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Confocal Microscope Laboratory

Capabilities

The Confocal Imaging Laboratory supports: 

  • Separation of up to four fluorophores in a single sample.
  • Fluorescence excitation of CFP, GFP, fluorescein, Fluo-3, YFP, rhodamine, and Cy5.
  • Ultraviolet laser excitation for blue-emitting fluorophores, and for photolysis of caged compunds.
  • Automated collection of optical sections, for three-dimensional reconstruction.
  • Electrophysiology interface to coordinat microscope operation with external events.
  • Lateral resolution of up to 250nm, axial resolution of up to 1 um.
  • Laser scanning Nomarski differential interference contrast transmission microscopy.

Location

The Facility for Confocal Microscopy is located in space provided by the Department of Physiology on the fourth floor of Howard Hall, 660 W. Redwood St, Room 433.

Hardware

Zeiss Laser Scanning Microscope 

Zeiss LSM410  

  • Axiovert 100 inverted microscope
  • Omnichrome Series 643 Model 50YB Krypton/Argon ion laser
    • 25mW of blue (488nm) excitation light
    • 25mW of green (568nm) excitation light
  • Coherent INNOVA Enterprise Model 652 Argon ion laser
    • 110mW of ultraviolet (351nm + 364nm) excitation light
  • Zeiss Helium/Neon internal laser with 6mW of red (633nm) excitation light  
  • Objectives: 63x/NA1.4 oil immersion plan-apochromat, 40x/NA1.2 water immersion C-apochromat, 25x/NA0.8 multi-immersion plan-neofluor, and 10x/NA0.45 plan-apochromat, affording resolutions of up to 250nm (lateral) and 1 um (axial).  
  • Nomarski differential interference contrast (DIC) optics and transmitted light detector  
  • Long working distance condensor (NA=0.55), oil immersion condensor (NA=1.4)  
  • Three Hammamatsu photomultiplier tube detectors
    • Two Model R6060 Peltier-cooled PMTs
    • One Model R4632 low-noise, green-sensitive PMT
  • Dichroic mirrors and filters for simultaneous or sequential observations of AMCA/Indo-1, fluorescein/fluo-3, rhodamine and Cy5 fluorophores, and for reflection confocal microscopy

Funding Sources

NSF Grant for the Confocal Facility

In April, 1994 the Division of Biological Instrumentation and Resources of the National Science Foundation provided $94,128 to establish the Facility for Confocal Microscopy. The grant was organized by R. Bloch, (Physiology), along with co-PIs A. Azad (Microbiology), B. Krueger (Physiology), G. Lewis (Microbiology), P. Luther (Physiology), D. Pumplin (Anatomy), J. Wade (Physiology), and P. Yarowski (Pharmacology). The University of Maryland School of Medicine and Graduate School contributed a total of $94,128 in matching funds.

NSF Supplement to the Original Grant

In July, 1995 the Division of Biological Instrumentation and Resources of the National Science Foundation approved a $20,000 supplement to the grant that established the Facility for Confocal Microscopy. With the additional $6,000 provided by the University of Maryland School of Medicine in matching funds, we were able to acquire a 40x/NA 1.2 plan-apochromatic water immersion objective for high-resolution studies of living cells; a high-transparency 63x/NA1.4 plan-apochromatic objective; a high-resolution oil immersion condensor and associated optics for laser scanning Nomarski microscopy; and an electronics interface to integrate electrophysiological signals with the imaging system.

NSF Grant for an Ultraviolet Laser

In January, 1996 the Division of Biological Instrumentation and Resources of the National Science Foundation made an award of $48,965 to add an ultraviolet laser and associated optics to the Facility for Confocal Microscopy. This grant was organized by Paul W. Luther (Dept. of Physiology, PI) along with co-PI's Joseph P.Y. Kao (Dept. Physiology), Mike G. Klein (Dept. Biochemistry), W. Jon Lederer (Dept. Physiology), William R. Randall (Dept. Pharmacology), and Martin F. Schneider (Dept. Biochemistry). In addition, 14 others made contributions as minor users. The University of Maryland School of Medicine and Graduate School contributed a total of $48,965 in matching funds towards this project.

The addition of this laser to our confocal microscope allows us to: 

  • Apply fluorescent probes that require ultraviolet excitation (such as ratiometric ion indicators, nuclear stains, and neuronal tracers).

  • Take advantage of an additional fluorescence channel to examine the distribution of four macromolecules in the same sample.

  • Examine biological specimens at a higher resolution than is possible with visible light.

  • Combine photolysis of caged compounds with confocal microscopy.

Research Equipment Enhancement Fund (REEF)

In March, 1998 the Facility for Confocal Microscopy received an award of $10,000 from the University of Maryland School of Medicine. The PI of the application was Paul W. Luther (Dept. Physiology), and the funds were used to acquire Peltier-cooled, low noise photomultiplier tube detectors for the LSM410.