Microscope Light Properties and Optics

Module 1 Objectives

• Understand light properties, wave theory, light transmission through a specimen, and microscope image magnification.
• Learn how to obtain contrast in stained and unstained cells, optical components, aberration rectification, improving structural details, and resolution
• Learn how to determine microscope resolution, identify optical aberrations in microscopic images, and match objective lenses to specimens.

Introduction to Light

• Electromagnetic waves have different wavelength, microwaves, infrared, visible light, ultraviolet light, x-ray, and gamma ray; shortest to longest, respectively.
• Energy of electromagnetic waves increases as their wavelength decreases.
• Visible light wavelengths are between 400 and 700 nm
• Electromagnetic waves consist of electrical and magnetic vibrations that oscillate in perpendicular waves.
• Wavelength is the distance between two crests or troughs.
• Frequency is how often a wave cycle passes through a given point.
• Wavelength distance decreases as frequency increases; they have an inverse relationship.
• Amplitude is the vertical distance from the rest point to the crest, or rest point to the trough.
• Higher amplitude results in higher intensity or brightness of light.

Visible Light Details

• Energy of an electromagnetic wave is proportional to frequency.
• Low-frequency radio waves and high-frequency gamma rays.

Properties of Light

• Light bends or refracts as it moves from air to water.
• Shorter wavelengths, like blue light (500nm), bend more than longer wavelengths, like red lights (700nm)
• Light is reflected off the back of a droplet and refracted again upon exiting to form a rainbow.
• Reflection - Light strikes the plane's medium without penetration
• Refraction - Light shifts when passing through a medium, bending the light.
• Absorption - Light is absorbed by the material and undergoes energy transfer.
• Transmission - Light hits a transparent object, penetrates, and travels through.

Law of Refraction and Snell's Law

• Refraction shifts light when it passes through a medium, bending the light.
• Snell's Law explains the relationship between the path of light as it crosses a boundary between two media. The path is affected by phase velocities.
• Indices of refraction (RI) or numeric value of a light changes direction
• Refraction index of a pure vacuum = 1.
• Refraction index of any other substance > 1.

Diffraction and Interference

• Diffraction of light occurs when light waves pass near a barrier and spread out.
• Diffraction occurs in all waves (Ripples in ponds)
• If crests coincide in waves, wave amplitudes will add together, causing constructive interference and light fringe.
• If amplitudes are equal in waves, amplitude doubles.
• If crests of one wave coincide with the troughs of another wave, destructive interference and dark fringe occurs.
• Diffraction pattern is determined by the wavelength of the light and the aperture width.
• Diffraction will occur if the wavelength is larger than the aperture width.
• Shorter wavelengths interfere more with smaller objects and cause more light scattering.
• Diffraction of light limits the resolution with conventional light microscopy techniques.
• The light will pass through the back aperture

Light and Microscopy

• Compound microscopes have an objective lens to magnify as the first lens, and an ocular lens as the second lens.
• Upright microscopes have samples that are placed on a stage below the objective lenses.
• Inverted microscopes have samples that are placed above the objective lenses and are used for live-cell imaging.
• Transmitted Light Microscopy relies on light waves passing through a specimen.
• Reflection (fluorescence) microscopy utilizes reflected light path.
• Light is reflected off the specimen and collected in a camera/eyepiece.

Dimensions and Scale

• 2D images are taken at one focal plane (lateral XY dimension).
• 3D images capture with width (x), height (y), and depth (z), known as the axial dimension (XY or YZ).
• Species are observed over time (T) in 2D (XYT) or 3D (XYZT).
• Typical optical microscope resolves structure down to 0.1 to 0.3 mm.
• Image smaller biological structures require super-resolution (optical) or electron microscopy

Introduction to Light Microscopy

• Antonie Von Leeuwenhoek (1632-1723) used a simple single lens microscopy at 200X magnification, observing erythrocytes, polymorphonuclear leukocyte in an unmounted and unstained blood smear, bacteria, and sperm.
• 18th century: Achromatic microscope objective (free of major chromatic aberration
• 19th century: Improvements in lens design due to quality of glass used for microscope optics
• In 1873 Ernst Abbe provided scientific basis for the production of powerful light microscopes
• In 1893 August Koehler standardize microscope illumination.
• 20th century: development of phase contrast, differential interference contrast.
• Fundamental principles established for fluorescence and single and two-photon microscopy
• 21st century: advance in optical microscopy and super-resolution microscopy
• Resolution is the minimum distance in microns between two points where the two points can be clearly distinguished from each other
• Abbé's formula: d= λ/2(n sinα)
• d is the value of the minimum resolved distance
• λ is wavelength
• n is refractive index
• α is half the angular intake of the lens
• The light microscope resolution is limited to half the wavelength of light.

Transmitted Light Microscope

• Ocular lens enlarges primary image, formed by objective lenses.
• Prism directs rays to ocular lenses.
• Objective lenses closest to the specimen form the primary image and can be several.
• Condenser lenses focus light rays through the specimen.
• Kohler illumination of the specimen is the most important variable in achieving high quality images in microscopy.
• Object illumination is a method where an image source is projected through the condenser.
• Condenser lens collects the light from the source for the entire view-field
• Condenser Lens provides a cone of light to objective
• The light cone must be properly adjusted to optimise the intensity and angle of light entering the objective front lens. Two identical NAs are required, the condenser NA exactly matching the objective NA (numerical aperture).
• Higher NA = collecting more lights
• A condenser of high NA must have its NA reduced by an iris diaphragm
• Wavelength of illumination at mid-spectrum wavelength of 550 nm.
• Low specimen contrast and improper illumination lower resolution.

Light Microscope Resolution

• Factors that may lower resolution are low specimen contrast and improper illumination
• Using the 20X S Plan Fluor ELWD objective, with numerical aperture (NA) equal to 0.45, the microscope resolutions at 550 nm and 400 nm wavelengths are 611 nm and 444 nm. Shorter the wavelenght, higher the resolution.

Magnification and Resolution

• Objective lens gathers light from the specimen and creates a magnified image of it, and are responsible for primary image formation, and its properties define image quality
• Magnification power (M) of a lens depends on the focal distance (f) and the distance of the image plane from the lens (h), therefore M =h/f
• A lens with a short focal distance will have a greater magnification power than a lens with a longer focal distance
• Working distance is the distance between the objective lens's front element and the closest surface of the coverslip when the specimen is in focus.
• Numerical aperture characterizes the range of angles over which the objective lens accepts light.
• High NA objective collect lights over a larger angle,accepting more light than a Low NA objective with smaller angle
• Immersion objectives allow more light collection as they reduce light scatter.
• Resolution of a microscope (σ) is the minimum separation distance between two points required to distinguish them.
• Resolution depends on the wavelength of detected light (λ) and the Numerical aperture (NA) of the objective as described by the Rayleigh criterion; σ =1.22 x λ / 2xNA
• Resolution is high and resolution distrance is small when image contains hight structural detail
• High NA lenses = smaller minimmum resolution = higher NA

Abbe Formula vs Rayleigh's Criterion

• Difference is determining resolution of the light microscope with the two methods.
• Abbe's formula assesses considers numerical aperture of objective lens and wavelength of lights
• Rayleigh's criterion assesses diffraction pattern (airy sac) produced

Refraction, Image Quality, and RI mismatch

• Refractive index mismatch occurs when light travels through materials that have different refractive indexes.
• RI mismatch occurs with Immersion objectives.
• Lens designs use immersion fluid with an RI that closely matches the glass components.
• Light rays passing through specimen encounter a homogeneous medium between coverslip and immersion oil and are not refracted as enter the lens.

Refractive Index

• The more light collected by an objective lens, the greater the teh image resolution.
• Differences in RI between the sample mounting medium, coverglass, and objective immersion medium causes refraction and loss of light.
• Spherical Aberration arises from refraction differences across a spherical lens, light entering near the edge is unfocused compared to center. There is an Rl mismatch between RI of immersion and specimen. Corrected lenses result in one focal point and clearer image.
• It's important to reduce refractive index mismatch when: Sample mounting medium, Glass coverslip, Mounting samples directly onto the coverslip
• High-quality oil immersion objectives perform optimally only with a 0.17mm coverglass
• The best coverglass thickness will be indicated on barrel

Correction for Aberrations

• Lens Types and Corrections for each lens
• Corrections for chromatic and spherical aberrations
• Flat field correction to account for field curvature
• Chromatic aberrations occur when a lens is unable to focus wavelengths of lights and are due to low-quality lenses and are seen as “fringes” of colours
• Types: Lateral and axial form

Chromatic Aberrations

• Lateral causes incorrect light focusing thats perpendicular to light path. Corrected with objective lens with crown and flint glass doublets
• Axial involves white light refracting at range of component wavelengths and will have a a different focal point. Corrected with multi-color light base.
• Field curvature correction fixes issue lenses not accounting for field curvature

Objective Lens Type and Correction

• Achromat (Achromatic Lenses)
• Correct chromatic aberration for red and blue wavelengths.
• Made of two lens elements (lens doublet) with different refractive properties and brings light near focal plane as secondary correction
• Also corrected for spherical aberration in the green region.
• Fluorite (Semi-Apochromatic Lenses)
• Use fluorospar or synthetic substitutes and improves spherical correct for 2-3 wavelengths
• Apochromat (Apochromatic Lenses)
• Highly corrected microcope and provides chromatic/spherical correction for up to 4 wavelengths.
• Typically constructed with two lens doublets and one lens triplet.
• Plan Objectives (Plan Achromat, Plan Fluorite, Plan Apochromat) use field curvature correction and prevents image perimeter from being out of focus compared to center.

Differential Stains

• Absorbs light at different wavelengths to provide differentiation between cell structure and cell types
• Gram stain distinguishes gram negative (unstained) and gram positive (stained) bacteria.
• Masson's Trichrome stain differentiates collagen fibres from both muscle and nuclei in tissue sections.
• Stain filter allows cetrain color to go through, while another color is absorbed

Phase Contrast

• adding specific stains kills the living cells
• Detect differences in refractive index and cell density and convert these phase shifts into visible differences in light intensity.
• Relies on ability to amplify phase changes as light passes through a specimen.
• Special ring condenser allows passage through a specimen and alignment with the phase plate in the objective lens.
• Objecticve phase plate allows for constructive/destructive interference that are detectable

Phase Contrast Elements

• For phase contrast microscopy, the objective lens must have a phase plate with achromat
• Before imaging, the condenser annulus and phase plate must be aligned using a centering telescope/ Bertrand lens.
• In phase contrast microscopy, bacteria and cell structures with high refractive indices, like nuclei, ribosomes, mitochondria, and nucleoli, appear dark, while less dense areas, like cytoplasm and non-cellular regions, look lighter. Bacteriologists use this technique.

Differential Interference Contrast (DIC)

• Detects changes in refractive index creating a 3D-like image of thin optical sections that minimizes interference, providing superior depth discrimination compared to phase contrast. Uses full condenser aperture for higher lateral resolution compared to phase contrast microscopy.
• Polarised light is separated into two,separated by a small distance by the Nomarski (or Wollaston) prism
• Phase Shift is detected as elliptically (circular) polarised light

DIC function

• Polarising Filter 1 polarizes which prepares path for prism 1
• Prism 1 separates incoming light by directing specific paths
• Specimen induces phase change in specific light. This phase difference makes eventually image
• Polarising Filter 2 adjusts re-combined light to optimum
• Prism 2 brigs the re-omised light's path back

Phase Contrast vs DIC

• DIC is better for thick samples as it gives higher resolution in all directions (X, Y, Z), creating clearer images with less blur compared to contrast
• DIC is better for thick samples as it gives higher resolution in all directions (X, Y, Z), creating clearer images with less blur. It also reduces the halo effect seen in phase contrast.
• DIC does not work well on plastic due to effects on polarization
• Examples like zeiss offer alternatives liike PlasDIC

Fluorescence Microscopy

• Fluorophores have unique spectral properties that are important
• Different fluorescence light sources require different filters
• It is important to use appropriate objectives
• Highly sensitive, specific and provides sharp contrast.
• It is excellent for the detection of intra/sub structure

Fluorescence Microscopy

• Energy to excite the molecule = Energy of lights + energy form heat
• Molecule with flourescene is calle fluorophore that causes Luminescene by abosoption of incident light radiation

Fluorophore excitation

• Fluorophore emits the excitation via absorption of lights
• Molcue reaches higher energy state that is unstable and cannot br sustained. The energy is released and molecule drops back to stable state

Quantum yield

• Is used to calculate radio of light via flouroscnece
• There is brightness form the molceule
• Stokes shift explains differnces between absorption wavelenghts

Flourecent Molecules

• Consits of crystal, organic fluorescein and protein
• The excited light via 395 and 475 nm emmits 508 nm
• Floureceent can be optimised and inert
• LED bulbs can be used due to energy range
• Light can be spearated to aborsption of light

Floureceent Filters

• Filters can be used to sepatrate light by blocking, absoriing, and reflectting
• the 3 fliter cubes can be used here

Dye

• Dye ands Stain long used to process image and visualise biological sample
• The molucuel is capable off excitement
• Moolceu will return to lower state energy state
• The molucuel will repeat its stimulation

Exitation

• The high wavelenght of light is more efficeitn with the shorter range
• Illumination range of the bandpass
• Use BroadBand filter

Floureceent Filters

• Use filter through blocks and allow transmits lights pass
• Violet is compact and can replace more expensive options for bios
• Lasers help by isolating floureceent

Flourohphore and Protein

• Is is used with smaller weight, larger proteins and molecules that jump to higher state
• Flourecetnt protein is combined of jellyfisg
• The GFP has beta helixs consiteding sheet

Fluoresecent filters

• An optical component the increases contraction
• combines with excitations filter
• Dichroic with short wave

Advantages

• There is high contraction and specificity
• Great on sub
• The add is easily addable
• It is simple and effective

Histology

• Looking at disease, dysregulation
• Modern technitiques extend to techniques knwledge

Fixiation

• Terminates cell and metabolism with decay
• It prevents autolysis and hardens tissue

Firatives

• Bining is differeent proteins and lysines
• Solubes are anchored to matrix
• Great at reducing solibility for presevation
• Preservinhg nucleid acid
• The sample will shrink

Sample

• The sample requires further disection
• And examination is a must

Processing

• The tissue biopsy contains cell fills with water
• Then dehydration willemty pt the fluid
• The cell space will refill with paraffin

Embedding

• The center is processed to especimes that are applied
• Mainitain orientarion

Sectioning

• With the paraffin cut to thickness
• Ensruing single cell layer is achieved
• Harde elemtns placeds of ice for support

Sectioning and drying

• Is is float in area to be picked and charged
• Water should be less then x6

Staining and mounting

• After staining, section covered for analysis
• Is to maintain contrasts
• snap freeznig is to improve benefits

Color

• Controls helps woth structure
• Probes help baseds on pH and polari

flourescence and auto

• There is some naturL emission on the biological
• can interfere if specific singles very din
• Grouth media can create auto

Antibiotics anti singl

• Single stauinh is best
• Control with specific antibody

Antibodies stains

• To step process involves conjugated fluorophore anti body
• dye combination is used