Webinar: Cryogenic fluorescence microscopy – how to optimise your experimental set-up for biological insights

Lunedì 5 Settembre 2022 - 6:00 pm CEST

La microscopia a fluorescenza in ambiente criogenico (Cryo-FLM) è spesso utilizzata, in ambito della crio elettro-tomografia (Cryo-ET), per identificare la regione di interesse (ROI) nella fase di preparazione delle lamelle.

Questa tecnica è estremamente utile nell’aumentare il successo di preparazione delle lamelle potendo tener traccia delle zone d’interesse sul campione.

I benefici derivanti dalla correlativa Cryo-FLM sono molteplici e ben noti, ma la sua utilità può essere compromessa qualora il design ottico del sistema non sia ottimizzato per la rivelazione dei segnali di fluorescenza emessi dal campione, soprattutto quando sono deboli o quando gli artefatti della misura non possono essere discriminati e quindi rimossi dall’analisi.

In questo webinar, Marit Smeets, Product Manager della Delmic, insieme al Dr. Stephen Carter, del centro di Virologia della MRC-University di Glasgow e al Dr. Alan Mullan product manager di Andor Technology per le camere dedicate alla microscopia, presenteranno lo stato dell’arte e gli aspetti cruciali della Cryo-FLM.

Gli esperti mostreranno alcune tecniche per ottenere sempre dati reali, attendibili e avulsi da artefatti; infine, risponderanno ad ogni domanda relativa a questa potente tecnica correlativa.

Il webinar prevede tre talk:

  • Avoiding excitation crosstalk in multi-channel fluorescence imaging - Ms Marit Smeets
  • Subtracting autofluorescence inherent to cryo-FLM - Dr Stephen Carter
  • Choosing a scientific camera for cryo fluorescence microscopy - Dr Alan Mullan

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Di seguito gli abstract dei tre talk:

Avoiding excitation crosstalk in multi-channel fluorescence imaging - Ms Marit Smeets

Cryogenic fluorescence microscopy (cryo-FLM) combined with scanning electron microscopy (SEM) makes the process of lamella milling in the cryo-electron tomography (cryo-ET) workflow more accurate and efficient. The benefit of cryo-FLM can however be compromised if the optical design of the system does not allow the users to easily overcome the artifacts inherent to fluorescence microscopy.

Excitation crosstalk is an artifact that can appear when two or more fluorescent labels with partially overlapping excitation and emission spectra are used to tag different types of biomacromolecules. In this presentation, we will discuss the cause of excitation crosstalk and how this can be prevented by selecting the correct optical components.

Subtracting autofluorescence inherent to cryo-FLM - Dr Stephen Carter

Cryogenic correlated light and electron tomography (cryo-CLEM/cryo-ET) is a powerful technique to study the complex relation between protein complexes and organelles inside cells. Typically, cryo-CLEM uses fluorescent proteins to initially locate a protein-of-interest inside large eukaryotic cells using cryo-fluorescence microscopy. This fluorescence information allows coordinates of a protein-of-interest to be mapped for precise localization of the same target inside the electron microscope and subsequent high-resolution structural interrogation using cryo-ET. A major challenge for researchers that can hamper the identification of the “real” fluorescent protein signal in the cryo-fluorescence microscope is the presence of bright “autofluorescence” at 80K.

In this talk, Stephen Carter will focus on how autofluorescence can be problematic for cryo-CLEM. He will provide practical examples detailing sources of autofluorescence in mammalian cells that have hindered his cryo-CLEM experiments and simple ways the autofluorescence problem can be overcome.

Choosing a scientific camera for cryo fluorescence microscopy - Dr Alan Mullan

Imaging cameras come in a wide range of different sensor formats and offer combinations of sensitivity, speed, and field of view. Furthermore, the same sensor may feature in a number of different cameras models, but the sensor can be implemented differently. These differences may appear to be quite subtle and thus unclear if they have any meaningful impact on imaging performance.

One difference between camera designs is how the sensor is cooled, and additionally how the sensor is enclosed. It is relatively well known that cooling the sensor reduces thermal noise (dark current) and this is important for applications like luminescence that require longer exposures into many minutes. But how valid is sensor cooling to the shorter exposures of typical fluorescence-based imaging that are measured in milliseconds, and how much cooling is needed?

In this presentation, we will discuss the topic of sensor cooling of sCMOS and CCD (and EMCCD) based cameras. We will look at how sensors may be cooled efficiently in terms of the overall camera design. As part of this, approaches to how the sensor is enclosed in the sensor chamber will be outlined. Then we will look at what parameters of the sensor are impacted by operating temperature and how this impacts sensor behaviour. From this we can determine if, and when we need to consider cooling for a given imaging application.

Contatti

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