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Leica SP8 Confocal/Multiphoton Microscope


Description

This upright laser scanning confocal microscope has 2-Photon capability, which provides the ability to perform intravital imaging and second harmonic generation (SHG) imaging. Intravital imaging is further facilitated by the four non-descanned hybrid detectors, resonant scanner, and special 20x 1.0 NA and 25x 0.9 NA dipping lenses on this system.

Applications

  • Intravital multiphoton imaging (isoflurane anesthesia available)
  • Second harmonic generation (SHG) imaging, backward and forward
  • Third harmonic generation (THG) imaging
  • Image tiling with precision motorized Scientifica stage
  • Fluorescence recovery after photobleaching (FRAP)
  • Intensity Förster resonance energy transfer (FRET)
  • Routine confocal microscopy and spectral scans

Leica SP5 Multiphoton Confocal

SP5 Multiphoton microscope

Scheduling Core Instruments

Registered users can access the PVIC online scheduler to reserve and use the Penn Vet Imaging Core instruments.

If you are not already registered, please contact the core manager.

Application Examples

Second Harmonic Generation (SHG) imaging of collagen

cross section of ascending aorta

The cross section of ascending aorta shown here was imaged by SHG microscopy to show fibrillar collagen (blue).

This image was taken on the Penn Vet Imaging Core’s Leica SP5 2-Photon microscope with a 20x (1.0 NA) water immersion lens. A Coherent Chameleon Ti:Sapphire laser was used to illuminate the tissue section with infrared light.

Second harmonic signal from fibrillar collagen, which is half the wavelength of the illuminating light, was then collected into non-descanned detector NDD1.

Additional images of tissue autofluorescence were simultaneously acquired in wavelengths within the ranges of 495-560 and 560-619 (NDD2 and NDD3; pseudocolored green and red, respectively).  Autofluorescence was present in all three channels and combined to form a grayish color in the image overlay shown here, differentiating the SHG signal (present only in the blue channel) from autofluorescence.

Image courtesy of James Monslow and Ellen Puré, Dept. of Animal Biology, UPenn School of Veterinary Medicine.

Second Harmonic Generation (SHG) imaging of collagen

skin SHG Volk-lab smFor this image, SHG microscopy was used to visualize fibrillar collagen (green) in a section of skin at the epidermal and dermal junction, with a hair follicle visible in the middle of the field.

This image was taken on the Penn Vet Imaging Core’s Leica SP5 2-Photon microscope with a 20x (1.0 NA) water immersion lens.  Fibrillar collagen present in the section combined 900 nm wavelength photons from a Coherent Chameleon Ti:Sapphire laser into light of 450 nm wavelength, which was then collected into non-descanned detector NDD1.

An additional image of tissue autofluorescence was simultaneously acquired for wavelengths within the range 495-560 nm (NDD2; pseudocolored red).  In the overlay shown here, autofluorescence collected by both detectors combined to form an orange color, whereas SHG signal was only detected by NDD1 and therefore appears as green in the image overlay. 

Image courtesy of Ashley Case, Becky Brisson, and Susan Volk, Dept. of Clinical Studies, UPenn School of Veterinary Medicine.

Confocal Microscopy and 3D reconstruction of Z-stacks

This movie shows a 3D reconstruction of scanning laser confocal images of a hydrogel (red) with silica particles (green).

The images were acquired on the Penn Vet Imaging Core’s Leica SP5 upright confocal/multiphoton microscope with a 20x (1.0 NA) water immersion lens. The 488 nm Argon laser and 543 nm HeNe laser were used to illuminate the sample and the fluorescence emitted was collected with the internal spectral detectors.

The image Z-stack was rendered into the 3D image shown here with Volocity software from Perkin Elmer, accessed through the core’s Volocity license server.

Image courtesy of Gaoxiang Wu and Shu Yang, UPenn School of Engineering and Applied Science.