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Objects for simplified STED-SPIM

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Nanometer sized fluospheres are used to check the spatial resolution enhancenement provided by the simplfied STED-SPIM method. Microfabicated objects and slices of mouse brain are also good candidates.

The "objects" to test the simplfied STED-SPIM.

100µm thick stack of 1µm diameter fluospheres
3D constructed from a 25 µm thick stack of 0.5 µm diameter fluosphere
Z profile of 2 touching fluospheres
Z profile of a 0.5µm fluosphere with and without STED
3D of microfabicated spring
Stack of a microfabicated 40µm long spring
Three dimensional micro-objects such as springs are obtained using 3D laser lithography based on two-photon induced photochemistry. In this approach, a laser beam (Q-switched Nd :YAG microchip laser) is coupled with an inverted microscope. The laser is focused into the sample, a drop of resin consisting in an organic-inorganic hybrid material (Ormocomp) containing 0,6 % Wt. photoinitiator and 0,01 % Wt. coumarin 490 to allow imaging. This drop is deposited onto a regular glass slide. During the fabrication the sample is translated through the laser focus by a 3-axis piezoelectric stage. The fabrication contours are calculated by a dedicated software that also controls the fabrication parameters, i.e. laser power, exposure time and 3D positioning. After writing the 3D structure the unreacted resin matrix is removed by washing with acetone leaving us with the 3D objects attached on the glass surface and surrounded by air
100X100X100 µm3 Stack of a microfabicated gelatin 3D structure
Confocal microscopy of a Gelatin 3D microstructure.
These kind of structures are promizing as scaffold for 3D cellular culture and superresolved 3D imaging.
Microscopy performed on the IAB platform
3D from a 100 µm thick Stack of mouse cortex
Astrocytes are antiGFAP / Cy3 stained. Fluorescence is excited by the 532 nm beam alone. The stack dimensions are : 140X100X100 µm3 in XYZ
The sample is cleared and embedded in a pluronic gel.
Astrocytes in a mouse brain block
The 3D animation comes from a stack of 100, 1µm thick optical slices (600µm X 600µm) in a block of a mouse brain embedded in a pluronic gel. The astrocytes are anti-GFAP/CY3 immunostained.