Confocal Microscopy

This system mates an upright microscope with four lasers: a UV Diode (405nm), an Argon laser (458nm, 476nm, 488nm, 514nm), a red Diode laser (543nm), and a HeNe laser (647nm). The system has a spectrophotometer scan head that allows the user to tune the four PMT detectors to any emission wavelength, and it allows simultaneous imaging with an additional transmitted light detector. It can also perform spectral scanning of fluorescence for generation of dye emission spectra and for separation of dyes with overlapping emission spectra. It also features a motorized stage that can mark and find multiple points and automate montage acquisition and stitching to generate high resolution images of large areas.

At left the Leica SP5 DM microscope. At right, image of Drosophila heart by Jianbo Na

Technical Specifications

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User Protocol

This system has an inverted microscope enclosed within a CO2, humidity, and temperature-controlled chamber for live cell confocal imaging. It has four lasers: a UV Diode (405nm), an Argon laser (458nm, 476nm, 488nm, 514nm), a DPSS laser (561nm), and a HeNe laser (647nm). Its spectrophotometer scan head uses up to five PMT detectors to detect five different tunable emission wavelengths, allows simultaneous imaging with an additional transmitted light detector, and can perform multiple types of FRET imaging experiments. Like the DM upright confocal, it can also perform spectral scanning of fluorescence for generation of dye emission spectra and for separation of dyes with overlapping emission spectra. But to further distinguish it from the DM, it also has a “resonance scan” mode that enables up to 25 512x512 frames per second imaging for time lapse experiments. The motorized stage contains adapters for different dishes and chamber slides to accommodate a wide range of experimental setups. It also can mark and find multiple points and automate montage acquisition and stitching to generate high resolution images of large areas.

The Leica SP5 DMI microscope with environmental control

Technical Specifications

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* available on request

User Protocol
Tiling Profocol

The newest Leica laser-scanning confocal microscope is also equipped with three depletion lasers to perform super-resolution microscopy. STED (stimulated emission depletion) microscopy allows subdifraction limited imaging achieving a resolution of 50nm in x and y. 3D super-resolution imaging: the 3D STED module allows to adjust the point spread function to improve on lateral or axial resolution to get the optimal resolution for each specific sample and scientific question.

This system is an inverted microscope. It has three excitation lasers: a UV Diode (405nm), an Argon laser (458nm, 476nm, 488nm, 514nm) and a White Light Laser (range from 470nm to 670nm). Its spectrophotometer scan head uses up to 2 PMT and 3 HyD detectors to detect five different tunable emission wavelengths which allows simultaneous imaging. An additional PMT allows detection of transmitted light. The depletion lasers (592nm, 660nm and 775nm) can perform STED imaging of up to three different dyes.

Like the Leica SP5 confocal (DM and DMI), it can also perform spectral scanning of fluorescence for generation of dye emission spectra and for separation of dyes with overlapping emission spectra. The motorized stage contains adapters for different dishes and chamber slides to accommodate a wide range of experimental setups. It also can mark and find multiple points and automate montage acquisition and stitching to generate high resolution images of large areas.

Technical Specifications

Objectives

* available on request

** suitable for STED imaging

This state of the art laser scanning confocal microscope is equipped with Airyscan. Airyscanning dramatically increases signal by using an array detector to capture light that is otherwise rejected by the confocal pinhole. The extra light can be used to increase signal to noise (capturing at the same speed) or resolution (Capturing at a reduced speed). Airyscan is a super-resolution strategy: 1.7x the resolution of a standard confocal microscope in the xy plane (~140nm) and about 30% improvement in the z-axis: ~400nm. Airyscan can be used with all of the fluorophores that can be imaged with this confocal.

Zeiss LSM880 with Airyscan and environmental control

The LSM880 system sits on an inverted AxioObserver microscope stand and is enclosed by a C02, 02, humidity and temperature-controlled chamber for imaging living samples. It has seven laser lines (see below) and is equipped with two fluorescence PMTs, one PMT for transmitted light, and a highly sensitive GaAsP detector that can capture 32 channels simultaneously. Up to 13 images can be captured per second at 512 X 512 pixels.  Linear scanning makes this microscope suitable for a variety of quantitative image measurements such as fluorescence correlative spectroscopy. The stage is motorized permitting multidimensional time lapse imaging at several sites and tile scanning to generate high resolution images of large areas. For live imaging, stage inserts can accommodate 35mm, 60mm and Labtek/IBIDI chamber slides or multiwell glass bottom plates. Hardware is controlled by Zeiss Zen Black software, which also incorporates some image processing options.

Technical Specifications

Objectives

* available upon request

User Protocol
Airyscan Protocol

The LSM780 system sits on an upright Axio Examiner microscope stand with a fixed stage. It has seven laser lines (see below) and is equipped with two fluorescence PMTs, one PMT for transmitted light, and a highly sensitive GaAsP detector that can capture 32 channels simultaneously. Up to 13 images can be captured per second at 512 X 512 pixels.  Linear scanning makes this microscope suitable for a variety of quantitative image measurements such as fluorescence correlative spectroscopy. Hardware is controlled by Zeiss Zen Black software, which also incorporates some image processing options. Microscope is enclosed by a C02, 02, humidity and temperature-controlled chamber for imaging living samples.

Zeiss LSM780 with a darkened environmental control box

Technical Specifications

Objectives

* available on request

User Protocol

This is a custom system comprised of a Zeiss Axio Observer Z1 inverted microscope, Yokogawa CSU-X1 spinning disk, and controlled by MetaMorph image acquisition software.  Its Prairie Technologies Aurora solid state diode laser launch has five laser lines: 405nm, 488nm, 515nm, 561nm, 640nm.  This microscope is ideal for long term, high speed, time lapse imaging.  It has multiple features to enable a wide range of live confocal imaging experiments, including:  a Tokai Hit enclosed stagetop incubator system with controlled temperature, CO2, and humidity, as well as lens heater; adapters for cell chambers, dishes, and slides; an encoded motorized stage that can mark multiple points within a sample or between different wells; a Piezo focus device for nanopositioning during high speed, high resolution z-stack imaging; and an on-site tissue culture facility to maintain cells prior to imaging.  For additional speed, the dual Hamamatsu EM CCD digital cameras enable simultaneous imaging of two fluorescent channels at a time.

Yokogawa spinning disk confocal microscope

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