Scenario 4:
You are trying to image some biological event that occurs upon ~10ms time scale in a population of cells or tissue sample. This could involve measuring changes in membrane potential or calcium concentration, or rapid changes in some other biochemical event. The sample is of course live, and cellular (~5μm) spatial resolution is required. The probes are generally reasonable bright but not sufficient that photobleaching and phototoxicity is not an issue. Issues to consider include acquisition time and the balance of signal collection efficiency and photo damage.
The scenario describes commonly encountered problems in applying fluorescence microscopy to study biological samples. I would like you to discuss ways of overcoming the problems at hand.
-What is the cause of the fundamental limitation(s) present in the problem when using a conventional fluorescence microscope system (wide field microscope or regular confocal microscope)? What are the tradeoffs (e.g. photobleaching, acquisition speed, spatial resolution, signal, noise and background, or other factors) that provide practical limitations?
-What type of optical imaging approach, technology or strategy could overcome these problems?
-What light source, optical detector, fluorophore and other key instrumentation/reagents will be needed and why? (you may want to provide specs for specific products where relevant).
-How will this strategy work and how will it generate new information about the object/process being studied: e.g. will fluorescence intensity be measured, or fluorescence lifetime, or scattering, some other property?
-What other limitations could there be in the approach planned? Can these also be minimized and how?
Scenario 4:
You are trying to image some biological event that occurs upon ~10ms time scale in a population of cells or tissue sample. This could involve measuring changes in membrane potential or calcium concentration, or rapid changes in some other biochemical event. The sample is of course live, and cellular (~5μm) spatial resolution is required. The probes are generally reasonable bright but not sufficient that photobleaching and phototoxicity is not an issue. Issues to consider include acquisition time and the balance of signal collection efficiency and photo damage.
The scenario describes commonly encountered problems in applying fluorescence microscopy to study biological samples. I would like you to discuss ways of overcoming the problems at hand.
-What is the cause of the fundamental limitation(s) present in the problem when using a conventional fluorescence microscope system (wide field microscope or regular confocal microscope)? What are the tradeoffs (e.g. photobleaching, acquisition speed, spatial resolution, signal, noise and background, or other factors) that provide practical limitations?
-What type of optical imaging approach, technology or strategy could overcome these problems?
-What light source, optical detector, fluorophore and other key instrumentation/reagents will be needed and why? (you may want to provide specs for specific products where relevant).
-How will this strategy work and how will it generate new information about the object/process being studied: e.g. will fluorescence intensity be measured, or fluorescence lifetime, or scattering, some other property?
-What other limitations could there be in the approach planned? Can these also be minimized and how?
