Confocal microscopy is a very powerful tool and has many advantages over wide-field fluorescent microscopy. One of these advantages is the ability to look at thin optical sections from a sample of cells or tissue without physically cutting the sample. This enables the user to get rid of the out of focus light associated with wide-field fluorescent microscopy.
Image diagram provided by Les.Boland Zeiss.co.uk
The above diagram represents the optical path on a laser point scanning confocal microscope. A small diaphragm, situated in a conjugated focal plane, prevents out of focus light reaching the detected. The diameter of this diaphragm controls the depth of the optical section. Images captured will have improved Z resolution and fine detail that is normally obscured by the out of focus blur assosiated with wide-field fluorescent microscopy will be seen.
System designed for high-resolution confocal imaging of both fixed and living cells. The FV1000 is built around a BX81 inverted microscope and is equipped with a SIM scanner that enables synchronization of specimen excitation and observation (while one laser stimulates, the second laser simultaneously provides high resolution imaging). This makes it the system of choice for FRAP, FLIP and photoactivation. It can view up to 3 fluorophores plus transmitted light and can be used for short-term timelapses (no incubation chamber attached at present - a small Pecon 37oC stage heater and CO2 are available).
The system is used for the following applications:
• Transmitted light detection for brightfield or DIC
• Photoactivation and photobleaching using the SIM scanner (50mW 405mn diode laser). This allows visualization of very fast protein dynamics (100-200ms recovery time)
• Standard multi labelled sample imaging. The system has a variable bandpass filter that allows for flexibility when choosing the wavelength of the emission fluorescence to be captured
Laser lines available: 405nm, 458nm, 488nm, 515nm, 559nm, 633nm
Objectives available: 10x/0.4, 20x/0.75 40x/0.9, 60x/1.35 oil, 60x/1.4 oil, 40x.0.8 water, 60x/0.9 water
Zeiss 710 confocal microscope
System has 7 excitation lines at 405nm, 458nm, 488nm, 514nm, 561nm, 594nm and 633nm, and is equipped with the Zeiss Quasar detector configured with 2, 3 or 34 channels. Built around a Zeiss Axioimager upright stand it is primarily for fixed samples and is suitable for a wide range of applications, including imaging of quadrupled labelled specimens, colocalization of proteins and 3D imaging experiments. It is operated with Zen software with the 'Smart Setup' function that allows you to easily set up dye configuration, making it easier for inexperienced users.
The system can be used for the following applications:
• DIC imaging using the transmitted light detector
• Spectral imaging/online unmixing
• Tile scanning
• Z sectioning and 3D imaging
• Visualizing multi-labelled samples
Zeiss 880 confocal microscope with 'Airyscan FAST' module
Offers users clear advantages over our standard confocal microscopes for certain samples and applications. The 'Airyscan' detector delivers increased resolution with high sensitivity at around 140nm laterally and 400nm axially with 488nm. The 'AiryscanFast' mode increases scanning speeds by a factor of 4. This allows you to reach speeds that have only been possible with the use of a resonant scanner without sacrificing sensitivity or resolution. It has a full cage environmental chamber for live cell imaging and is also suitable for photoactivation, FRAP and FCS.
Laser lines available: 350nm, 405nm, 445nm, 488nm, 512nm, 561nm, 594nm, 633nm
Zeiss 880MP 'AiryscanFAST' confocal microscope
Timelapses & Live Cell Imaging
Andor Revolution XD spinning disc system
Built around a Nikon Ti-E inverted microscope with PFS. This should be your system of choice for live cell imaging.
• Yokogawa CSU-X spinning disc confocal unit with a disk speed that supports up to 2,000 frames per second
• Laser Combiner with 488nm and 561nm lasers
• High resolution Andor Neo sCMOS camera (2560 x 2160 pixel, 16 bit, linear full well 30,000 e, read out noise 1 e, quantum efficiency > 55%, 100 frames/sec @ 2560 x 2160 pixel) for high speed/sensitivity/resolution)
• Cairn Optsplit for simultaneous duel fluorescence imaging
• Andor mosaic FRAPPA unit for FRAP and photo activation
• Okolab bold line stage top incubator with humidifier and CO2.
• Andor IQ acquisition software
More information about the system is available from www.andor.com
Essen Bio IncuCyte live cell analysis systems FLR, Zoom and S3
Consist of a microscope that is placed inside an incubator. They can accommodate various flasks and plates, can acquire up to 2000 images per hour and can be used for the monitoring cell growth, health and viability. Commonly they are used for cell migration assays and kinetic apoptosis assays but there are an increasing number of other applications. Depending on the system, you can image two fluorescence channels and phase contrast with a choice of x4, x10x and x20 magnification.
A fully automated, highly sensitive confocal microscope system. It has the capability to scan very rapidly (230 frames per second in a 512 x 64 pixel image and 30 fps at 512 x 512). This is due to its two scanning systems. Firstly, there is a conventional sawtooth scanning system – galvano, which is used for high-resolution imaging where speed is not essential. Secondly, there is a fast resonant scanner, which is used for applications where the need for speed outweighs the need for high resolution. The two scanning systems can be operated in rapid succession or simultaneously. This makes it possible to stimulate the specimen during observation. As a result, the rapid protein dynamics that occur immediately after the laser has stimulated the cell can be accurately captured and analysed. The A1R also has a spectral detector, based on a conventional 32 cathode PMT. The system is built around a Nikon Ti Eclipse microscope with PFS. This makes it the perfect confocal to use for live cell timelapse imaging.
• Scan head with regular galvanometer mirrors and resonant mirrors
• 4 laser system with AOTF 405LD 38mw, 561nmDPSS 25mW, 638nmLD 10mW and multi-line argon gas laser (457nm, 488nm, 514nm)
• XY, XYT, XYZ, XYZT and multipoint scanning. Galvano pixel max 4096x4096 pixels
• Resonant pixel max 512x512 pixels
• Simultaneous photoactivation/bleaching and image acquisition
• 4 channel PMT detector and 32-channel PMT spectral detector
• Okolab full environmental chamber
Nikon LTTL Microscopes
We have Nikon Ti eclipse timelapse microscope systems with PFS, which continuously corrects the focus at the plane of interest with its continuous optical offset feature. When the PFS is turned on the position of the glass bottom dish/plate is detected using a 777nm infra red light. The result is an end to out of focus drift. You should no longer have to adjust the focus knob after adding reagents or after stage movement. The PFS is designed to work with glass bottom dishes. We have used plastic dishes but have had mixed results. If you are using plastic dishes or plates our advice is to switch off the PFS and use the system in the conventional way. The system is equipped with a Sutter lambda 10-3 controller, ASI MS2 XY stage with linear encoders, Photometrics CoolSnap HQ or Prime CCD camera and Metamorph 7.6 software.
PhaseFocus VL21 live cell imaging system
The Phasefocus Virtual Lens® provides non-toxic phenotypic screening for live cells, enabling long-term timelapse assays without the need for fluorescent labels. This is an important consideration for the screening of sensitive primary cells, and stem cells. More information can be found on the PhaseFocus website: http://www.phasefocus.com/
FLIM & FRET
The LaVision TRIM microscope is a high-resolution multiphoton system able to image small groups of cells down to the level of subcellular structure and generate 3D reconstructions. It is used to image neurons of the central nervous system and epithelial cells within the crypts of the colon. Its scan head is mounted on a Nikon Eclipse inverted microscope. Illumination is provided by a Chameleon ll Ultra fast laser (680nm-1080nm 3.3W peak), which also pumps an APE optical parametric oscillator (1050nm-1320nm, 500mW peak) to provide excitation of red fluorescent proteins.
Applications: offers fluorescent and second harmonic generated imaging of samples from cell culture through to whole tissue samples via intermediate systems such as organotypic invasion assays (artificial skin).
Example samples: cells on glass / CDM / collagen invasion assays; organotypic samples that are a mix of collagen type 1 and fibroblasts, providing a model of stroma with both fluorescent cultured cells and fluorescent fibroblasts possible. This allows the 3D visulization of the interaction between tumour cells and the host stromal fibroblasts. The nature of this assay easily allows the direct genetic and/or drug modification of cellular behaviour to be studied over a course of approximately a month; sample slices – the quantity of collagen can be quantified in various locations; tissue samples from genetic models, transplant generated models.
Techniques available: intensity fluorescence and second harmonic generation; full range of fluorophores accessible to imaging from DAPI through to far-reds; fluorescence lifetime imaging (FLIM) across all fluorophores and autofluorescence, e.g. NAD(P)H; Z-stack, tile scanning, time course.
This information and more about the system can be found on the LaVision website.
LIFA FLIM microscopes
We currently have two LIFA FLIM systems from Lambert Instruments. The LIFA (Lambert Instruments FLIM attachment) for lifetime imaging microscopy is a dedicated system that allows rapid image acquisition and generation of lifetime images. It can be attached to any widefield fluorescence microscope and works in the frequency domain. To achieve this the system uses a modulated light source and a modulated image intensifier as a detector. Quantitative lifetime data can be generated in seconds and the advanced software can analyse your data and present the calculated fluorescence lifetimes visually. The LIFA system is attached to a Nikon TE 2000 inverted microscope. One of our systems is equipped with an Omicron, a 50mw 445nm laser for CFP lifetimes and a 488nm 60mw laser for GFP. This system has a Yokogawa CSU22 spinning disk unit. The second system is coupled through our custom TIRF condenser to make it a FLIM-TIRF system. This system can also be used for widefield with a range of LED light sources (407, 445, 453, 469 and 535 are available).
Nikon TIRF microscope
The facility has one total internal reflection fluorescence (TIRF) microscope and one TIRF-FLIM system. TIRF is a powerful technique that uses an evanescent wave to excite flurophores that are present at the bottom of the specimen adjacent to the glass water interface. The optical section that is excited is in the range of 100nm-150nm. It is often employed to study cellular membrane activities, the dynamics of actin, cellular adhesion, cell movement, single molecular events, vesicle and protein tracking. TIRF is so named because it involves total internal reflection of the excitation light. The excitation light must strike the glass-medium interface at the critical angle (the angle at which no light passes from the glass slide to the medium). Instead, the light is reflected and this results in an evanescent wave being produced. One of the advantages of TIRF is that the background and out of focus signals are dramatically reduced, which allows you to view very dim fluorescent events.
Above image courtesy of Juliana Schwarz showing TIRF optical path
The system is appropriate for live cell fluorescent protein imaging and is built around a Nikon Eclipse inverted stand with custom optics for illumination of GFP and RFP at 473nm and 561nm, respectively. Red and green fluorescent proteins are imaged simultaneously onto a high sensitivity Photometrics Evolve 512 camera using a DualView DV2 emission splitter. The system is also equipped with a 405nm laser for photo-activation and FRAP.