607SM - TECNICHE AVANZATE DI INDAGINE MICROSCOPICA 2023

Section outline

• Select section General

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  General

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  • Select activity Annunci

    

    Annunci Forum
• Select section Lecture 1 Cojoc Introduction

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  Lecture 1 Cojoc Introduction

  • What types of microscopies will we discuss about ?
  • Length scales and spatial resolution limits
  • Why Optical Microscopy is important vs Electron Microscopy?
  • Micro-spectrometry techniques: Raman + InfraRed - why are them important ?
  • Micro and nano particle manipulation without mechanical contact; how and why?
  • Force spectroscopy - application to biomedicine
  • SUMMARY of the COURSE !
  • Review of some important concepts in Optics: light as wave, reflection and refraction, diffraction and interference, photoelectric effect

  
  

  
  

  
  

  
  

  • Select activity Lecture 1 Cojoc

    

    Lecture 1 Cojoc File PDF
• Select section Lecture 2 Cojoc Optical Microscopy

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  Lecture 2 Cojoc Optical Microscopy

  1. Optical Microscopy – Physical Principles

  1.1.  Basics (Image formation, magnification, resolution, image quality)

  1.2.  Digital camera (image acquisition, formats, properties)

  1.3.  Phase imaging (qualitative and quantitative)

  1.4.  Dark field and Polarization microscopy

  
  

  • Select activity Lecture 2 Cojoc

    

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• Select section Lesson 3 Thalhammer

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  Lesson 3 Thalhammer

  OPTICAL MICROSCOPY

  
  

  1.How a microscope works

  
  

  2.Magnification vs resolution

  
  

  3.Numerical aperture and working distance

  4.Objectives
  

  5.Point-spread function and Airy disk

  
  

  6.Optical abberations

  
  

  • Select activity lesson 3

    

    lesson 3 File PDF
  • Select activity optical imaging techniques in microscopy

    

    optical imaging techniques in microscopy File PDF
• Select section Lesson 4 Thalhammer

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  Lesson 4 Thalhammer

  CONTRASTING TECHNIQUES
  

  •Brightfield

  •Darkfield

  •Phase Contrast

  •Polarization Contrast

  •Differential Interference Contrast (DIC)

  •Fluorescence Contrast

  
  

  • Select activity Lesson 4 Thalhammer

    

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• Select section Lecture 3 Cojoc Non Linear Optical Microscopy

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  Lecture 3 Cojoc Non Linear Optical Microscopy

  1.5. Non Linear Optical Microscopy

  - Electric Polarization, Susceptibility and complex Refractive Index in linear optics / materials

  - Dispersion and Absorption

  - Electric Polarization in non linear materials - macroscopic and microscopic views

  - Two Photon Excitation Fluorescence TPEF microscopy

  - Second Harmonic Generation SHG and Third Harmonic Generation THG microscopy

  - Spontaneous Raman Scattering RS and Coherent Raman Scattering CRS microscopy  

  • Select activity Lecture 3 Cojoc

    

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  • Select activity Reference 1

    

    Reference 1 File PDF
  • Select activity Reference 2 Review NLO Applications Bio

    

    Reference 2 Review NLO Applications Bio File PDF
  • Select activity Reference 4

    

    Reference 4 File PDF
• Select section Lecture 4 Cojoc Super resolution Optical Microscopy

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  Lecture 4 Cojoc Super resolution Optical Microscopy

  1.6. Super Resolution Microscopy / Nanoscopy

  - Total Internal Reflection Fluorescence TIRF microscopy

  - Confocal vs STED, PALM/STORM and MINFLUX microscopy

  • Select activity Lecture 4 Cojoc

    

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  • Select activity Lecture 4 update

    

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• Select section Lecture 5 Cojoc X-Ray Microscopy

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  Lecture 5 Cojoc X-Ray Microscopy

  2. X-Ray Microscopy

  - Contrast mechanisms and Image formation in X-ray microscopy

  - The components of an X-ray microscope vs component optical microscope

  - Soft and Hard X-ray microscopies, spatial resolution

  - X-ray sample interaction - spectroscopy

  - Synchrotron Radiation and Free Electron Laser imaging and diffraction

  - Example of X-ray microscopy applications to cell and tissue imaging

  
  

   

  • Select activity Lecture 5

    

    Lecture 5 File PDF
  • Select activity Note on X-ray wavelength energy relationship

    

    Note on X-ray wavelength energy relationship File PDF
• Select section Lecture 6 Cojoc

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  Lecture 6 Cojoc

  Optical Tweezers (OT) and their applications to biological systems

  • Principle of optical trapping and manipulation by light
  • Optical trapping of biological samples
  • Force spectroscopy - single molecule and single cell with OT 
  • OT to implement local delivery of molecules in cell biology experiments
  • OT to apply small controlled forces in mechanotransduction exps

  • Select activity Lecture 6 Cojoc

    

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• Select section Lecture 7 Cojoc - AFM Andolfi

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  Lecture 7 Cojoc - AFM Andolfi

  Atomic Force Microscopy AFM, with applications to biotechnology

  • Principles of AFM imaging and force measurement
  • Types of AFM imaging modes, characteristics for biological samples
  • Single molecule and single cell force spectroscopy with AFM
  • Applications in biotechnology 

  
  

  • Select activity Lecture 7 - AFM Andolfi

    

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• Select section Lesson 10- Thalhammer

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  Lesson 10- Thalhammer

  • Select activity Lesson 10- fluorescence microscopy

    

    Lesson 10- fluorescence microscopy File PDF
• Select section Lecture 8 Cojoc - Electron Microscopy Simone Dal Zilo

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  Lecture 8 Cojoc - Electron Microscopy Simone Dal Zilo

  • Why Electron Microscopy - resolution, contrast
  • Image formation in EM
  • Comparison optical microscope - electron microscope
  • Comparison between TEM and SEM
  • Electron beam - matter interaction
  • Sample preparation
  • Cryo EM, FIB principles

  • Select activity Lecture 6 Electron Microscopy

    

    Lecture 6 Electron Microscopy File PDF
• Select section lesson 12 - Thalhammer

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  lesson 12 - Thalhammer

  fluorescence microscopy part 2: fluorophores, filter combinations

  advanced microscopy techniques:

  - confocal  microscopy

  - 2 photon microscopy

  - superresolution  microscopy techniques (SIM, STED, STORM, PALM)

  - TIRF, FRET, FRAP

  
  

  • Select activity lesson 4 advanced microscopy techniques

    

    lesson 4 advanced microscopy techniques File PDF
• Select section lesson 13 Thalhammer

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  lesson 13 Thalhammer

  live imaging techniques part1

  - challenges

  - incubation

  - contrasting techniques

  - phototoxicity

  - live labelling

  - changes in fluorescence

  -resolution vs speed vs sensitivity

  - immersion media

  - signal to noise

  • Select activity lesson 5 live imaging part1

    

    lesson 5 live imaging part1 File PDF
• Select section lesson 14 Thalhammer

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  lesson 14 Thalhammer

  changes in fluorescence intensity:

  - changes in ion concentration:

                  Calcium imaging, genetically encoded calcium dyes

                  Imaging of vesicle release

  
  

  changes in fluorescence localisation: 

  - trafficking of organelles

  • Select activity lesson 6 live imaging part2

    

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• Select section lesson 15 Thalhammer

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  lesson 15 Thalhammer

  - pixel

  - digital resolution

  - sampling theory

  - pixel depth (bit)

  - quantization

  - quantitative microscopy

  - questions of quantitative microscopy

  - example for image processing program: FIJI

            histogram, LUT, channel composite, noise, filter, threshold

  • Select activity lesson 7 image processing and analysis

    

    lesson 7 image processing and analysis File PDF
• Select section lesson 16 Thalhammer

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  lesson 16 Thalhammer

  use example confocal stack in Fiji

  opening and saving of files

  image processing tools: LUT, filter, brightness&contrast, histogram, threshold

  binary images: erode, dilate, skeletonise, generation of masks

  quantitative measurements: particle size, number and intensity

  
  
• Select section PAPERS FOR EXAM - Dan Cojoc

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  PAPERS FOR EXAM - Dan Cojoc

  Students should choose a paper (each student a different paper) and prepare a short presentation (max 10 slides) to be presented in 10-12 minutes.

  The presentation should contain:

  - the principle of the microscopy technique used in the paper 

  - which is the bio-application and why this technique was used

  - which is the most relevant result obtained

   

  • Select activity Papers

    Download folder

    • Papers
      • 2003_OT_OpticalTweezers_Cytoskeleton.pdf
      • 2006_PAM_PhotoAcoustic Microscopy microvascular imaging.pdf
      • 2012_FIB-cryoEM Focused ion beam micromachining of eukaryotic cells for cryo EM.pdf
      • 2014_AFM Atomic Force Microscopy_Glia cell adhesion mechanical properties.pdf
      • 2015_STED nanoscopy_Subcortical Cytoskeleton Periodicity in Living Neurons.pdf
      • 2015_X-Ray Micro_rapid probing of features in exposed cells.pdf
      • 2018_OT_Mechanotrasnduction in neuronal cells.pdf
      • 2018_PALM_Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesion.pdf
      • 2020_NLO Non Linear Optical microscopy _ Nonlinear Imaging of Cellular Models.pdf