Confocal microscopy is a fluorescence microscopy technique developed to improve resolution by removing of out of focus light, which allows thicker samples to be imaged. Historically, the only way to effectively image deeper samples was to use sectioning to divide sample into very thin slices. Not only is this difficult to do, but it also prevents the imaging of live specimens.
The technique was first developed in the 1950's by Marvin Minsky and has subsequently been built upon with developments in laser and computer technology to become a key technique in life sciences.
Confocal image of tubulin (green, microtubules) combined with SIM image of Cos7 cells stained with TOM20 (red, mitochondria marker), DNA (blue, DAPI), SiR (grey, actin) (scale bar: 10µm)
A confocal microscope typically uses mirrors to focus light on a specific focal plane within the sample. The laser light source emerges through an excitation or illumination pinhole, before being directed towards the sample. In doing so, a narrow beam is created and only the fluorophores close to the focal plane are excited. The reflected light is then fed back through a semi-transparent mirror, towards the detection system, which it protected by a pinhole aperture.
The pinhole aperture on the camera only allows a small central portion of the light to continue on its way to the detection system and this is accurately calibrated so only light from the required depth is captured. All other light, reflecting from different depths in the sample, is blocked out, resulting in a higher resolution image from a very specific depth. This is known as optical sectioning.
The Nanoimager uses a digital approach to confocal microscopy and by doing so, it becomes a much more versatile microscope. The digital approach also allows it to provide multimode SIM microscopy (mSIM) and a range of other super-resolution techniques including dSTORM, PALM, single-particle tracking and smFRET. It is this versatility that sets the Nanoimager apart from confocal-only devices and indeed, a single cell within a sample can be imaged by multiple different techniques. For example, if not all fluorescent labels are compatible with dSTORM, then a combination of confocal microscopy and dSTORM be used for multicolor imaging.
Not only is the Nanoimager more versatile, its unique design means it has unrivalled stability. It naturally dampens vibrations so no optical table is required and it doesn’t require a dark room. It also comes with whole-body heating up to 42℃ to support live-cell imaging.
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