“Super Resolution Microscopy Techniques“

1. 4Pi Microscopy
2. STED
3. STORM
 Features

1. Optical: Development of Spatial Resolution beyond the

diffraction barrier.
2. Biological: Visulaization of the elucidate Biological
Functions & Phenomena, functioning of liveing cells and
Cellular Environment.

https://www.mrgscience.com/uploads/2/0/7/9/20796234/relative-sizes-2_orig.jpg
 Key Point

 To exploit Photoswaitching of Fluolorecent Probs OR

Non-Linear responce in the Fluorecence Emission.

 How to achieve this,

• Resolution =
1/Diffraction limit (d)
d = λ/(2 N.A.)
• Where,
λ = Wavelenght
N.A. = Numerical
Aperture
https://static3.olympus-
ims.com/data/Image/IM/OpticalMicroscopes/numerical_ape_01.gif?r
ev=38F4
 Key Point

RESOLUTION DIFFRACTION LIMIT

d = λ/(2 N.A.)

I. Lower the Wavelength: UV objects (e.g Fluorescent Dyes)

II. Higher the Refractive Index: Use larger Reflective Index
Glass (e.g. UV to IR)
III. Higher the Incident angle: More reflection can be collected
= More information. (4Pi or I5M Method)
 4Pi Microscopy

 Ideally,

• Light collected by each

Lens = Solid angle = 2π

• So, by Combining both

lenses,
Total Collected light = 4π
(Whole Sphere)

• Three Type:
Type-A, Type-B & Type-C https://www.ks.uiuc.edu/Research/mi
croscope/images/4Pi.jpg
 4Pi Microscopy

Objective 2

Mirror 1 Mirror 2

Objective 1

• https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQtiWXA9xZbOPgJo
L4yqoKfsV5QSlL6ww4zsks3HS-7sqZsLUMz
 4Pi Microscopy
 Higher resolution along the optical axis. (about 100nm)
 2PE reduces the side lobe height due to the quadratic
dependence on the illumination intensity.
 The simultaneous use of coherent excitation and
detection aperture expansion leads to the difference
between two wavelengths, reducing the spatial overlap
of the side lobes of PSFs.

http://physics.bu.edu/images
/research/15_4pi_axial.jpg
„Stimulated Emission Depletion Microscopy“
(STED)
 Nano Scale 3D Optical Imaging.
 Stimulated Emission

E1 < E2

http://hyperphysics.phy-astr.gsu.edu/hbase/imgmod/qpro2.gif
 STED Microscopy

200 nm

λ STED= 770 nm
http://youtu.de/Dkz00plB3g0

 1st Lens based Fluorescence Microscopy.

 Integration of Fluorophore properties in Image
formation.
 STED Microscopy

A. Without STED Beam B. With STED Beam

http://youtu.de/Dkz00plB3g0 http://youtu.de/Dkz00plB3g0

Where, Isted = STED Intensity

Isat = Saturated Intensity
 STED Microscopy

https://www.researchgate.net/profile/Xiangping_Li9/publication/321226952/figure/fig4/
AS:667766004649995@1536219230444/a-c-Spatial-overlapping-between-the-depletion-
and-the-excitation-beam-checked-by-PSF_Q320.jpg
Principle: STED Microscopy

https://www.youtube.com/watch?v=nFaGOEbBkyk
http://youtu.de/nFaG0EbBkyk
Advantages: STED Microscopy
• High resolution ( < 50 nm)
• Multiple areas can be probed
• Resolution depends on the
Laser Intensity ( FWHM = f(
Intensity))
• Resolution is independent of
Wavelength.
• Can image Live biological
samples.
• Optical system is simple to
understand and use.
• Can scan the sample in z
direction also. (3D image
formation)
• Modiffication of Abbe’s Law: