Assessment of the LSMTech InverterScope® and its application to in vivo 2-photon brain imaging

TY - CONF

T1 - Assessment of the LSMTech InverterScope® and its application to in vivo 2-photon brain imaging

AU - Marrison, Joanne

AU - Brackenbury, William John

AU - Chawla, Sangeeta

AU - Hunt, Mark Jeremy

AU - Whittington, Miles Adrian

AU - O'Toole, Peter J.

PY - 2016/5/24

Y1 - 2016/5/24

N2 - We use an inverted multiphoton microscope in a multi-disciplined Department of Biology for in vivo liver and spleen imaging. However, some sample types cannot be readily addressed using an inverted microscope and we now have a need to use the same microscope for in vivo brain imaging.To address this problem we recently purchased an LSM Tech InverterScope® objective inverter to convert our inverted system to an upright configuration. After testing we found this was not optimized for 2-photon imaging of green fluorescent protein (GFP). LSM Tech was very proactive and further customized the InverterScope® to maximize both the excitation and emission transmission. After testing the customized InverterScope® we found the excitation power exiting the objective for five wavelengths (405, 488, 561, 633 and 920nm) was minimally affected and there was also no impact on evenness of illumination across the field. Using a standard fluorescent sample there was only a 15% loss in emission at the internal detectors after 488nm excitation and a 7% loss in emission at the non-descanned detectors after 920nm excitation compared to without the InverterScope®. On the basis of these results we used the customised InverterScope® for in vivo imaging of microglia through a cranial window of a sacrificed CX3CR1-GFP mouse. We successfully imaged to depths of 400µm using laser powers between approximately 3mW-16mW (as measured at the objective) dependent on imaging depth. This was comparable to settings used without the InverterScope® but easier and quicker to set up, meaning contact with the animal was minimized, an important consideration for imaging an anaesthetised animal.In conclusion our data shows the customised LSM Tech InverterScope® to be an effective alternative to purchasing a new system, at least in the preliminary proof of concept phases of this type of work and has enabled us to progress work that would classically have required an upright/fixed stage multiphoton system. For a multiuser facility, this approach is proving highly effective.

AB - We use an inverted multiphoton microscope in a multi-disciplined Department of Biology for in vivo liver and spleen imaging. However, some sample types cannot be readily addressed using an inverted microscope and we now have a need to use the same microscope for in vivo brain imaging.To address this problem we recently purchased an LSM Tech InverterScope® objective inverter to convert our inverted system to an upright configuration. After testing we found this was not optimized for 2-photon imaging of green fluorescent protein (GFP). LSM Tech was very proactive and further customized the InverterScope® to maximize both the excitation and emission transmission. After testing the customized InverterScope® we found the excitation power exiting the objective for five wavelengths (405, 488, 561, 633 and 920nm) was minimally affected and there was also no impact on evenness of illumination across the field. Using a standard fluorescent sample there was only a 15% loss in emission at the internal detectors after 488nm excitation and a 7% loss in emission at the non-descanned detectors after 920nm excitation compared to without the InverterScope®. On the basis of these results we used the customised InverterScope® for in vivo imaging of microglia through a cranial window of a sacrificed CX3CR1-GFP mouse. We successfully imaged to depths of 400µm using laser powers between approximately 3mW-16mW (as measured at the objective) dependent on imaging depth. This was comparable to settings used without the InverterScope® but easier and quicker to set up, meaning contact with the animal was minimized, an important consideration for imaging an anaesthetised animal.In conclusion our data shows the customised LSM Tech InverterScope® to be an effective alternative to purchasing a new system, at least in the preliminary proof of concept phases of this type of work and has enabled us to progress work that would classically have required an upright/fixed stage multiphoton system. For a multiuser facility, this approach is proving highly effective.

M3 - Abstract

T2 - 16th ELMI Congress

Y2 - 24 May 2016 through 27 May 2016

ER -