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Confocal Laser Scanning Microscope Leica SP8 (1)

Confocal laser scanning microscopy enables optical sectioning of multilayer fluorescent specimens with high contrast by applying a pinhole in the optics. It is therefore possible to image a thin optical slice out of a thick specimen (up to 100 µm) that represents under optimal conditions a slice of approximately 500 nm. Moreover, conventional contrast methods such as the differential interference contrast (DIC) can be used and in reflection mode particles or surfaces can be analyzed.

The CLSM SP8 (1) based on a DMI6000CSB allows precise detection of standard fluorophores in your sample. The system excites via diode laser at 405 nm, Argonlaser combined with AOBS at 458, 488, 514 nm, diode laser at 561 nm and a He/Ne laser at 633 nm. The SP8 (1) has two standard GaAsp photomultipliers (PMTs) and two extremely sensitive Hybrid detectors (HyD), combining PMT and avalanche photodiode (APD) technology. An optional incubation chamber allows imaging of living cells.

The system is almost fully automated and offers with its broad excitation spectra, its automated system parts, the galvo stage and the optional incubation chamber a variety of possible uses.

Immersion objectives are optimized for oil- / glycerol immersion.

Detailed specifications CLSM Leica SP8 (1)

Microscope:

  • Leica DMI6000CSB stand (inverted)
  • Motorized scanning stage
  • Heated incubation chamber with CO supply from LIFE IMAGING SERVICES

Lasers:

  • 405 nm Diode laser
  • Argon/2 (458, 488, 514nm)
  • 561 nm Diode laser
  • HeNe 633nm

Conventional fluorescence filters for eyepiece vizualization:

  • Blue (A = xBP340-380, dichroic 400, LP425)
  • Green (I3 = xBP450-490, dichroic 510, mLP515)
  • Red (N2.1 = xBP515-560, dichroic 580, mLP590)

Condenser:

  • Smart condenser S28/0.55 w/o flip

Objectives:

  • 5x/0.15 HCX PL FLUOTAR WD 13.7 mm
  • 20x/0.75 HC PL APO Imm Corr (oil, water, glycerol) WD 0.68 mm
  • 63x/1.4 HC PL APO CS2 OIL WD 0.14 mm
  • 63x/1.3 HC PL APO CS2 Glyc WD 0.30 mm

Dichroic(s):

  • AOBS

Detectors:

  • All spectral detectors: four channels - two PMTs, two HyDs - high sensitivity Hybrid Detectors - HyD1 | PMT2 | HyD3 | PMT4
  • Motorized transmitted light detector

Computer:

HP Z420 Workstation, Windows 7-64 bit, LAS X

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Confocal Laser Scanning Microscope Leica SP8 (2)

Confocal laser scanning microscopy enables optical sectioning of multilayer fluorescent specimens with high contrast by applying a pinhole in the optics. It is therefore possible to image a thin optical slice out of a thick specimen (up to 100 µm) that represents under optimal conditions a slice of app. 500 nm.

The CLSM SP8 (2) based on a DMi8 CEL Compact allows precise detection of fluorophores in your samples. The system excites via diode lasers at 405, 488, 552, or 638 nm. The SP8(2) has two standard GaAsp photomultipliers (PMTs) and one extremely sensitive Hybrid detector (HyD), combining PMT and avalanche photodiode (APD) technology.

The system is optimized for confocal microscopy with standard dyes (DAPI, Alexa488, Alexa555/Cy3, Alexa648/Cy5, etc.) and gives, in combination with its strong and stable diode lasers, convincing results with the given limitations in excitation. The microscope is comparably easy to use as it is pretty stripped down to the components necessary for confocal microscopy in combination with the newest generation of Leica microscopes with a user-friendly touchscreen. All immersion objectives are optimized for oil immersion. „Conventional“ light-microscopy with the Trans-PMT or oculars, respectively, is rather sophisticated as, unlike for the confocal detection, objectives, aperture and filters have to be set manually. The system has no incubation chamber.

Detailed specifications CLSM Leica SP8 (2)

Microscope:

  • DMi8 CEL Compact stand (inverted)
  • Semi-Motorized scanning stage (manual xy)

Lasers:

  • 405 nm Diode Laser
  • 488 nm Diode Laser
  • 552 nm Diode Laser
  • 638 nm Diode Laser

Conventional fluorescence filters for eyepiece visualization:

  • Blue DAPI LP Excitation: BP 360/40 Emission: LP 425
  • Green FITC LP Excitation: BP 470/40 Emission: LP 515
  • Red RHOD LP Excitation: BP 540/45 Emission: LP 590

Condenser:

  • S1/S28; Head S28/0.55

Objectives:

  • 10x/0.30 HC PL FLUOTAR WD 11 mm
  • 40x/1.30 HC PL APO Oil CS2 WD 0.24mm
  • 63x/1.40 HC PL APO Oil CS2 WD 0.14mm
  • 20x/0.75 HC PL APO Imm Corr (oil, water, glycerol) CS2 WD 0.68 mm

Dichroic(s):

  • SP8 LIAchroics Compact RGB

Detectors:

  • Three channels - two PMTs, one HyD - high sensitivity Hybrid Detectors - | PMT1 | PMT2 | HyD1
  • All spectral detectors (AOTF)
  • Motorized transmitted light detector

Computer:

  • HP Z420 Workstation, Windows 7-64 bit, LAS X with LAS X Dye Finder and LAS X 3D Visualisation
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TIRF microscope Zeiss Axiovert 200M

Total internal reflection fluorescence microscopy (TIRF microscopy) is one example for surface-sensitive microscopy and is therefore only applied to samples that are in contact with a solid, transparent interface (e.g., the mounting glass slide). In TIRF microscopy, the excitation light is (in contrast to conventional fluorescence microscopy) guided to the interface at the angle of total internal reflection. Hence, the excitation light is almost completely reflected, but creates an evanescent (light-)field at the interface, which excites only those parts of the sample that are in close vicinity to the interface (typically the closest 100 to 150 nm). This spatial confinement decreases the noise background (and thus improves the signal-to-noise ratio), but also increases the optical resolution along the optical axis (with respect to usual wide-field or confocal microscopy solutions). Typical applications include imaging of cell structures that are closely located to the cell membrane (e.g., the cytoskeleton) or the transient attachment of biological nanoparticles (such as viruses/virions, vesicles, exosomes) to cell membranes.

The Zeiss Axiovert 200M is an inverted fluorescence microscope equipped with a VisiTron laser-based TIRF extension (excitation at 488 and 561 nm) and a CoolSNAP CCD camera. It provides conventional fluorescence microscopy, FRET and TIRF.

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