Understanding Light: Properties and EM Spectrum

Introduction to Light

• Electromagnetic waves are pervasive, utilized extensively, and increase in energy as their wavelengths shorten.
• These waves travel through free space at the speed of light, encompassing a spectrum from radio waves to gamma rays.
• Visible light falls within the 380-750nm range.
• Electromagnetic waves consist of electrical and magnetic vibrations oscillating in perpendicular waves, originating from a source.
• Wavelength defines the distance between two crests or troughs, while frequency measures wave cycle passage through a point.
• Amplitude is the vertical distance from crest to trough, indicating intensity or brightness.
• Wavelength and frequency have an inverse relationship; energy is proportional to frequency.
• Visible light spans 380-750nm, transitioning from red (750nm) to violet (380nm).

Properties of Light

• Light bends/refracts entering a water droplet, with shorter wavelengths (blue) bending more.
• Light reflects off the droplet's back and refracts again upon exiting, enhancing color dispersion and creating a rainbow.
• Reflection occurs when light strikes a medium's surface without penetration.
• Refraction involves light bending as it passes through a medium.
• Absorption happens when light is taken in by a material, leading to energy transfer.
• Transmission occurs when light penetrates and travels through a transparent object.
• Snell's Law explains light's path when crossing between two media, noting a direction change from air to water.
• The ratio of sines of incidence and refraction angles equals phase velocities in the two media.
• Snell determined refractive indices (RI) based on light direction changes; RI in a vacuum is 1, with all other materials >1.
• Diffraction occurs when light waves spread out near a barrier.
• Thomas Young's double-slit experiment demonstrated interference.
• Constructive interference results in a bright fringe when crests coincide, adding amplitudes.
• Destructive interference yields a dark fringe when a crest coincides with a trough, canceling each other out.
• Diffraction patterns are dictated by light wavelength and aperture width; diffraction does not occur if the wavelength is smaller than the aperture.
• The interference pattern is wavelength-dependent, with shorter wavelengths more likely to interfere with smaller objects, causing greater light scattering.

Light and Microscopy

• Transmitted light microscopy relies on light waves passing through a specimen.
• Bright-field, phase-contrast, and differential interference contrast (DIC) are the main types of transmitted light microscopy.
• Inferences about object structure are drawn from changes in light properties.
• Reflection (fluorescence) microscopy uses a reflected light path, capturing light information from the specimen in a camera or eyepiece.
• Compound microscopes magnify the image first through the objective lens, then further with an additional lens.
• Upright microscopes have the sample on a stage below the objective lenses.
• Inverted microscopes have the sample on a stage above the objective lenses, for live-cell imaging.
• Compound microscopes can support transmission and/or reflectance (fluorescence) light microscopy.

Dimensions and Scale

• 2D data is an image taken at one focal plane with 'x' (width) and 'y' (height) dimensions, known as the lateral XY dimension.
• 3D data captures XY dimensions and Z (depth), known as the axial dimension (XZ or XY).
• Time series data allows observation of specimens over time (T) in 2D (XYT) or 3D (XYZT).
• More dimensions provide more information.
• Optical microscopes can resolve structures of 0.1-0.3mm.
• Super-resolution (optical) microscopes or electron microscopy is used for smaller images.

Magnification and Resolution

• The objective lens gathers light from the specimen and creates a magnified image.
• Objective lens is responsible for primary image formation and helps determine image quality.
• Key properties inscribed on the objective lens barrel: Magnification, working distance, Numerical aperture.
• Magnification power (M) depends on focal distance (F) and the distance of the image plane from the lens (h), expressed as M=h/f.
• At constant (h), a shorter focal distance lens has greater magnification than a longer one.
• Working distance is the distance between the front of the objective lens and the coverslip when the specimen is in focus.
• Numerical Aperture (NA) is a dimensionless number characterizing the range of angles over which the objective can accept light.
• A higher NA objective collects light over a larger angle and accepts more light than a low NA objective.
• Immersion objectives allow more light to be collected by reducing scatter.
• Microscope resolution (δ) is the minimum separation needed to distinguish two points as separate.
• Resolution depends on detected light wavelength (λ) and objective NA, per the Rayleigh criterion (a modification of the Abbe resolution limit) δ=1.22λ/NA.
• Smaller δ indicates higher resolution.
• Higher NA objectives provide better resolution.
• Shorter wavelengths result in smaller/greater resolution.
• Abbe's formula and the Rayleigh criterion are different methods for determining the resolution of a light microscope.
• Abbes method takes the NA and wavelength, Rayleigh uses the diffraction pattern

Refraction and Image Quality

• Refractive Index (RI) mismatch occurs when light travels through materials with different refractive indexes.
• Abbe's formula states resolved distance (d), where NA = (nsinα), and n = RI: d=λ/2(nsinα).
• Higher n (RI) values improve resolution (smaller fraction).
• Using the correct immersion objective avoids RI mismatch and resulting spherical aberration (distortion).
• RI mismatch leads to artifacts and aberrations in the final image.
• Reducing RI mismatch involves growing/mounting cells directly onto a coverslip.
• Use glycerol-based mounting mediums with glycerol/oil objectives.
• Water immersion objectives are used with aqueous samples, especially live samples.
• Use No. 1.5 (0.17mm thick) glass coverslips when using objectives with an NA higher than 0.4.
• Outcomes of RI mismatch include loss of signal, limited optical resolution, degraded scattering, decreased image contrast, and optical aberrations (primarily spherical).
• Spherical aberrations occur due to refraction differences across a spherical lens, causing light entering near the edge to not focus at the same point.
• This is due to a mismatch between RI of immersion and specimen media.
• Live cultured cells in aqueous media require a 40x water immersion objective.
• Blood smears mounted in glycerol require a 100x glycerol immersion objective.
• Dry samples mounted without a coverslip require a 20x dry objective.
• Optimal cover glass thickness is indicated on the objective barrel.
• High-quality or immersion objectives perform best with a 0.17mm coverglass.
• Incorrect thickness reduces contrast and resolution.

Lens Types and Corrections

• Modern objectives account for multiple correction factors, including chromatic and spherical aberrations.
• Infinity correction allows additional components to be introduced into the light path.
• Flat field correction accounts for field curvature.
• Chromatic aberrations occur when a lens can't focus all wavelengths of light to the same point.
• Lateral chromatic aberration is the focusing of light colors occurs in an incorrect manner and is perpendicular to the light path. Is reduced with an achromatic lens.
• Axial chromatic aberration occurs from light made up of different component wavelengths, which are refracted to different extents after passing through the convex length. It is corrected using an achromat lens.
• Spherical aberrations occur due to lens structure and geometry.
• Light passing through the periphery of a spherical lens is refracted more.
• Also occur due to RI mismatch.
• Standard Achromat lenses don't account for field curvature, where the perimeter is out of focus.
• Plan achromats are corrected for field curvature.
• Achromat/Achromatic lenses correct chromatic aberration for red & blue component wavelengths.
• Achromat/Achromatic lenses brings red & blue wavelengths to the approximate same focal plane as green wavelengths
• Achromat/Achromatic lenses made of 2 lens elements (lens doublets) with different RI and dispersive properties
• Achromat/Achromatic lenses are also corrected for spherical aberration
• Fluorite/semi-apochromatic lenses correct chromatic and spherical aberration for 2-3 component wavelengths.
• Fluorite/semi-apochromatic lenses are made using glass formulations that contain fluorspar in addition to lens doublet
• Apochromat/Apochromatic lenses correct chromatic and spherical correction up to 4 wavelength
• Apochromat/Apochromatic lenses are made with 2 lens doublets and a lens triplet
• Apochromat/Apochromatic lenses are most highly corrected available
• Plan objectives (inc. plan achromat, plan fluorite, plan apochromat) are additionally corrected for field curvature

Different Stains

• Staining or optical techniques introduce contrast as samples are transparent are hard to be seen under bright-field microscopy: the use of differential stains, phase contrast techniques, differential interference contrast (DIC), fluorescence microscopy
• Differential stains can absorb different wavelengths of light, like Gram stain and Masson trichrome stain.
• Absorption of wavelengths is based on additive and subtractive color theory.
• Stains act as filters to absorb particular color wavelengths to achieve contrast with differential staining.
• Magenta stain absorbs green, lets through red & blue
• Cyan stain absorbs red, lets through blue & green
• Yellow stain absorbs blue, lets through green & red
• Stains can be used alone or in combination.
• Using all 3 stains above results in black.

Phase Contrast

• Contrast methods are required when a differential stain can compromise the sample (e.g., stains could ball we tissue)
• Phase Contrast and DIC are 2 microscopy techniques that help visualise phase changes that occur when light travels through a specimen
• Optically dense objects (high refractive index) in the sample can induce phase changes
• Phase and DIC techniques can used to detect the differences in RI and cell density and convert these phase shifts into visible differences in light intensity
• Phase contrast is particularly useful for live cell imaging
• Phase contrast microscopy relies on being able to detect and amplify the phase changes that occur as light passes through a specimen
• Light from the lamp source needs to pass through a phase ring in the condenser and deviate in such a way so that the light that passes through the specimen will be collected in line with the phase plate
• The objective phase plate allows for further phase changes in the light
• Use a lens with a phase plate, In addition, the objective lens has achromatic doublets and triplets for color correction, and a meniscus lens for flat-field correction
• The condenser annulus and phase plate must be aligned using a centering telescope or Bertrand lens before Imaging
• Bacteria or sub-cellular organelles that have high refractive indices will appear dark while less optically dense regions appear lighter (cytoplasm, non-cellular regions)
• As bacteria induce very strong phase changes it is the method of choice for bacteriologists
• Phase contrast has Halo effect → meaning can't focus edges

Differential Interference Contrast (DIC)

• Most modern research-grade microscopes are already configured to include DIC
• DIC is Prefered contrast method for live imaging of larger eukaryotic cells
• DIC detects gradients of refractive index change rather than detecting high refractive index objects, resulting in a 3D-like image.
• DIC can produce thin optical sections free of obscuring disturbances from specimen features positioned beyond the immediate focal plane
• DIC has better depth discrimination (axial resolution) than phage contrast
• Because the aperture of the condenser is fully utilised (no phase ring), the lateral resolution obtained with DIC is also greater than the phase contrast technique
• In DIC polarised light is separated into 2 wavefronts that are separated by a small distance by the (condenser) Normarski (or Wollaston) prism
• The 2 wavefronts pass through the specimen and are retarded to varying degrees the wavefronts enter the objective Normarski (Wollaston) prism and are recombined
• A phase shift is then detected as elliptically polarised light, and non-phase shifted light is removed by the analyser
• DIC light path: light Source → Polarising filter 1 → Prism 1 → condenser lens → Sample and slide → Objective lens Prism 2 → Polarising filter 2

Phase Contrast vs DIC

• DIC is more useful for thick specimens (due to higher resolution in 3 dimensions).
• DIC has Increased resolution giving a sharper image.
• DIC minimises the halo effect.
• DIC may not be suitable for cells grown on plastic or adhered to plastic as plastic can interfere with light polarization.

Historical Context and Key Figures

• Von leeuwenhoek created the first microscope with ×200 magnification to see RBC,WBC
• Ernst Abbe determined the minimum resolved distance with d=λ/2(nsinα).
  • d = Minimum resolved distance
  • λ = wavelength
  • n = RI
  • α = half the angular intake of the lens.
  • (nsinα) = numerical aperture
• Resolution is limited to half the wavelength of light.
• Optical microscope = 200 nm; therefore, can't resolve anything below 100 nm (e.g., viruses, proteins).
• Objective lens is most important, responsible for primary image formation.
• Condenser lens makes a cone of light and projects evenly onto the sample.
• Condenser lens' NA can be adjusted with a condenser aperture to match the objective lens NA.
• Kohler Illumination is the most important variable for high-quality images.
• When in focus should see an Image of the field diaphragm with In focus edges and centered

Fluorescence Microscopy

• Fluorescence Microscopy provides high sensitivity, specificity and contrast images of specimens.
• Fluorescence Microscopy can provide greater detection and imaging of intracellular or sub-cellular Structures and processes, compared to conventional light microscopy.
• In fluorescence microscopy light originates from the object rather than passing through it.
• Sir George Stokes created fluorescence in 1852.
• Fluorescence is luminescence caused by the absorption of incident light radiation at one wavelength, followed by the near immediate emission at a Longer wavelength and that ceases almost immediately when the incident radiation light stops
• A Molecule that can emit fluorescence is a fluorophore, which is a molecule with aromatic ring structures
• The excitation of a fluorophore by a particular wavelength (e.g. blue light) results in the emission of a longer wavelength (e.g. green light)
• Quantum Yield is the ratio between the number of photons emitted and the number of photons absorbed
• Quantum Yield is a value between 0 and 1, with 1 being the highest level and 0 being the lowest
• Brightness of a fluorophore is determined by quantum yield and its extinction coefficient
• For experiments it is recommended to select fluorophores that high extinction coefficient (i.e absorb more light) and a high quantum yield
• Stokes Shift is the difference between the maximum absorption wavelength and the maximum emission wavelength of a fluorophore.
• Stokes Shift is important as it influences the choice of fluorescence filters that you use in order that the strong excitation of the microscope is separated from the weak emitted light of the fluorophore

Fluorescence Mechanism

• Excitation: Light is absorbed and causes energy to rise from the ground state to an excited state.
• Loss of energy: the excited state is unstable so energy is lost to semi-stable
• Emission: Light is emitted at a Longer wavelength
• The range of light that can be absorbed is the excitation spectrum, and the highest amount of absorbed light is excitation max
• The range of light emitted is the emission spectrum, and the most emitted wavelength Is emission Max
• Shinging light from either side of the excitation max causes lower-intensity emission
• Stokes shift is the difference between the absorbed wavelength and emitted wavelength

Fluorophores

• Fluorophores are molecules that can re-emit light upon light absorption (excitation)
• Fluorophores are mostly small