Center for Advanced Light Microscopy (CALM)
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Wide field

pDV

Technique

In wide field fluorescence microscopy, the entire field of view is illuminated with excitation light that is reflected by a beam splitter into the microscope beam path. The objective of the microscope works as a condensor in this layout. Image information (emitted light) is sampled by the microscope objective in a classical way. The emitted light has to pass the beam splitter that was used to reflect the excitation light into the beam path. Therefore, this beam splitter has to fit the properties of the investigated fluorophore. Light that can excite the fluorophore has to be reflected, whereas the emitted light has to pass this optical element. Finally, a fluorescent image is acquired by a camera. The technique is diffraction limited and provides no optical sectioning.

Use

First choice for a new sample or stain. Relatively simple technique that can acquire images with high speed. Data can be interpreted quantitatively. Depending on the light source and available beam splitters, all fluorophores for fluorescence microscopy can be used. Moderate bleaching and phototoxicity.

Configuration

Manufacturer: General Electric (GE)

Illumination
Insight SSI (LED)

Optics
60x1.42 oil-immersion objective PlanApo U (Olympus)
10x0.4 UPlanSApo (Olympus)  temporary mounted in the STED microscope
4x0.13 UPlanFLN (Olympus)

Camera
Photometrics Cool-Snap camera, 12 bit, 1024x1024 pixel

Filter
Polychroic Beam Splitter (suitable for DAPI, FITC, RD-TR-PE, Cy5)
Filter EM457/50
Filter EM528/38
Filter EM617/73
Filter EM685/40
Filter EM360/40
Filter EM490/20

Filter EM555/28
Filter EM640/20
Polychroic Beam Splitter (suitable for DAPI, FITC, Alexa594, Cy5, GFP/mCherry)
Polychroic Beam Splitter (suitable for CFP/YFP/mCherry)
DAPI 435/48
CFP 475/24 GFP/FITC 525/48
YFP 548/22
TRITC 597/45
mCherry/Alexa594 625/45
Cy5 679/34 e imaging with the OMX.