Basic Optics

• 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.

or 

 

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://imb.uq.edu.au/research/facilities/microscopy/training-manuals/microscopy-online-resources/image-capture/nyquist-conditions

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.