Live Cell Imaging (a movie is worth a thousand pictures i.e. ~ a million words)
We are, first and foremost, cell biologists. This is why our primary experimental
approach in the lab is live-cell imaging. We use a number of cell biological tools such as FRAP, FLIP, FRET, photo-conversion (PhoC), and bi-molecular fluorescence
complementation (BiFC) to visualize our biochemical processes of interest in living cells.
Our aim is to resolve, in time and space, dynamic processes such as protein folding, misfolding,
ubiquitination, and aggregation.
Microscopes available in the lab
We welcome collaboration and are happy to share our imaging capabilities with other labs, especially ones at the Silberman Institute of Life Sciences. We are especially eager to introduce students at the Institute to advanced imaging, and to train them to use the appropriate equipment. Since this equipment is our life-line, outside use of the microscope is a very serious matter for us. Anyone interested is invited to read our Imaging Collaboration Policies and to get in touch with Dan.
Our microscopy set-up is divided up into several modules for different kinds of imaging. All of the modules are compatible with each other in terms of objectives, exitation and emission filters, and operating software (meaning that we can move all of these things around from one microscope to another).
diSPIM Selective Plane Illumination Microscope
We are the first lab in Israel to install a SPIM imaging system. This microscope uses 4 simultaneous laser sheets to rapidly image cells and C. elegans in 3D. The advantage of the SPIM system is speed (sub-second 3D stack of 40-100micron volume) and low to non-existent phototoxicity.
Nikon A1R-si Confocal Microscope
The A1R confocal is great for a number of applications, particularly spectral resolution (working with fluorophores whose emission spectra are close together), rapid imaging (such as transport within the cytosol), F-techniques (such as FRAP, FLIP, FRET, and Photoconversion). Ours has four solid state lasers : 405nm 50mW CUBE laser; 488nm 100mW Obis laser; 561nm 50mW Sapphire laser; and 640nm 40mW CUBE laser. Important features of the A1R includes a dual scanner system that enables simultaneous bleaching/photoactivation and acquisition (this is important for monitoring the movement of rabidly diffusing particles in live cells. The resonant scanner images very rapidly with lower resolution, and the galvano-scanner images more slowly at higher resolution. The A1R also has nice spectral resolution using a combination of a spectral detector and a 4 PMT filter-based detector system including two GaAsP PMTs with ~45% photon efficiency.
In addition to the applications outlined above, the main application of a confocal microscope is resolution in 3D (confocality is the result of a pin-hole that keeps out "irrelevant" light that comes from Z-regions other than the one you want to focus on). We also have a live-cell incubator built around the microscope, enabling long-term live-cell time-lapse studies. We also have a Piezo stage that can fly through a Z-stack in under a second, enabling rapid 3D time-lapse.
Nikon nSIM Super-resolution Microscope
As part of our microscopy system we also have an nSIM Structured Illumination Microscopy module. This system is not confocal - it uses a TIRF 100x objective and two lasers (488nm and 561 nm). An optical grid produces the SIM pattern, and the light is detected by a super-high sensitivity IXON EM-CCD camera. Because of the need to overlay grid illumination patterns a SIM image takes at least about a second to acquire - this requires fairly stable samples, but live-cell imaging is possible. This is a very specialized application that is designed to achieve very high resolution (~100nm), potentially in live cells. Experiments require alot of preparation and planning. We usually put 100nm beads into the sample that we are trying to image, in order to measure the point spread function of the objective in the same environment as our sample.
Nikon TiS Tissue Culture Fluorescent Microscope
Great for snapping pictures, checking tissue-culture transfections and yeast fluorescence. Samples are excited by a mercury lamp combined with standard exitation/emission filters (DAPI, CFP, FITC, Cy3, Cy5).
In order to enable a diverse number of imaging applications we have equipped our microscopes with some specialized objectives (in addition to what is standard).
100X CFI Apochromat TIRF - Oil NA1.49
Mostly for SIM but is also great for confocal imaging. It is an Oil TIRF objective so its ideal for working distances that are very close to the cover-slip (within ~100 into the sample).
60X CFI Plan Apo Lambda DM - Oil NA1.4
Oil objective that provides the greatest resolution/brightness trade-off for molecules not too far from the coverslip.
60X CFI Apochromat IR - Water NA 1.27
Also mostly for SIM since we have another water objective. This one has chromatic correction. Water objectives are great for 3D imaging (relatively) deep into an aqueous sample (such as a cell), without some of the spherical abberation that you see with oil objectives.
60X CFI Plan Apochromat - Water NA 1.2
The water objective that we use for confocal imaging.
100X CFI Plan Apochromat - Air NA 0.9
Has lower resolution than a water or oil objective, but has the advantage of easy mobility below the cover-glass with no need to worry about adding oil or water. Good for high-throughput imaging of 96-well plates and for movies of multiple positions on a place.
A super-resolution image of the IPOD
3D image of an H4 cell with labelled nucleus and actin
A 3D image of yeast ER and JUNQ
JUNQ asymmetric inheritance
IPOD asymmetric inheritance
Stress Foci inheritance
JUNQ and IPOD formation in 3D!!!
C. elegans moving in 3D