Basic Optics
Initial Training for Optical Microscope Users
- Data Storage
- Fluorescence Imaging
- IGB Core Instrument LSM 880
- IGB Core Instrument LSM 900
- IGB Core Instrument V16
- IGB Core Instruments Axiovert 200M
- IGB Core Instruments LSM 700
- IGB Core Instruments Zeiss LSM 710
- Objectives
- Optics
- Sampling
- Working in the IGB Core
Data Storage
Store data on the data drive on the local machine:
D\Data may be deleted at anytime. Do not leave your data here!
The core staff will clean the computer drives occasionally. This means that your profile, desktop and data from the data drive will all be removed. We will also remove other files that look like they are not needed.
Core-Server:
The core-server was set up by the IGB Computer Network and Research Group (CNRG) as a place for core users to store their data long term and move it back to your office. You do not need to bring thumb drives or other devices that could bring a computer virus into the core when you come to collect data. Move your data off of the local machine onto the core server at the end of each imaging session. Your PI will have a folder on the core-server and you can make a sub folder for your work. you will have access to all of the sub folders in your PI's folder but not other PI's folders. All of the data in your PI's folder was paid for by your PI and belongs to him/her.
Your PI will be charged $8.75/terabyte every month Email help@igb.illinois.edu for information on charges and tape backup for long term storage.
Long Term Storage
CNRG provides tape backup for long term storage for $200/ terabyte
Fluorescence Imaging
Why Fluorescence:
We can label what we want to see
Excitation and Emission
Dapi
Widefield vs Confocal
https://www.journals.uchicago.edu/doi/full/10.1086/689588
IGB Core Instrument LSM 880
IGB Core Instrument LSM 900
405nm, 488nm, 561nm, 640nm excitation.
Zen Blue
file:///C:/Users/gfried/Downloads/EN_poster_Beampath-LSM-900_A1.pdf
IGB Core Instrument V16
IGB Core Instruments Axiovert 200M
Axiovert 200M
Cameras
cMOS
IGB Core Instruments LSM 700
LSM 700
http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/lsm700/indexflash.html
IGB Core Instruments Zeiss LSM 710
Light Path
https://www.gu.se/en/core-facilities/lsm-710-nlo
Seven visible excitation lines: 405nm, 458nm, 488nm, 514nm, 561nm, 594nm, 633nm.
Tisaphire laser 700nm to 980nm
Spectral Unmixing http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html
Multiphoton Microscopy http://zeiss-campus.magnet.fsu.edu/referencelibrary/multiphoton.html
Fluorescence Lifetime Imaging Microscopy (FLIM) http://www.iss.com/microscopy/components/FastFLIM.html
Objectives
Optics
Properties of light
Wave particle duality:
Light is a wave
Absorption and Emission
Beer's law
Refraction
Ray tracing
Resolution
Resolution: The ability to separate two objects
A definition of Numerical Aperture
Fourier transform:
Transform from real space to frequency space
Now we look at a real square wave and frequency space
Look at a simple optical system:
Mathematical prediction of the Point Spread Function (PSF)
on the left we have the mathematical point source know as a delta function.
were
the intensity at the Fourier plain can be found by taking the Fourier transform of this function.
this has the same intensity at all points inside the aperture and zero outside. The second lens is now taking a Fourier transform on a box function the width of the aperture.
or
Substituting in the definition for NA
Now we go back to Resolution. How close together we can position two points and still distinguish them
More intuitive approach
Notes from:
http://web.mit.edu/2.710/Fall06/2.710-wk12-b-sl.pdf
https://links.uwaterloo.ca/amath353docs/set11.pdf
https://www.thefouriertransform.com/pairs/box.php
http://www.phys.unm.edu/msbahae/Optics%20Lab/Fourier%20Optics.pdf
How to Chose the Optimal Objective Dr. Sebastian Gliem
Super Resolution Techniques
Sampling
How does digital sampling affect resolution
Look at imaging these object with a digital camera
How close together do pixels need to be?
Sampling over time:
How often do you need to image a moving sample
Nyquist theory states that you should sample more than 2 X the frequency that you expect.
Over sampling
Nyquist sampling
Under sampling causes aliasing
Optical Transfer Function MTF
When objects get close together the contrast decreases.
MTF = Image Modulation/Object Modulation
MTF = 2(φ - cosφsinφ)/π and
φ = cos-1(λν/2NA)
The Optical Transfer function is the Modulation transfer function times a phase component.
OTF = MTF × eiφ(f)
Camera bit depth
0 or 1
00 or 01 or 10 or 11
000 or 001 or 010 or 011 or 100 or 101 or 110 or 111
and so on
Jpg is 8 bit Tiff can be 16 bit
references
https://microscopy.berkeley.edu/courses/dib/sections/02images/sampling.html
https://ocw.mit.edu/courses/mechanical-engineering/2-71-optics-spring-2009/video-lectures/lecture-22-coherent-and-incoherent-imaging/MIT2_71S09_lec22.pdf
Working in the IGB Core
Expect to walk into a room with a fully functional instrument
Let a core staff person know if you see a problem
Clean up when you leave
Acknowledge the IGB Core as:
“Core Facilities at the Carl R. Woese Institute for Genomic Biology"
Let us know when you publish
Collaborations with the core facilities staff can be beneficial in the development of unique methods or capabilities.