Lecture 12 (Super Resolution Microscopy)

Light is a wave so it is

subject to diffraction

Numerical aperture lens can only

focus the light into a “point cloud”, but not a single point

Dimension of XY

200-300nm in super resolution microscopy

Dimension of Z

500-800nm in super resolution microscopy

Abbe’s Diffraction Limit is?

Diffraction limits what

the resolution of light microscopy

point spread function (PSF)

describes the response of a focused optical imaging system to a point source or point object

Is this resolvable?

Yes

Is this resolvable?

No

What does single molecule microscopy require?

Well isolated molecules before imaging

Single Molecule Microscopy limit

Who was awarded the 2014 Nobel Prize in Chemistry for Super Resolution Microscopy

Eric Betzig, Stefan W. Hell, and William E. Moerner

What is PALM

Photoactivated Localization Microscopy

Principle of PALM

location of each molecule is first determined by fitting the expected molecular image given by the PSF of the microscope [(G), center] to the actual molecular image [(G), left], the molecule can be plotted [(G), right] as a Gaussian that has a standard deviation equal to the uncertainty σx,y in the fitted position.

What does STORM stand for?

Stochastic Optical Reconstruction Microscopy

What is STORM (Explanation)

STORM is a super-resolution microscopy technique that utilizes the stochastic switching of fluorescent molecules to achieve high-resolution imaging, allowing for precise localization of individual molecules in biological samples.

STED definition

STED (Stimulated Emission Depletion) is a super-resolution microscopy technique that uses a pair of laser beams to achieve resolution beyond the diffraction limit of 0.2um by selectively depleting the fluorescence of molecules in specific regions, thereby allowing for high-resolution imaging of biological samples.

Abbe’s Equation modified for fluorescence

STED did what?

reduced the PSF to break diffraction limit

n is

the refractive index of the medium, which affects the resolution in microscopy

Alpha is

the half-angle of the cone of light entering the objective lens, impacting the numerical aperture and resolution

n sin alpha is

the numerical aperture of a microscope, influencing its resolution capabilities

Higher NA equals

smaller diffraction limit and better resolution

How does STED work?

by using a focused laser beam to excite fluorescence in a sample while a second, donut-shaped laser beam depletes the fluorescence surrounding the focal point, narrowing the observation area and enhancing resolution.

STED Diffraction Limit

STED what does l (x) stand for?

Local intensity of the STED depletion laser at position x

STED what does lsat stand for?

Saturation intensity, the STED intensity at which fluorescence is reduced to half its depleted value under a simplified steady-state model

STED what does S= l/lsat stand for?

Saturation factor, a dimensionless measure of how strongly the STED beam suppresses fluorescence