L12: Microscopy II

What is darkfield microscopy useful for?

low contrast, absorption, and high diffusion

What type of light is collected in DF microscopy?

scattered light

What is the difference in DF and BF ability to produce an image that accurately represents the morphological information of an object?

BF mogs

What is DF best for?

revealing outlines, edges, and boundaries

In DF which regions appear bright?

scattering regions

In DF, what regions appear dark?

those that don’t scatter

How can DF microscopy be performed?

both in reflection and in transmission

How is the objective andmicroscope set up so light illuminating the sample isnt collected by the objective?

there is a light stop that blocks the central rays

only oblique illumination reaches the sample

What makes sodium chloride optically isotropic?

its cubic crystalline lattice

What makes calcite optically anisotropic?

its anisotropic crystalline lattice

What characterizes an amorphous polymer?

no periodic crystalline structure or repeating unit

What is birefringence?

double refraction of light

What causes birefringence?

orientation dependent differences in a compound’s refractive index

What type of material exhibits birefringence?

transparent, molecularly ordered material

Describe the geometry of the axis of isotropic crystal (salt)?

all axes are the same length and perpindicular to one another

salt has a single refractive index

Describe the geometry of an anisotryopic crystal’s (quartz) axis.

3 equal horizontal axes at 220° to each other, and 1 vertical axis, perpendicular to the others

How many refractive indices does Quartz have?

two

Describe the orientation of refractive indexes in an optically isotropic material?

constant throughout

How does light travel through optically isotropic materials?

one single velocity

What determines how an anisotropic material interacts with light?

it depends on the orientation of the crystalline lattice w respect to the incident light

What happens after light enters a non-equivalent axis in an optically anisotropic material?

it is refracted into two rays and each is polarized w their vibration directions oriented perpendic to each other and traveling at different velocities

Describe the steps of polarized micrsocopy.

1. a linear polarizer placed before the objective polarizes light parallel to its axis

2. polarized light enters anistrropic crystal

3. light is refracted and divided to 2 components vibrating parallel to the crystallographic axes & perpendic to eachother

4. ligh reach a 2nd polarizer that is oriented perpendic to the first polarizer

5. retardation of one ray w respect to the other causes interference to occur between the waves as they pass through analyzer

Birefringent samples acquire a spectrum of color when observed through crossed polarizers.

What is the main result of polarized microscopy?

enabling the quantifying birefringence

Why does collagen exhibit birefringence?

its ordered helical structure

fiber has dif refractive indexes along it length vs across it

What is the assembly protocol for polarized microscopy targeting extrinsic birefringence?

• aq silk fibroin is mixed w crosslinkers

• mechanical tension is introduced

• during drying, silk fibroin undergoes βsheet transformation and the formation of a nanofibrillar network

What is birefringence useful for w/ respect to mechanical properties?

quantifying stress-induced alignment of silk fibrils in aerogels

What else is observed in correspondence with minor nanofibril alignment?

low birefringence, in correlation to low-stress regions

What is the main mechanism fluorescence microscopy uses to increase contrast of an object being imaged?

absorption of light, quantum yield of fluorophore

What are some biomedical applications of fluorescence?

• detecting individual fluorescent molecules

• studying macromolecules distribution and dynamics inside tissues

• study of rxn mechanisms by fluorescence energy transfer

• dynamics of single tagged molecules

• determ of ionintracell concentration

  • study of cell viability and cell functions

What are different ways a specimen can fluoresce?

intrinsically fluorescent

conjugation between tagged macromolecule and fluorochrome

What are the different parts of a widefield fluorescence microscope?

eyepieces, camera, fluorescent filter set, objectives, specimen, xyz stage, epi-illumination source

What does an excitation (Ex) filter do in a widefield fluorescence microscope?

selectively transmits a band of short wavelengths for exciting a specific fluorophore in the specimen

What does a dichroic mirror do in a widefield fluorescent mircroscope?

reflects the Ex light towards the objective and specimen, while transmitting returning Em fluorescent light towards the detector

What does an emission (Em) filter do in a widefield fluorescence microscope?

transmits Em light and it removes unwanted background light

What type of objectives are preferred?

color corrections (apochromatic) and high NA

What is the problem that confocal microscopy addresses?

thick fluorescent specimens, bright fluorescent signals from objects lying outside focal plane increase backgroun and yield low-contrast images

How does confocal microscopy address the low contrast issue for thick fluorescent specimens?

rejects signals from sources above and below the focal plane

1. illuminates specimen w a focused scanning laser beam

   1. places a pinhole aperture in the image plane in front of an electronic photon detector

How does confocal microscopy work?

uses a stepper motor that changes the microscope focus in small steps along the z-axis

Are confocal images generated in a microscope?

no, built up electronically

What does the photomultiplier transducer do in confocal microscopy?

transduces the intensity of the photons at the pinhole in a voltage

signal is digitizeed and converted into pixels that form an image

How many pinholes can be used in some confocal configurations?

2! additional pinhole allows transmission only through a small area

What role does raster movement of the laser play in confocal microscopy?

one mirror scans along x-axis

the other on y-axis

both are coordinated to generate a pattern of a raster on the specimen

What role does electronic magnification play in confocal microscopy?

magnification can be adjusted electronically by varying the area scanned by the laser (zoom factor) w/o having to change the objective

How does decreasing the size of the pinhole affect thickness of the focal plane along the z-axis and contrast?

reduces thickness of focal plane along z-axis

improves contrast by excluding out of focal plane sources of light

What is another factor that can affect spatial resolution?

scan rate

effects of different parameters

What are the advantages of confocal over widefield?

sharp image

dont have to physically slice the sample

can investigate the spatial arrangement

composite or projection view

3D views

5D views (x,y,z, timed, multiple colors)

List a biomedical application of confocal microscopy.

disease development monitoring