The Essentials of a Setup for Optical Studies

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The laboratory setup appropriate for fluorescence measurements requires the following components. Note that the user must define the types of intended experiments before making final decisions about a number of these.

Software

• A computer with imaging software used to control wavelength changer, shutter, camera, frame grabber, experiment sequencing and data storage.

hardwares

• Epi-illumination fluorescence microscope, either inverted or non-inverted.
  
• Objective lens; make sure this can transmit and collect enough light at near-ultraviolet wavelengths if dyes such as fura-2 are to be used.
  
• Excitation light source (not the bright-field source used for visualization): an arc lamp containing either xenon, mercury or a mixture.
  
• Excitation or emission optical filter and dichroic mirror. These are usually available in a filter cube specifically designed for the microscope and a particular dye. For ratiometric use of fura-2, for example, a commercial filter cube will contain the appropriate emission optical filter and dichroic mirror. The two excitation filters needed are installed in the wavelength changer described next.
  
• Wavelength changer and shutter. The former moves a chosen optical filter into the excitation or emission light path. The latter shutters the light to prevent deleterious effects of light, which can inactivate the dye (bleaching) and damage the preparation.
  
• Vibration isolation devices may be needed if trying to perform a simultaneous patch-clamp and imaging experiment, or if very high gain is needed in the photodetector. If a mechanical wavelength changer is used, it may be coupled to the microscope by a fiber optic light guide to reduce mechanical shock.
  
• Light detector, either a photometer or a camera; see below.
  
• If imaging is used, a video (analog) or digital camera is required, including a camera, an optional controller and a computer plug-in circuit board to acquire the camera data to host memory. If a photometer is used, a data acquisition interface is needed to digitize the photometer output.
  
• Data storage device; image data can take a lot of storage space because each full image occupies a quarter of a megabyte or more.
  
• A computer with imaging software used to control wavelength changer, shutter, camera, frame grabber, experiment sequencing and data storage.