Alberto Diaspro

Image scanning microscopy (ISM) with a single photon avalanche diode (SPAD) array detector

If a scanning illumination spot is combined with a detector array, we acquire a 4 dimensional signal. Unlike confocal microscopy with a small pinhole, we detect all the light from the object, which is particularly important for fluorescence microscopy, when the signal is weak. The image signal is basically a cross-correlation, and is highly redundant. It has more than sufficient information to reconstruct an improved resolution image. A 2D image can be generated from the measured signal by pixel reassignment. The result is improved resolution and signal strength, the system being called image scanning microscopy. A variety of different signal processing techniques can be used to predict the reassignment and deconvolve the partial images. We use an innovative single-photon avalanche diode (SPAD) array detector of 25 detectors (arranged into a 5× 5 matrix). We can simultaneously acquire 25 partial images and process to calculate the final reconstruction online.

Imaging of Endocytosis in Paramecium by Confocal Microscopy

Endocytosis is the general term for internalization of fluid, solutes, macromolecules, plasma membrane components, and particles by the invagination of the plasma membrane and the formation of vesicles and vacuoles through membrane fusion. The means by which food material enters the body is to a great extent dependent on the size of the particles involved [1, 2]. One type of endocytosis is phagocytosis where large (>250 nm) particles are taken up by cells. In protozoa, phagocytosis is a feeding mechanism. Particles are brought into the cell in large endocytic vesicles called phagosomes (food vacuoles). The phagosomes fuse with lysosomes and digestion of the ingested particles occurs. In multicellular organisms, phagocytosis is a behavior seen only in certain specialized cells (for example, macrophages). The process is essentially the same whether it is phagocytosis of particles or other organisms or pinocytosis of molecules. Both endocytic processes are affected identically by inhibitors of aerobic metabolism and by low temperatures.