Inverted and Upright Confocal Scanning Laser Microscopes at the Center for Biofilm Engineering, Montana State University-Bozeman
- Confocal Laser Scanning Microscope
Confocal microscopy is widely used for high-resolution imaging predominantly in combination with biological samples as this technique can control the depth of field, eliminate/reduce background signal, and enables the collection serial optical sections. These instruments are equipped with 405, 488, 561 and 633 nm laser excitation lines, tandem and resonant scanners, heated stages with environmental control chambers capable of controlling temperature, humidity and gaseous headspace.
The CSLM is capable of imaging biofilms on opaque surfaces, so a wide variety of materials can be used in the experimental flow cells. As biofilm formation proceeds in an experiment, representative areas of the colonized surface are scanned with the use of the automatic stage. Digital data is collected from sequential scans, and stored data can be viewed in the x, y, z coordinates to yield a 3-dimensional image of the biofilm architecture. Quantitative and qualitative information about biofilm architecture can be retrieved easily from examination of CSLM data, in both the x-y and x-z planes, and the existence or absence of structural features, such as microcolonies and water channels, can be determined.Applications include:
- Imaging (size, shape, morphology)
One is an inverted confocal microscope with 405, 488, 561 and 633 nm laser excitation lines. It is equipped with a tandem scanner, so it can be switched from standard scanning mode to operate in Resonant Scanner mode, which enables scanning at exceptionally high frequencies for fluorescent imaging. This faster scanning is necessary for most live cell imaging (note: "live cell imaging" doesn't generally refer to imaging bacterial cells, but rather mammalian cells and processes). This inverted SP5 also includes a heated stage with an environmental control chamber (i.e. it can be used to provide an enclosed CO2 atmosphere), and a motorized stage with Mark-and-Find and image tiling capabilities.
The second SP5 is an upright confocal microscope, also with 405, 488, 561 and 633 nm lasers, a motorized stage, Mark-and-Find, and tiling capabilities. This upright has a removable heated chamber that encloses the entire microscope, so that larger, incubated flow cell systems can be accommodated over long periods of time. This enables high-resolution time-lapse monitoring of biofilm development, treatment and detachment phenomena. Additionally, this microscope is equipped with Fluorescence Lifetime Imaging (FLIM) capability, which is also referred to as Single Molecule Detection.