Teach & Tech

Before starting the acquisition of your image data, one need to check some basic setting on the setup to use, to make sure that you benefit the most of the advanced equipment you are to use.

• Köhler Illumination procedure
• Objectives: what to consider (NA, immersion, Refractive Index) & IOF cleaning protocols

Fluorescent proteins and dye properties: you can find detailed information about your favorite fluorescent proteins and filters – or make panels to prevent cross excitation and/or bleed-trough. Make sure that the microscope that you plan to use can effectively excite your fluorophores and separate the emission spectrum precisely

• FP-base
• Spectraviewer
• IOF Applicaion Note: How to perform a Color Shift Correction

Sample preparation is to be tuned to both the sample and imaging application. Here we only list some points of attention and specific technologies to provide a first glance:

• • Cover slips and mounting glasses: cover slip thickness is indicated with a grade number – for example, No. 1, No. 1.5, No. 2. The most commonly used cover-slip for optical microscope is is No. 1.5. High resolution imaging application require high precision cover-slips (smaller tolerances). In addition, new cover-slips are still contaminated with impurities. It is highly recommend to clean cover-slips before use, especially for high resolution imaging applications. 
  • Mounting media: different mounting media are on the market – where there is a clear separation for non-fluorescent samples and fluorescent samples. Mounting media also might have different refractive indices – that need to be matched with the immersion used!
  • Expansion Microscopy: Enhanced imaging of biological specimens with fine details by physically making them bigger through an isotropic chemical expansion process that preserves nanoscale details → http://expansionmicroscopy.org
  • Cleared tissue imaging:the natural opacity of organ tissue prohibits in depth analysis of thick tissues. A multitude of tissue optical clearing (TOC) protocols have been established for various tissues to overcome this limitation by turning organs into transparent, light-permitting specimens → a brief overview of some tissue clearing resources
  • Refractive index matching: Iodixanol is a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids (Boothe et al., 2017). Our ‘ Automated Rental System‘ hosts a Refractiometer, amongst other optical tools.

Working with light requires to prevent any obstruction in the light path, to obtain the highers performace out of any optical system. Proper cleaning of all optical components is critical to reach this goal.

MICROSCOPY MAINTENANCE & CLEANING OPTICS

• How to clean objectives, according to Nikon protocol
• Removing immersion from objectives, by Olympus
• Cleaning optical components, according to Leica
• Microscope cleaning and maintenance, according to Zeiss

COMPANY RECOMMENDED MICROSCOPE SANITIZING ROUTINES

• • Leica how-to-sanitize-a-microscope
  • Nikon recommended microscope disinfecting procedures
  • Olympus: how to clean & sterilize your microscope
  • Zeiss: microscope virology application disinfection routing

Please also see our equipment page for a short description of the available instrumentation product types:

Different super-resolution applications are able on the following facility super-resultion instumentation

• Airyscan: multi-array detector, allowing whole airy-disk imaging.  principle of the Airyscan detector (Huff et.at., 2017; ‘Let me google that for you‘) – airyscan instrumentation
• SORA: Super-resolution via Optical Re-Assignmentis (SoRa) a magnification changer for Yokogawa CSU-W1 spinning disks confocal microscopes enabling optimum resolution across different objective magnifications → resolution xy: 120nm resolution; z 300 nm (after deconvolution). ( Azuma & Kei, 2015; ‘Let me google that for you‘) –
• SRRF: Super-resolution radial fluctuations (SRRF) is a super-resolution imaging application that can acchieve up to 150nm resolution (Gustafson et.al., 2016; ‘Let me google that for you‘) – available equipment
  
• dSTROM: Direct Stochastic Optical Reconstruction Microscopy (dSTROM) is a ‘single molecule super-resolution imaging application that can be used with standard fluorophores (van de Linde et al., 2022; ‘Let me google that for you‘

Before applying these technologies, make sure to check the required details about your favorite fluorescent proteins and filters on FP-base or Spectraviewer

• FLIM: Fluorecent Lifetime Imaging Microscopy (FLIM) is a technique based on the exponential decay of a fluorescent sample.  Lifetime imaging has many applications including: i) Multi component separation, ii) Auto-fluorescence removal, iii) Detection of molecular interactions (e.g. FLIM-FRET), iv) Environmental sensing (e.g. pH). More information →  our FLIM resource page – FLIM instrumentation
• FRAP: Fluorescent recovery After Photobleach (FRAP)
• FRET: Fluorescence (Förster or ) resonance energy transfer (FRET) is distance-dependent (10–100 Å) involving an  energy from an excited donor molecular fluorophore to a neighboring acceptor fluorophore by ‘inter-molecular long-range dipole–dipole coupling’. FRET efficiency can be used as a metric of protein configuration or interaction. (let me google that for you)

• • Fluorescent Correlation Spectroscopy (FCS) → Zeiss LSM880 up Airyscan
  • Laser ablation & micro-dissection: see ‘Force Probing Assays’-section
  • Lifetime-Gated Acquisition: An specific ‘Life-time imaging application’ allowing to separate emitted signal based on the photon arrival time, e.g. separating fluorophore signal from auto-fluorescent signal – often applied in chromoplast containing (plant) samples. (let me google that for you) – FLIM instrumentation
  • Photo-Patterning Applications: i) 2D photopatterning on substrate, ii) Photopatterning on 3D microstructures, iii) Microfabrication, iv) Hydrogel polymerization → Nikon Ti2E-03 (PRIMO)
  • Wavelengts (LAMBDA Λ / lambda λ) scans: When either excitation- (Λ) or emission (λ) spectra are not available, and excitation / emission spectrum scan can be perfomed – using tunable lasers (excitation) and dynamic emision detectors (λ) → Λ / λ scan instrumentation

• Laser ablation & micro-dissection: UV laser-assisted micro-dissection and ablation → Andor Spinning disk
• D.P.A.: Dual pipette aspiration assay  → Leica Stelaris 5 inverted confocal
• Magnetic force probing: Magnetic tweezer → Leica Stellaris 5 inverted confocal
• AFM: Atomic Force Microscopy – Attaching beads to a cantilever

• • Leica Sample navigator & Life Data Mode
  • Nikon Automated Water Immersion dispensing (long-term imaging) → Nikon Ti2E-02, Nikon CSU W1-01/02/03
  • Zeiss ZEN Connect & Image Blocks: multiscale imaging and navigation → instrumentation
  • Sample Stabilization: (Root) tracker: stabilization of moving/drifting objects + tracking → available on all Zeiss LSM800/900 confocals
  • Multi-blocks made easy: Zeiss experiment designer macro to run consecutive acquisition blocks → available on all Zeiss LSM800/900 confocal microscopes

As of 2022, all IOF (teaching) activities are listed in our IOF Events Calendar that can be found on our News & Events Page. Previous courses and events are stored on our courses & workshops  and Equipment demonstrations & Workshops pages. Courses are further organized using the ISTA Moodle platform.