffraction-limited distribution of intensities

• Features a bright central maximum surrounded by diffraction rings

• The resolution is limited by this diffraction, as it determines how close 2 Airy disks can get before they merge, and information is lost

What is maximum theoretical resolution?

Defined by when the first maximum of an airy disk coincides with the first minimum of another airy disk.

• Dotted lines are the maximum theoretical resolution

What is a point spread function (PSF)?

• Describes how the optical system blurs a point source

• It characterises resolution in 3D (in Z direction too), and the PSF determines the minimum size you can resolve

• Objects smaller than the PSF appear the same size as the PSF

  • Therefore, the smaller/narrower the PSF, the better the resolution, but it depends on what the experiment requires

What effect does a confocal pinhole has on the PSF?

The pinhole blocks out-of-focus emission, thus resulting in a narrower PSF, and a better resolution.

• The wider the pinhole, the more light reaches the detector, and the opposite occurs

Why can you image a single fluorescent protein but NOT measure its size?

• Because the protein is much smaller than the PSF (~250–300 nm).

• The image shows only the Airy disk of the optical system, not the actual size of the molecule.

• The PSF sets the measurement floor.

Spherical aberration?

• Occurs when the focus at the centre of the lends differ from the periphery

• Can be caused by a mismatch in refractive index between immersion medium and sample

• Results in: blurry image, loss of resolution, loss of brightness, Z-position inaccuracy

Chromatic aberration?

• Occurs when different wavelengths of light focus at different focal lengths

• Happens as the lens has a wavelength-dependent refractive index

• Results in: color around objects and multi-color images that are out of focus relative to each other (shifted)

No matter how advanced the microscope system is, there are 3 parameters that we add as users, what are they?

• Sample

• Immersion media

• Coverslip

What is the refractive index mismatch, and what aberration does it cause?

• Happens when the refractive index of the sample medium differs from the objective immersion medium

  • (e.g., aqueous sample n=1.33 with oil objective n=1.5).

• Light rays bend differently at the interface, causing mainly spherical aberration

Describe 3 contrasting enhancing techniques in Brightfield microscopy?

1. Phase contrast:

   • Due to cells having different thicknesses, and thus refractive indices, the light gets delayed, creating a phase shift

   • Phase contrast converts the invisible phase shift into visible brightness differences

   • Parts of the cell appear darker/brighter than the background

   • Optimal for unstained living cells (like bacteria) or thin samples

     • Not optimal for thicker samples

2. Differential interference contrast (DIC):

   • Pseudo 3D method, with extremely thin focal depth

   • Based on polarisation microscopy, where polarisers are positioned perpendicularly to block out background light

   • Uses Normanski prism to split light into 2 beams that passes through the sample

   • If one beam passes through a thicker/denser area, and the other beam through a thinner, they are delayed differently, which is converted to intensity contrast by DIC

   • Optimal for unstained cells, edges, organelles, fine

3. Darkfield:

   • Provides an image formed entirely from diffracted light with a black/dark background

   • Blocks out direct transmitted light from entering the objective, so only light scattered from the sample enters

   • Optimal for viewing samples without labelling, like pollen, and for small objects