Center for Advanced Light Microscopy (CALM)
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Point scanning confocal

SP5

Technique

The excitation beam path of a laser scanning confocal microscope focuses the excitation light to one diffratction limited spot in the specimen. Fluorescence light that is emitted from this spot is spatially filtered by a pinhole. In consequence, only light from a small 3D volume (voxel) that can be as small as 250nmx250nmx500nm (x,y,z) reaches the detector. By scanning this optical arrangement over the specimen, 2D optical sections can be aquired. Aquiring several 2D sections spaced in z allows to image 3D objects.

Use

Gold standard for imaging of optical sections, high-resolution 3D images and colocalisation studies. Living specimens often do not tolerate the high photon exposure. Reduced speed of image acquisition due to the point scanning principle.

Configuration

Manufacturer: Leica
Type: SP5

Illumination
Laser
Diode laser 405 nm kpl Coherent (25 mW)
Argon Laser (458 nm, 476 nm, 488 nm, 514 nm), 5mW
DPSS Laser kpl 561 nm MG (10 mW)
HeNe Laser 594 nm, Uniphase (2.5 mW)
HeNe Laser 633 nm, Uniphase (10 mW)

Heating chamber covering the microscope

Optics
HCX PL Fluotar 10x 0.3 dry
HCX PL APO Lambda Blue 20x 0.7 imm
HCX PL APO Lambda Blue 63x 1.4 oil
HCX PL APO 63x 1.3 glycerol 37°C
HCX APO LU-V-63x 0.9 water

Detectors
Photomultiplier tube
HyD detector