Microscopy Exam 1

4 ways we describe light

monochromatic vs polychromatic

linerarly polarized vs nonpolarized

coherent vs noncoherent

collimited vs divergent

monochromatic light

light having a single wavelength or frequency

polychromatic light

Light of many colors (wavelengths) usually referred to as white light.

polarized light

light that vibrates in only one direction

Photons can oscillate on any axis (in 2 dimensions) as it travels

coherent light

Light of only one wavelength that travels with its crests and troughs aligned

Our cones detect

red, blue, green

when photons interact with an object, they can ____

Reflection

Refraction

Diffraction

Absorption/energy transfer/Fluorescence

Birefringence

If n2 > n1:

Light rays 'bend' towards the normal

If n2 < n1:

Light rays 'bend' away the normal

critical angle

the angle of incidence that produces an angle of refraction of 90 degrees

refractive index is _____ dependent

wavelength

real image

An upside-down image formed where rays of light meet.

virtual image

an image that forms at a location from which light rays appear to come but do not actually come

photoelectric effect

refers to the emission of electrons from a metal when light shines on the metal

diffraction

Occurs when an object causes a wave to change direction and bend around it

double slit experiment

proved that electrons have wave like properties (wave length)

airy disk

A bright central point surrounded by rings of light and dark caused by the pattern of interference of spherical wavefronts converging at the focal point.

What we use to probe objects influences the image we generate, smaller probes improve ______

Resolution of Space

spherical aberation

a loss of definition in the image arising from the surface geometry of a spherical mirror or lens.

Numerical Aperture of an objective is ______

a measure of its ability to gather light

conjugate focal planes

A set of image or focal planes

Microscopes have ____ 'sets' of conjugate planes.

two

Koehler Illumination method

provide uniform light on the specimen plane while putting the light source on a separate conjugate plane

infinity light

parallel light between the objective and tube lens

How to define Imaging performance

Resolution of Time

Resolution of Space

Resolution of Light Sensitivity

Resolution of Signal (vs noise)

resolution of time

How well a phenomenon can be sampled across time

resolution of space

How accurately we can capture the dimensions of a phenomenon (target)

smallest distance between two points on a specimen that can be identified as two separate entities

Resolution of light sensitivity

The dynamic range (gray scale) of an image

resolution of signal

Clarity and visibility of objects in the image

We often 'stain' samples to increase contrast, thereby enhancing the Resolution of Signal

Rayleigh Criterion

2 points are resolved when they are 1 Airy disk radius apart (first maxima to first minima)

2 points are resolved when they their half maximum intensity touch (R = 0.51 λ/NA)

Airy disk is elongated along the _____ plane

z

Light intensity I

the rate of flow of energy per area per time

proportional to the amplitude of the wave squared I ∝ A^2

Contrast

the difference in intensity between the object (specimen) and background

Sampling

the act of creating discrete captures of continuous data (sound, light, etc). A camera samples light in both space and time

undersampling can lead to

Aliasing: a distortion artifact that creates patterns in periodic structures

Nyquist Sampling Theorem

The radius of the Airy Disk has to be captured by at least 2 pixels for us to resolve them

steps of microscope use

Posture

Adjust Light

Adjust ocular/Eye piece distance

Put sample on stage and coarse focus with the lowest magnification setting

Adjust ocular focuses

Adjust Condenser height

Repeat focusing steps when changing magnification

Dark current

Thermally generated electrons that are indistinguishable from photo-generated electrons

Charged Coupled Device (CCD) Cameras

pixels that are arranged in parallel registers and inputted serially

The sensor is exposed to light for a specific amount of time (exposure time, a camera setting)

Pixels are shifted off the parallel register onto the serial register

Pixels are 'read' one at a time (the output amplifier boosts the signal and assigns an analog voltage before assigning a digital value)

Complementary metal-oxide-semiconductor (CMOS) Camera

pixels that each have their own electron to voltage converter, simultaneous processing of pixel information

CCD vs CMOS

CCD: Pixel transfer is slower so the capture rate is lower aka slower image acquisition rate and lower maximum frame rate

CMOS: Pixel to pixel variability is higher, aka higher noise

Analog, continuous signals are converted to

digital images that our computers can store

Digital values are

discrete, integer values with a finite range (gray levels or scale)

Signal to Noise Ratio

the amount of signal present compared to the amount of noise

Pixel size and light sensitivity

Larger pixels can capture more light (from your target)

So you increase light sensitivity while decreasing resolution Large pixels can be 'made' via pixel binning (A camera setting)

Exposure time

The duration of time you let light hit the camera before you digitize it

Inversely related to maximum frame rate (number of images you can acquire per second)

Increasing the exposure time increases the total light (both signal and noise)

Gain

amplifies the brightness of all photons, but can increase signal more than background/noise)

Increasing the digital gain reduces the number of electrons assigned per gray level in a gray scale

i.e. if you normally have 10 electrons per gray level, doubling the gain will make it 5 electrons per level

Trade off between Dynamic range and SNR

Dynamic Range

The range from background level to the brightest signal

why polarized sunglasses are useful

birefringence

where there are 2 (or more) refractive indexes usually based on polarization

Fun pic of double polorization

Birefringence causes

an elliptical wave front with a "slow" and "fast" axis

When linearly polarized light passes through a birefringent material

the fast and slow waves become out of phase.

The analyzer

combines the two waves and creates an interference color based on the difference between the fast and slow waves

The color is therefore dependent on the thickness of the material, and the refractive indexes of the fast and slow wave