Imaging Techniques

2.5.1 Live-Cell Imaging

Confocal fluorescence microscopy was carried out on transfected cells in glass- bottomed 35mm dishes (section 2.3.2) in a Zeiss XL incubator (37°C, 5% CO2).

Several different imaging systems were used for experiments.

A Zeiss LSM510 Axiovert confocal microscope with a Spectrophysics pulsed multiphoton MaiTai laser was used for multiphoton excitation of samples, in addition to a standard Argon ion laser and 561nm diode laser for imaging with standard wavelengths. A x40 1.3NA oil immersion apochromat objective was used for imaging. Standard filtersets were available for imaging using ECFP, EGFP, EYFP and DsRedXP as well as UV excitation such as DAPI.

A Zeiss LSM 710 Axiovert with Confocor FCS module was generally used for UV excitation and FCCS data collection. UV excitation was carried out using a 405nm laser while an Argon ion laser and 561nm diode laser were used for imaging with specified standard wavelengths. A Plan-Apochromat 40x/1.4 Oil DIC M27 objective lens was used on this system for imaging. A wide range of spectral options were available due to the 34 channel QUASAR detection unit. This allowed superior spectral separation of fluorophores.

A Zeiss LSM780 with GaAsp detectors was used for imaging as well as FCS and FCCS with the wavelength specified in each section. A Fluar 40x 1.4NA oil immersion objective lens was used on this system for imaging studies.

Live cell imaging was conducted using the zeiss Zen MTS macro which allowed the creation of complex imaging scenarios including multifield time series experiments with bleaching steps and autofocus to control for minor temperature changes causing focal changes through expansion and contraction of the dish. All of these functions would not have been available in both AIM and Zen2010B software suites without the use of this macro. Live cell time lapse imaging data sets were analysed using Cell Tracker, a program specifically developed for use with such datasets (Shen, Nelson et al. 2006). Both cytoplasm and nuclear compartments were tracked over multiple frames for each field in a time series experiment. Data sets were then exported to Excel for further analysis (Microsoft, USA).

2.5.2 FRET: Optimised Illumination Strategy

While optimisation of an illumination strategy is discussed at length in Chapter 3, two distinct illumination strategies were finalised for the majority of biological experiments. They differ by the use of either multiphoton excitation or UV excitation of Dronpa to promote activation. Switching Dronpa into the off state was achieved using the 488nm line of an Argon Ion laser to simultaneously image and switch Dronpa into the off state.

Table 2-2 Time lapse imaging parameters 488nm Excitation 458nm Excitation Switch parameters Image

Properties Lightpath settings

LSM510E Multiphoton 4.5% 5%, 810nm 2.5% 25it 512x512 stack 12bit 458/466/810nm beam split 515LP IAA/IDA Band pass IDD Band pass LSM710 405nm UV 4% 3.5% 405nm 1% 5it 256x256 stack 8bit 2.55µs pixel dwell

458/488 vis beam splitter 405 invis beam splitter

IAA range

IDD Range

IDA range

Separation of ECFP and EYFP fluorescence was carried out using the linear unmixing algorithms in Ver. 3.0 of the Zeiss LSM510 software (Zeiss). This was carried out through comparison with reference spectra taken from cells expressing ECFP or EYFP alone or nothing (background reference). The fluorescence spectrum was separated into ECFP, EYFP and background signals. FRET was assayed by acceptor (EYFP) photo-bleaching. For bleaching the entire cell assayed was exposed to 50 iterations of 514nm laser light set at 100% power. Once signals were unmixed into respective channels, fold-change in donor fluorescence, relative to an unbleached cell in the same field of view, were used as a measure of FRET. Multiple cells were averaged and data were expressed as mean change in donor fluorescence +/- SD.

2.5.3 FCS and FCCS

FCS and FCCS was carried using either a Zeiss LSM780 with or Zeiss 710 with confocor 3 mounted on an Axio observer Z1 microscope with a 63x C-apochromat, 1.2 NA water-immersion objective. Zen 2010B was used for data collection and analysis. EGFP fluorescence was excited with 488 nm laser light and emission collected between 500 and 530nm dsRed-express was excited with 561 nm laser light and emission collected between 580 and 630nm. The protocols as outlined in Kim et al. (Kim, Heinze et al. 2007) were followed, with 10 x 10 s runs used for each measurement. The lateral beam dimension was estimated to be 229 ± 6.3 nm using Rhodamine 6G as a known calibration standard. A structural parameter value of 5, which is the ratio of the axial to lateral beam dimensions, was assumed. Free EGFP in cells was measured to have a diffusion rate of 27 ± 5.8 μm2

s-1, agreeing with previous measurements (Baudendistel, Müller et al. 2005).

The intensity fluctuations recorded and their auto and cross-correlation function calculated on ZEN 2010. Measurements (10x10s) were carried out in cytoplasm or nucleus, with binning time of 200ns. The data was fitted into a mathematical model describing one or two component diffusion; the appropriate model was selected based on the Chi2 value describing each fit. The cross-correlation function included correction for triplet state transitions of fluorophores and assumes Brownian diffusion of molecules.