Fluorescence Microscopy

Brightfield Microscopy

We can only see and detect changes in intensity and color. Magnification and resolution are useless without contrast. This type of microscopy does not have a ton of contrast so it can be hard to read unassisted

Phase Objects

They alter the phase of light, so its kinda hard to tell what they are without any contrast.

Phase contrast

Adds color to the thinsg that are changing the direction of light so we can better understand them

Differential interference contrast

Makes the image look more 3D so you can tell from there

Fluorophore

A molecule that cn absord light of one particular wavelength or energy and emit light of another wavelength

What is fluorescence?

A particle gets charged by a certain wavelegnth and in reponse will return to its original state but this time at a lower wavelegnth

Characteristics of fluorescence

1. Excitation peaks are BROAD: due to the number of rotational, vibrational and electronic states

2. Each molecule has a characteristic excitation and emission spectrum

3. The lifetime of the excited state is approximately 10^-15 seconds

4. Because the excited state is HIGHLY REACTIVE it can “bleach” rendering the fluorophone non-fluorescent

5. Stokes Shift - emission is always LOWER energy and therefore a LONGER wavelength

In the visible light spectrum, which has the shortest wavelength

Purple which has Higher energy, more harmful to cells, more easily scattered, higher resolution

In the visible light spectrum, which has the longest wavelength

Red, which has lower energy, less harmful to cells, less easily scattered, and lower resolution

What are the 3 ways you could label your sample?

Organelle specific stain

Antibody laveling

Genetic tagging

What are Antibody labeling typically refered to?

Alexa fluor dyes! have a bunch of different possible colors

Green fluorescent protein

It is from jelly fish! it allows you to genetically encode a fluorescent marker onto a gene, and have it transcribe it. No inserting flourescent protein or antibody misbinding can happen!

Widefield / Epi Fluorescence microscopy

It is an inverted microscope, and it provides an illumination source, typically a LED.

You put your sample on the stage, and the objective is used to illuminate the sample and collect the fluorescence

What all is in the filter cube?

excitation, excitation filter, dichronic mirror, emission, and an emission filter!

How does an epi microscope work?

Sends an excitation into the sample, soem of it goes to our eyes while the other goes the opposite way towards a CCD camera!

Spectral Overlap

With broad excitation and emission spectra and collection parameters, fluorescence from more than one fluorphore can appear in a single channel

They can overlap and skew your results!

How does imaging multiple colors work?

The fluorphone does not have a specific color associated with it, so a computer will assign the contrast it sees as a color.

How do confocal microscopes work?

It is able to target a specific focal plane and not get light from other planes. Allows for higher resolution pictures

What is the confocal principle?

Laser is scanned across sample, pinhole is placed at conjugate sample plate, light from an object in the focal plane is focused at the pinhole, and makes it through to the detector, light from an object out of the focal plane is defocused at the pinhole, and does not make it through.

Optical sectioning

Changing focusing (z) through the sample allows for serial optical sectioning (z-stack)

Filters in Laser Scanning Confocal

Excitation is laser - single wavelegnth

Still requires dichroic and emission filter as in widefield

What are the key features of an objective lens?

Magnification

Numeric Aperture

Working distance

Immersion media

Numerical Aperture

NA = n sin(μ)

n= index of refraction of media between sample and objective

μ: ½ the angular aperture

Resolution

R = 0.61λ/NA

R: resolution, smallest resolvable distance between two objects

Empty Maginification

The image is enlarged but no additional detail is resolved

Why is resolution important?

It allows you to determine if something is co-localized or not. it could be they are so close that the microscopes resolution cannot tell them apart

Convential microscope resolution limit

Right around genes ~100nm

What is STORM?

STochastic Optical Reconstruction Microscopy

This and PALM, STED, and SIM, allowed the resolution to increase allowing to see the interaction between smaller things and smaller distances

How do you achieve high resolution with STORM?

Blinking of the fluorophore on and off allows for spatial resolution of ‘localized’ points, which can then be reconstructed