Wide-field fluorescence microscopy is one of the most basic fluorescence imaging techniques. The whole specimen of interest is illuminated with the light source, resulting in that fluorescence signals from all focal planes are detected. The emitted fluorescence is visualized through eye pieces or captured by a camera. This technique enables the identification and visualization of cells, cell components and proteins. The wide-field fluorescence microscope in BIF is capable of multi-color fluorescence imaging with filters for DAPI, CFP, YFP, FITC/GFP, TRITC/RFP and Cy5. It also has a motorized XYZ-axis control for fluorescence image stitching and 3D imaging.
Equipment and Software:
Olympus BX53 microscope
Short Arc Mercury lamp for reflected/fluorescence excitation
CellSens Dimension
Sample Preparation:
It must be fluorescent and the intrinsic fluorescence of sample (autofluorescence) can be used.
Fluorescent stains
Immunofluorescence
Fluorescent proteins
Filter Cube of BX53:
| Filter Cube Position | Excitation (nm) | Emission (nm) |
| 1. BF (Bright Field) | Empty | Empty |
| 2. DAPI | 361-389 | LP435 |
| 3. GFP (FITC) | 450-470 | 500-550 |
| 4. TRITC (RFP | 535-557 | 574-607 |
| 5. Cy5 | 590-650 | 663-738 |
| 6. CFP | 426-446 | 460-500 |
| 7. YFP | 490-510 | 520-550 |