1 Module 2 overview lecture lab 1 Introduction to the module 1 Start up protein eng 2 Rational protein design 2 Site directed mutagenesis 3 Fluorescence and sensors 3 DNA ampli cation 4 Protein expression 4 Prepare expression system SPRING BREAK 5 Review gene analysis 5 Gene analysis induction 6 Puri cation and protein analysis 6 Characterize expression 7 Binding af nity measurements 7 Assay protein behavior 8 High throughput engineering 8 Data analysis 2 Lecture 3 Fluorescence and sensors I Basics of uorescence A Important applications B Energy levels and spectra C Emission quenching and energy transfer II Fluorescent calcium sensors A Properties of calcium sensors B Applying Ca2 sensors in cells C In vivo limitations and remedies D Advantages of genetically encoded sensors 3 uorescence luminescence microscopy H Lehmann S von Prowazek 1913 www zeiss com laser scanning microscopy Denk Svoboda 1997 Neuron Courtesy of Elsevier Inc http www sciencedirect com Used with permission functional imaging anatomical imaging histology Nagai et al 2001 PNAS Livet et al 2007 Nature Courtesy of National Academy of Sciences U S A Used with permission Source Nagai T et al Circularly Permuted Green Fluorescent Proteins Reprinted by permission from Macmillan Publishers Nature Ltd Engineered to Sense Ca2 PNAS 98 no 6 March 6 2001 3197 3202 Expression of Fluorescent Proteins in the Nervous System Nature 450 2007 56 62 2007 Copyright 2001 National Academy of Sciences U S A Source Livet J et al Transgenic Strategies for Combinatorial 4 5 www caliperls com in vitro assays Courtesy of Caliper Life Sciences Inc Used with permission 6 Introduction to uorescence Fluorescence arises from transitions among molecular energy levels Jablonski diagram electronic energy levels correspond to visible wavelengths vibrational energy levels correspond to infrared wavelengths with rotational energy levels are coupled to vibrations and account for the smooth appearance of absorption emission spectra 7 Absorption abs em uorescein Public domain image 350 400 450 500 550 Wavelength nm 600 Fluorescence emission Stokes shift Fluorescence spectra for a typical uorophore 650 Image by MIT OpenCourseWare small organics like uorescein are the most common uorophores in general the larger the aromatic ring system the longer the wavelength for excitation and emission quantum dots are 10 nm particles that exhibit narrower emission bands and less bleaching than organic dyes some atoms lanthanides exhibit uorescence as well 8 tetramethylrhodamine isothiocyanate em 580 nm 9 Decay of excited electrons can occur by radiative and nonradiative processes If N is the fraction of uorophore in the excited state and and k are radiative and nonradiative decay rates respectively dN k N dt such that N N0 e N e t 0 k t where is the uorescence lifetime incorporating both and k 1 k 0 describes the uorescence lifetime in the absence of nonradiative decay The ef ciency of a uorophore is quanti ed by its quantum yield Q Q k 0 One of the main routes of nonradiative decay is a process called quenching which results in environmental sensitivity for many uorescent molecules and underlies the mechanism of several sensors Fluorescence resonance energy transfer FRET can take place when the absorption spectrum of an acceptor overlaps with the emission spectrum of a donor and geometry favors dipolar coupling between the uorophores The distance at which 50 of excited donors are deexcited by the FRET mechanism is de ned as the F rster radius usu 10 100 R0 8 8 1012 2 n 4 QYD J Courtesy of Life Technologies Carlsbad CA Used with permission FRET ef ciency is de ned as E 1 r R0 6 1 1 6 10 11 Fluorescent calcium sensors A wide variety of uorescent calcium dyes are available They differ along several axes calcium af nity absorbance and emission properties structural properties e g protein vs small molecule membrane permeability binding and localization Indicators with each set of properties may be suitable for speci c experiments 12 A typical calcium sensor consists of a calcium sensitive component attached to one or more uorescent moieties BAPTA Fluo 3 EGTA dichloro uorescein Fluo dyes visible absorption emission wavelengths virtually no emission in absence of Ca2 range of calcium af nities Courtesy of Life Technologies Carlsbad CA Used with permission Fluo 3 Spectra 13 14 Sensors with different calcium af nities Kd values may be appropriate for different applications spontaneous Ca2 uctuations in Xenopus embryo Image removed due to copyright restrictions See http www invitrogen com site us en home support Research Tools Image Gallery Image Detail 2404 html calcium transients in dendritic spines Image removed due to copyright restrictions See http tools invitrogen com content sfs gallery high g001633 jpg www invitrogen com 15 Calcium Green related dyes What is the signi cance of having a dye with high uorescence intensity or dynamic range SNR Courtesy of Life Technologies Carlsbad CA Used with permission 16 How are calcium dyes applied to cells single cell injection Image from Svoboda K et al In vivo Dendritic Calcium Dynamics in Neocortical Pyramidal Neurons Nature 385 no 6612 1997 161 165 Macmillan Publishers Ltd Nature All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse AM esters multicellular loading or bath application Courtesy of National Academy of Sciences U S A Used with permission Source Stosiek C et al In vivo Two Photon Calcium Imaging of Neuronal Networks PNAS 100 no 12 2003 7319 7324 Copyright 2003 National Academy of Sciences U S A Courtesy of Life Technologies Carlsbad CA Used with permission 17 limitation effect on experiments solution dye binds to intracellular proteins and does not function loss of uorescence responses alteration of calcium sensitivity alter localization solubility e g using dextran conjugate dye leaks from cell or is sequestered into inappropriate cellular compartments loss of uorescence responses higher background uorescence use dextran conjugates or targeted indicators ratiometric imaging loss of uorescence responses lower imaging duty cycle select dyes with low bleaching ratiometric imaging dye bleaches over the course of experiments Ratiometric measurements 18 Suppose you measure uorescence intensity from a cell but you don t know either how much dye is present or what the calcium concentration is you have one equation in two unknowns L tot and Ca2 Ftot