Stimulated Emission Depletion (STED) microscopy is a super-resolution technique that allows structures smaller than the diffraction limit (approximately 200nm) to be visualized. The technique is very similar to confocal microscopy, except for the fact that a second laser shaped like a donut is aligned with the excitation beam. It is this second laser that allows super-resolution. The STED 3X is equipped with a pulsed white-light laser excitation source, so any fluorophore within the visible spectrum (470nm – 670nm) can be excited. Besides that the system consist of 3 high-power STED lasers: a 592nm continuous wave, a 660nm continuous wave and a pulsed 775nm laser. These 3 lasers allow the depletion of almost any fluorophore. A lateral resolution of 30nm using the pulsed 775 STED laser has been demonstrated. Moreover, the STED 3X also allows super-resolution in the axial direction using the 3D donut. Advantages of the STED 3X over other systems include: super-resolution in XYZ (30nm lateral, 120nm axial), spectral freedom and less photo toxicity using the pulsed excitation source, therefore better live-cell and in-vivo capabilities and high detection sensitivity using the Leica Hybrid Detector technology.
Confocal microscopy allows optical sectioning of the sample and therefore 3D reconstruction. The Nikon A1R+ HD is aimed at delivering fast results by using a HD resonant scanner, which allows recording up to 15 frames per second (1024×1024 pixels) and 30 fps in 512×512. Decreasing the number of lines in Y allows even faster recording of up to 420 fps (512×32 pixels). This is especially useful for imaging calcium fluxes or other highly dynamic cellular processes. The AR1+ HD is equipped with 7 laser lines, allowing excitation of fluorophores ranging from near UV up to far red. Detection can be performed using 2 GaAsP PMTs and 2 regular PMTs. The system is also equipped with a live cell incubator, allowing 37°C and 5% CO2.
2P Dual-photon microscopy allows the user to look deeper into tissue. This technique is especially useful in in-vivo experiments as infrared light is less absorbed by the tissue and therefore causes less photo toxicity. The penetration depth of the 2-photon microscope can be as much as 2mm and therefore allows structures to be visualized which could not be seen before. The system relies on a high-intensity laser (Coherent, Chameleon, 700nm – 1000nm adjustable) which aims to deliver 2 photons at the same time to the fluorophore. These two photons are then able to excite the fluorophore into the S1 state, just like in epifluorescence or confocal microscopy. The main advantages of the 2 photon microscope include: deep penetration depth, better in-vivo capabilities due to the less phototoxic wavelength being used and the fact that common fluorophores can be used.
3D Light-sheet microscope
Light sheet fluorescence microscopy (LSFM) is used to image large samples of up to a cubic centimeter. Typical samples include: whole brains, mouse embryos and zebra fish. The Light sheet setup is equipped with a white-light laser excitation source and 8 filtercubes, so that any common fluorophore ranging from 430nm to 660nm can be excited. Depending on the thickness of the light sheet beam, the maximum lateral resolution of the microscope is about 1 micrometer. Advantages of LSFM compared to other techniques such as confocal microscopy include: fast scanning (up to 100 times faster than point-scanning methods), therefore large 3D structures can be studied in a relatively short amount of time. Another advantage of LSFM is that there is also less photo bleaching compared to epifluorescence due to the fact that only fluorophores within the sheet are exited.
Neurotransmitter vesicles are critical for the communication between neurons in the brain, but these vesicles are too small to be individually studied by light microscopy. At the VU/VUmc EM facility we specialize in quantitative electron microscopy analysis of secretory vesicles: synaptic vesicles in neurons and dense core vesicles in neurons and endocrine cells. We have uncovered essential steps in their transport to the secretion site by using genetic models and semi-automated image analysis. We are now taking the next step by combining these tools with state of the art cryo-fixation and 3D electron tomography analysis.