High-Resolution High-Power Fluorescence In Vivo Imaging

Client Story:
One of our clients in North Western University (Chicago, IL, USA) wrote us: “I am looking for the ability to observe fluorescent cello behaviors in mouse brain with cranial window installations. Ideally, I can see eGFP, mKate2 [Far Red, EX:588nm, EM:633], and cerulean [EX:433nm, EM: 475nm] labeled stem cells in brain tumor xenografts under the cranial window cover glass. Blood vessels will be counterstained with far red fluorophores, so 4 color cubes distinguishing these colors would be ideal. Since we do have multiphoton confocal microscopy at the NU core, we do use it for some live animal imaging, but there is requirements on the fluorophores and only 20x waterline available. I would like the 8x zoom you have for greater detail and also larger field of views.

What software and camera resolution would be used for the image acquisition?

Later he added: …The FOV for light microscopy should be capable of this Nature Protocol article (Goldey et al., Nature Protocols 9, 2515–2538,2014).

Fig. 8b, c, g, h are the ideal FOV and resolution if we can achieve this, 75% of the images will be collected at this level of magnification/FOV/detail. I would love to collect images that are 4X more in magnification, but if the distance is an issue, then I can sacrifice. In order to keep the mouse head steady during anesthesia (to avoid position changes due to breathing/waking etc.), I have a stereotaxic frame to keep the mouse stable, shown here in the model and brand: Stoelting 51625.

The mouse skull will be the focus, and it is usually 1.5cm additional height from the top of the stereotaxic frame platform, which itself is 4.25” tall per product spec of 51625. So, the clearance between lens and the microscope bottom should be between 10-12cm.
Solution:
We prepared this package of BL240 which has LED fluorescent attachment with UV, Blue, Green and Far Red LEDs and its relevant filters, used 2x, 4x, 5x, 10x, 20x and 40x obj lenses with a camera and proper c-mount adapter which gives the 7mm x6mm FOV at 2x obj lenses while he can also ge high resolution images at micron level using 40x plan fluorescence lens. In addition we customized the microscope stand which allows a large sample height up to 100mm, a fine/coarse focus and a great XY stage to easily move the sample (a live mouse mounted on a stereotaxic).

In addition, the price of the package was way economic and the images were extremely  satisfactory. Just read in the customer reviews what he thought about this.