www.fluorescencemicroscopy.it

Images acquisition

In any microscopy technique - brightfield, DIC, fluorescence ..., optical media recording to be used must be carefully selected to offer the best possible picture quality for the application. This selection is often not trivial, since the researcher must consider a myriad of variables, most of which are mutually exclusive. Often, the use of a variable is positive for one reason and negative for another. For example, in most situations involving live cell microscopy, the primary task becomes the balance between the health of cells and image quality, taking into account factors such as cell viability, signal to noise ratio and resolution request or the speed of acquisition. The aims and objectives of the type of investigation should determine the order of priority of these factors.

The options for recording media include color CCD cameras to capture images in brightfield or for samples containing multiple fluorophores, infrared cameras often used in imaging mode DIC, cooled CCD cameras with a sensor to capture low level signals as fluorescent sensors for high resolution black and white for TIRF and other techniques and many others.

Shift and multiband filter set

When you make a photograph with multiple exposures or more digital images of samples labeled with different fluorochromes, using different filters in a standard microscope-cube, there are inevitable changes in registration between exposures.

These changes contribute to the following factors:

-) variability of the peaks in the emission filter and dichroic filter;
-) variations in thickness and alignment of the dichroic filter;
-) mechanical vibrations that occur when the filters are changed on cube.

Although for some applications these effects can be reduced to acceptable levels, many others require a design-cube filters more sophisticated and more complex optical devices. Table 1 lists a few methods available for applications involving the use of multiple investigations. These include multi-band filter set and various configurations using both multi-band filters that single-band filters

Methods for applications that use multiple investigations

All these methods are designed to eliminate registration errors mentioned above. Method 2 offers the ability to visually analyze up to three colors simultaneously. (Methods 3 to 6 also have this possibility if they are added to the filter set of multiband filters.)