Core Facilities
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Confocal Microscopy

Instruments

 Nikon-A1

Nikon A1 and Nikon A1-RS Confocal Microscopes

At the heart of the Nikon A1 hybrid confocal scan head is the dual scanning system that encompass a standard-speed scanner and a high-speed resonant scanner. They may be used individually or simultaneously. Applications include:

For the standard speed scanner: imaging of all fixed specimens and many live specimens, including FRET and ion ratio imaging (e.g.: calcium, pH);

High speed resonant scanner: Maximum frame rate can exceed 400 frames per second.

Simultaneous use of standard and resonant scanners: Useful for FRAP and photoactivation specimens that require stimulating with one scanner while simultaneously imaging with the other.

Three Detector Ports are Standard

  1. Standard Detector. The standard filter-based detector allows simultaneous or sequential detection of up to four channels of fluorescence.
  2. Spectral Detector. With the 32-channel spectral detector, up to eight individual overlapping fluorophores can be unmixed simultaneously and acquired at a maximum frame rate of 16 frames per second.
  3. A transmitted light detector allows simultaneous capture of a DIC image while capturing confocal fluorescence images.

Laser Platform:

405nm diode laser, 561nm diode-pumped-solid-state (DPSS) laser, and a 638nm diode laser.

An argon-ion gas laser is the fourth laser. It produces four lines: 457nm, 477nm, 488nm, and 514nm line.

Nikon Perfect Focus System (PFS) is the solution to keep cells in focus over prolonged time-lapse imaging sessions. Pick the focal plane and PFS monitors the focus every 5ms to maintain focus indefinitely.

Accessories: Motorized XY Stage and Piezo Z stage for rapid sectioning.
Stage top incubator for temperature, humidity, and CO2 atmospheric control.

 leica sp5

Leica sp5

The Leica SP5 Confocal has a filter-free spectral detector system with five individually tunable channels. The spectral separation is accomplished by a unique prism design. 

Each detector is freely tunable over the entire visible spectrum.  The SP5 has a prism base beam splitter (AOBS) that allows for free selection of up to 8 Lasers from UV – IR.  The spectral range of the detector optics covers 400 - 800nm.

The scanner can be run in either conventional or resonant modes. The conventional scanner can be scanned unidirectional or bi-directional with a speeds ranging from 10Hz to 2800Hz.  The resonant scan can also be scanned unidirectional or bi-directional at 8000Hz or 1600Hz respectively with a maximum frame rate of 250.  

The system is equipped with a Z-galvanometer that allow for XZ scanning as well as fast XYZ scanning.  The scanning stage allows for stitching together images from large fields of view as well as imaging multiple positions as XYZT images.

The laser lines available on the SP5 are 405, 458, 476, 488, 514, 561, 594 and 633.  Of the 5 detectors on the system 3 are conventional PMTs, 2 are Hybrid detectors (HyD).  Leica HyD detectors provide highly sensitive photon detection.

There is a Warner chamber for temperature control of live experiments.

perkinelmer-spinning-disk-1_crop

Multi Modal Nikon TIRF/Spinning Disc Confocal Microscope

This microscope has Total Internal Reflectance Fluorescence (TIRF), spinning disc, epi/BF, and ablation capabilities.

The microscope base is a fully motorized Nikon Ti-E with Perfect Focus System (interferometric based focus maintenance (PFS)).  The system also has a piezo stage with 100 um range capable of up to 100 steps/sec, mounted in a fully motorized XY stage.

An Agilent high power MLC400 provides;

150 mW         647 nm

70 mW           561 nm

30 mW           488 nm

25 mW           405 mW

A Yokogawa spinning disc (CSU-22) is mounted with a triple dichroic accommodating 488, 561, and 647 lasers.  The spinning disc detector is a Hamamatsu ImageM back thinned EMCCD (512, 93% QE, 30 full fps).

A TIRF illuminator is mounted accommodating all laser wavelengths, with mag optic and ¼ wave for super resolution localization applications.

A 100 mW 285 MicroPoint ablation module is mounted., capable of producing 365 nm ablation, 405 nm for PA/PC, and 435 nm for FRAP experiments.

The detector for TIRF/epi/BF/SR applications is an Andor iXon Ultra back thinned EMCCD (512, 93% QE, 60 full fps).

Temperature and CO2 control is available for this system.

prairie-mp-2_crop 

Prairie ultima multi-photon system

2 channel imaging using 2 GaAsp detectors per channel. A Coherent Chameleon Ultima II pulse laser, tunable from 680 to 1080 nms.
Dual scanning system - galvo scanners and an
AOD scanner with scan speeds up to 25 fps at 512 x 512 resolution
Fast Prior piezo z-stage with 500 microns of travel
405 laser for ablation, photo-activation and photo-conversion
Environmental chamber for live cell mammalian imaging. Temp. Range 25 to 45 degrees C.\
 
Objective lenses: 1.0 NA20 x and 40 x Plan Apo's, 1.3 NA 40 x Plan Apo
0.2 NA 5X Plan fluor and 10 x 0.6 NA Plan Apo

   

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Contact Us

Lewis Thomas Laboratory at Princeton University

119 Lewis Thomas Laboratory
Washington Road, Princeton, NJ  08544-1014

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Fax: (609) 258-3980
Website:  molbio.princeton.edu