High-Sensitivity Probes for Immunofluorescence

Immunofluorescence is a microscopy-based technique which permits the visualization of many components in any given tissue or cell type through combinations of antibodies bound to fluorophores. Consequently, the possible applications in research and patient care are numerous. For immunofluorescence evaluation, a variety of samples can be applied: It can be performed on cultured cells or cell suspensions, on specific targets in tissue samples or entire organisms.

A distinction needs to be highlighted between direct and indirect immunofluorescence. In direct immunofluorescence, the primary antibody is directly conjugated to a dye, allowing for easy handling and rapid analysis under the fluorescence microscope. In indirect immunofluorescence, a secondary dye-conjugated antibody, directed against the primary antibody, is used to analyze the structures of interest.

One drawback of fluorescence microscopy is that fluorochromes lose their capacity to emit light when irradiated for a long time due to photobleaching. Also, although the use of fluorescent reporter proteins enables analysis of living it might cause phototoxicity, particularly when a short wavelength is applied. Additionally, fluorescent dyes tend to generate reactive chemical species once illuminated, increasing the phototoxic effect. An additional issue is that only specific structures fluorescently labeled can be observed and consequently studied.

Most fluorescence microscopes in use are epifluorescence microscopes in which both observation and excitation come from above the sample and, consequently, the fluorescence signal is detected through the same objective used for excitation. In epifluorescence microscopy, secondary fluorescence emitted by the sample often occurs through the excited volume and compromises resolution of features that occur in the objective focal plane. The problem is related to thicker specimens (> 2 µm), which usually show a high degree of fluorescence emission causing the loss of the finest details. Through confocal microscopy an improvement is offered in both axial and lateral resolution, in addition it is possible to eliminate secondary fluorescence in areas removed from the focal plane from resulting images. In the confocal microscope, the illumination and detection optics are focused on the same diffraction-limited spot in the sample, which is the same spot imaged by the detector during the scan. To obtain the whole image, the spot must be moved along the specimen and data collected in a point by point manner. An important advantage deriving from the use of the confocal microscope, is the possibility to optically section the sample and, consequently, reconstruct it in a 3D way starting from high-resolution stacks of images.