First National FLIM Workshop

Posted 6 February 2017

Facility: Biomedical Imaging Facility

The aim of this workshop is an in-depth introduction to Fluorescence Lifetime Microscopy (FLIM). The program includes lectures about the principals of FLIM, practical sessions and research talks showcasing FLIM application in the Life Sciences.
 

Practical Sessions

Practical sessions will be divided into multiple topics. Firstly the focus will be on sample preparation and data acquisition. Like in any microscopy if the sample or the microscope is not set up properly the data will not make any sense. We will concentrate on common pit falls in fluorescence lifetime measurement including the correct selection of laser repetition rate for lifetime experiments and avoiding pulse pile-up effects.
We will cover the most common applications of FLIM including measurements of autofluorescence and its applications, we will demonstrate the effect of environment on fluorophore lifetime (polarity, pH, solvent etc), and Fluorescence resonance energy transfer (FRET) and Biosensors. 
In the second part of the practical session we will focus on data analysis. We will cover correct decay fitting, phasor analysis, and also pattern matching analysis. In this section we will cover the principles of the analysis methods and look at some real applications.
 
You will get a chance to see 4 FLIM platforms that are available in the BMIF.
Horiba FluroMax-4 Spectrofluorometer with DeltaHub time - Correlated single photon counting (TCSPC) controller and Deltadiode excitation at 295, 370 and  470 nm
 
BYO Sample: Wednesday 22nd February - Should you be interested in analysing your own samples please contact us.

 

Lectures

The FIRST FLIM workshop will start with a plenary lecture form A/Prof Trevor Smith from Melbourne University. 
 
Time-resolved emission microscopy – Methods and extended applications
Trevor A. Smith, Ultrafast and Microspectroscopy Laboratories, School of Chemistry & ARC Centre of Excellence in Exciton Science, University of Melbourne
Time-resolved emission microscopy is a powerful approach to provide additional information to that achievable by conventional intensity-only imaging.  In addition to providing maps of fluorescence decay behaviour (fluorescence lifetime maps), these methods can be extended beyond “fluorescence lifetime imaging” (FLIM) to map rotational mobility (local viscosity) by collecting emission as a function of polarisation.  Phosphorescence can also be used to map the environment of certain lumophores.  
There are several methods that can be used to achieve time-resolved emission microscopy when coupled to various modes of microscopy.  The various methods will be discussed along with how these methods can be extended to provide additional information.  The advantages and disadvantages of each technique will be assessed in relation to implementing these extensions to the technique.
We will report on experiments in which we have applied a range of time-resolved emission microscopy techniques, including FLIM, “phosphorescence lifetime imaging” (PLIM) and fluorescence anisotropy imaging (FAIM) to a wide variety of samples.  This includes the intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes, aggregation-induced emission compounds, and non-cellular systems.
 
Plenary lecture will be followed by an interactive lecture given by Dr. Alex Macmillan UNSW Sydney on principles and pit falls of FLIM (see below in practicals).
 
The Workshop will conclude with a series of research talks showcasing not only FLIM application about also summarising the knowledge from the workshop.
 
Dr Elizabeth New – University of Sydney
Using FLIM to enhance the output of responsive fluorescent sensors
While there are now many sophisticated imaging techniques to study biological systems, chemical tools are needed to gain an understanding of what is happening in the cell, on a molecular level. We are interested in designing small molecule sensors to probe sub-cellular molecular species, especially those with a fluorescence or magnetic resonance output. In particular, we are developing redox-responsive sensors that are able to report on reversible oxidative events in biological systems, and can distinguish transient oxidation events from oxidative stress. We also have an interest in imaging essential and therapeutic metals within cells using selective fluorescent sensors. In our research, we have utilised fluorescence lifetime imaging microscopy in order to observe ratiometric information about chemical systems using our fluorophores.
 
Dr Sean Warren – Garvan Institute of Medical Research
Studying cell signalling in vivo with FLIM-FRET
Genetically expressed Förster resonance energy transfer (FRET) biosensors are a powerful tool to study the activation of signalling proteins and pathways in living cells. There is a growing awareness, however, of critical differences in the behaviour of cell in native, 3D contexts and 2D cell culture. Our group uses genetically engineered mice expressing FRET biosensors to study signalling in vivo with FLIM. In this talk I will discuss our work using mice expressing biosensors for the small GTPases RhoA and Rac1 and how we address some of the challenges associated with intravital FLIM such as sample motion and tissue autofluorescence using our analysis software FLIMfit. To investigate the spatio-temporal relationship between the regulation of these proteins, which act in concert to regulate cell motility and migration, we have crossed the RhoA and Rac1 mice to generate a dual biosensor mouse. Using a custom multichannel FLIM system we can resolve the activation level of RhoA and Rac1 in the dual mouse on a subcellular level using a novel spectral-lifetime fitting approach.
 
Dr Ales Benda – BIOCEV, Czech Republic
 
Dr Kirstin Elgass -  Monash Micro Imaging
 
Blake Smith – Murdoch Childrens Research Institute 
 
Ephrem Sitiwin – UNSW Sydney

 

Registration

Registration fee – GST incl
LMA Members: $165 
LMA Non-Members:  $220 (includes LMA membership)
LMA student Non-Members:  $192.50  (includes LMA student membership)
LMA membership: (select LMA membership)
 
Cost includes  - USB with lectures and practicals, 
Morning tea, Lunch and Afternoon tea for both days
Dinner at Coogee Rooftop Pavilion– optional $55 (includes meal and drinks) 
 
 
CONTACT – Iveta Slapetova i.slapetova@unsw.edu.au