From f1b370cd824e71bdf5fdf99f5cab59d78e54da0d Mon Sep 17 00:00:00 2001 From: "lorenzo.zolfanelli93" Date: Fri, 28 Aug 2020 04:00:59 +0000 Subject: [PATCH] Update on Overleaf. --- chapters/3-methods.tex | 49 ++++++++++++++++++++++++++++++++++++++--- images/alexa.png | Bin 0 -> 74105 bytes 2 files changed, 46 insertions(+), 3 deletions(-) create mode 100644 images/alexa.png diff --git a/chapters/3-methods.tex b/chapters/3-methods.tex index dd11d49..da4c035 100644 --- a/chapters/3-methods.tex +++ b/chapters/3-methods.tex @@ -595,10 +595,53 @@ vicine alla finestra di emissione del fluoroforo possono portare a un aumento della luce retrodiffusa dal campione nel cammino di raccolta, aumentando significativamente il rumore all'aumentare della potenza del laser. Per questo motivo, visto che le sorgenti che abbiamo a disposizione -sono economici - - +sono economici laser a diodo con spettri di emissione non particolarmente +\textit{puliti}, è necessario utilizzare dei filtri di \textit{clean-up} +in modo da sopprimere con fattori di estinzione elevati ogni emissione spuria. + +Ugualmente importante è massimizzare l'efficienza di raccolta: +il rilevatore utilizzato, un sensore EMCCD, può operare in regime di +\textit{conteggio fotoni}, con una sensibilità molto elevata anche a +brevi tempi di integrazione. +Infatti, a differenza di quanto avviene nei comuni sensori CCD, +gli elettroni accumulati in ciascuna cella del sensore attraversano una +regione di moltiplicazione, dove, grazie a un'elevata differenza di +potenziale, viene massimizzata la possibilità di generare nuovi elettroni +per ionizzazione da impatto. Il segnale (sotto forma di cariche accumulate) +che a questo punto viene trasferito allo stadio di integrazione e lettura +del CCD, presentà già un elevato guadagno senza dover far ricorso a +ulteriori stadi di amplificazione elettronica. +Per operare correttamente il sensore EMCCD deve essere raffreddato +a temperature $< \SI{-60}{\Celsius}$, in modo da ridurre il più +possibile gli elettroni prodotti termicamente (corrente di buio), +che possono subire lo stesso processo di moltiplicazione dei +fotoelettroni. + +Oltre al rumore dovuto all'apparato sperimentale è importante tenere conto +del fatto che, quando si usa il classico schema di illuminazione a campo +largo, il fascio di eccitazione attraversa per intero il campione. +Quindi la presenza di fluorofori liberi in soluzione provoca un aumento +consistente del rumore dovuto alla fluorescenza fuori fuoco. + +I fluorofori che si intende utilizzare per lo studio della +meccanotrasduzione appartegono alla famiglia Alexa Fluor (Molecular Probes). +Si tratta di molecole organiche sintentica particolarmente fotostabili +e con resa quantica elevata. +In commercio si trovano versioni funzionalizzate legate a svariate molecole +e kit di coniugazione che consentono di legare semplicemente e rapidamente +i fluorofori a proteine e peptidi. +Tenendo conto delle caratteristiche dell'apparato è possibile utilizzare +i fluorofori Alexa Fluor 647, 532 e 488. In figura \ref{fig:alexa_spectra} +si riportano gli spettri di eccitazione e emissione. +\begin{figure} + \centering + \includegraphics[width=0.8\linewidth]{images/alexa.png} + \caption{Spettri di eccitazione (linea tratteggiata) ed emissione + (linea continua) dei fluorofori Alexa Fluor + 488, 532 e 635.} + \label{fig:alexa_spectra} +\end{figure} diff --git a/images/alexa.png b/images/alexa.png new file mode 100644 index 0000000000000000000000000000000000000000..9e9a66471b8e1519f23364615c119efdbec158f0 GIT binary patch literal 74105 zcmeFZby$>b*FK60(%mpJGW1A;IJ9&M49tK?DXk(5Asx~^ltYMwlmjTK0#edLNC^T; zhXMvt5)%6!eIFm+I7TAO4a`>Z`AcNoZ?sFRTO`Y=85 zQ8uS*O4h;iLx2C*inTHKX(P%o8f6mBpZ|zOhJ^mUsQf<{od4h01FqJC!?hp>mWUg_ z{-i&&9V@eV$rjJY0HWsuGsYim?EqqkC-ELG^CG-~MV8Jzd$q+UEgR9|cig(AQk2!DuRCo< zIojw)vtJpFX18wK>RtDL{;{&;9kjvl3uA-jm%C0SFG$Tw?zBXXDzu|-$yQXq)AKj` zVzy=#zJ_Qro+#cZ?vcV!Bd3jgH0)A(&SFfEmT=SjjpUw9=s<}e(notq3qu6mM8qKN zOLR>#o`@^iJ?T+_wjm?1ljXx!g$dd&7!Igcv4`PGu3LT29cT^qnPHt$VMfm=G#<%@ 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