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Ray: Confocal and Micropatterning microscope

Ray, c2 and primo setup

Summary

Ray is a multimodal workhorse. Ray uses a Galvo scanner for line scanning and is equipped with two different detector units. One detector unit contains 3 high sensitivity PMTs, therefore this system is ideal for everyday confocal imaging experiments. The other detector is a spectral detector and contains 2 GaAsP PMTs with tunable emission filters, this enables spectral imaging and FRET imaging. The spectral detector is also ideal for ‘exotic’ and large Stokes shift fluorophores. Ray is suited for fixed and live cells. Complete automation in combination with perfect focus system make this an ideal system to acquire entire slides and 96 well plates in an unattended fashion. Besides imaging, you can also “micropattern” a custom bioengineered microenvironment using the PRIMO (Alvéole). You can create microstructure substrates and control the surface biochemistry on which you’ll cultivate your cells.

Image modalities

  • Laser scanning confocal microscopy
  • Laser scanning spectral imaging
  • Brightfield
  • Micropatterning

Applications

  • Immunofluorescence.Imaging entire slides is easy and straightforward using tiling and stitching functions in the NIS-Elements software. You can control the orientation of the scanner and thereby control the orientation of your sample within the image.
  • Live cell imaging. The stage top incubation chamber regulates temperature (30 – 40 ⁰C), CO2 and humidity.
  • Hypoxia conditions for live cell imaging. The stage top incubator controls O2 (0.1 – 18.0 %) and as well as CO2 (5.0 – 20 %).
  • FRAP on live cells. You bleach, activate or stimulate with the 405 nm, 488 nm, 561 nm or 640 nm laser using the stimulation function of the NIS-Elements software. Cells can be immediately analyzed after or even during the acquisition with the time measurement tool.
  • Spectral imaging. The spectral range is 400 – 720 nm with a bandwidth of 10 or 20 nm.
  • Filter FRET / ratiometric FRET. Filter FRET using blue, green, red and far red fluorescent dyes or fluorescent proteins can be performed.
  • Spectral FRET. You can excite with 405 nm, 488 nm, 561 nm or 640 nm laser and measure the spectrum from 400 – 720 nm with a bandwidth of 10 or 20 nm resolution.
  • Image ‘exotic’ and large Stokes shift fluorophores. The tunable emission width in combination with 405, 488, 561 or 640 nm lasers provide many acquisition options for a wide variety of fluorophores.
  • Substrate structuration. Create microstructured substrates with controlled topography and stiffness.
  • Surface functionalization. Control the biochemistry of the in vitro cell microenvironment on stiff or soft cell culture substrates. You can even combine substrate structuration with surface functionalization. 

Technical description

The Eclipse Ti2-E (Nikon) is equipped with automated stage (Nikon), perfect focus system (Nikon), stage top incubator (Tokai Hit) to control the temperature (30 – 40 ⁰C), CO2, N2, and humidity (including objective heater and temperature sensor). The following Nikon objectives are available for this system: Plan Apo lambda 10x NA 0.45 air, Plan Apo lambda 20x NA 0.75 air, S Fluor 40x NA 1.30 oil, Plan Apo lambda 60x NA 1.40 oil, and Plan Apo lambda 100x NA 1.45 oil. All imaging functions are integrated in the NIS-Elements software (High content analysis package).

For laser scanning confocal mode (C2, Nikon), the sample is excited with 405 nm, 488 nm, 561 nm, and 640 nm laser from the laser unit (LU-N4, Nikon). Galvano scanner can zoom and rotate the scan direction, maximum pixels 2048 x 2048. The excitation light passes an adjustable pinhole and is directed to the sample by a dichroic mirror 405/488/561/640. There are 3 high sensitivity PMT detectors (C2-DU3, Nikon), allowing to acquire 3 channels simultaneous, but 4 channels sequential. This detector unit contains fixed emission filters, 3 bandpass filters (445/35 nm, 525/50 nm and 600/50 nm) and a long pass filter (660 nm LP).

For laser scanning spectral imaging mode, the excitation and scanning is similar as for laser scanning confocal, but the detection differs. A stack of images is acquired each image is acquired at a different emission wavelength. The spectrum is acquired using a GaAsP PMT detector (C2-DUVB, Nikon). The spectral range can be set from 400 – 720 nm with maximal 32 channels each channel with a band width of 10 nm or 20 nm. This mode only allows maximal 1024 x 1024 pixels.

For laser scanning imaging with tunable emission filters, the excitation and scanning is similar as for laser scanning confocal, but the detection differs. There are 2 GaAsP PMT detectors (C2-DUVB, Nikon), which allow acquisition of two channels simultaneous or more than two sequential. This mode only allows maximal 1024 x 1024 pixels.

For micropatterning, the PRIMO (Alvéole) is connected to the microscope. This device includes a 360 nm laser for micropatterning. The objective S Plan Fluor ELWD 20x 0.45 NA (long working distance) is available. The PCO.edge 4.2 bi sCMOS camera (PCO) is mounted on the microscope for micropatterning. Micropatterning is performed with the Leonardo software (Alvéole) and virtually any pattern can be created.

Location

Leichtag building, Room 470