Methods and systems for identifying and isolating stem cells and for observing mitochondrial structure and distribution in living cells

Abstract

Methods and systems for a) identifying and isolating stem cells, b) assessing mitochondrial distribution and structure in living cells and c) performing fluorescence microscopy on living cells while the cells remain within a condition-controlled cell culture chamber.

1 . A fluorescence microscopy system for performing fluorescence microscopy of living cells, said system comprising:
 a fluorescence microscope;   a microscope stage; and   a cell culture chamber associated with the microscope stage such that living cells within the cell culture chamber may be positioned on the microscope stage and viewed by the microscope without requiring removal of the cells from the cell culture chamber.   
     
     
         2 . A system according to  claim 1  further comprising apparatus for maintaining at least one predetermined condition within the cell culture chamber. 
     
     
         3 . A system according to  claim 2  wherein said at least one condition is selected from the group consisting of: temperature, humidity, pH, osmolarity, nutrient levels, ion levels and ambient gas composition, partial pressure or concentration of CO 2 , partial pressure or concentration of O 2  and partial pressure or concentration of N 2 . 
     
     
         4 . A system according to  claim 2  further comprising a fluid circulator for circulating cell-containing culture medium over the microscope stage. 
     
     
         5 . A system according to  claim 2  further comprising a camera useable for obtaining photomicrographs through the fluorescence microscope. 
     
     
         6 . A system according to  claim 5  further comprising a timer that causes the camera to obtain photomicrographs at desired time points. 
     
     
         7 . A system according to  claim 5  further comprising a programmable controller for controlling at least the camera so as to obtain photomicrographs at desired time points. 
     
     
         8 . A method for observing structural and/or functional attributes of mitochondria in living cells, said method comprising the step of:
 (a) detecting intrinsic reduced pyridine nucleotides in the mitochondria.   
     
     
         9 . A method according to  claim 8  wherein Step (a) comprises causing mitochondrial pyridine nucleotides to autofluorescence and then detecting the autofluorescence emitted thereform. 
     
     
         10 . A method according to  claim 9  wherein Step (a) is carried out by autofluorescence microscopy. 
     
     
         11 . A method according to  claim 9  wherein the autofluorescence distribution is detected. 
     
     
         12 . A method according to  claim 9  wherein the intensity of autofluorescence is detected. 
     
     
         13 . A method of  claim 11  wherein perinuclear concentrations of autofluorescence are detected. 
     
     
         14 . A method according to claim  34  wherein a histogram of autofluorescence distribution is prepared. 
     
     
         15 . A method according to  claim 8  wherein Step (a) is performed by autofluorescence using exitation by light at a wavelength that does not damage or kill the cells. 
     
     
         16 . A method according to  claim 15  wherein the excitation wavelength is about 365 nm. 
     
     
         17 . A method according to  claim 15  wherein the exitation wavelength is outside of medium and short UV wavelengths that are capable of causing cellular damage. 
     
     
         18 . A method according to  claim 8  wherein Step (a) is performed by autofluorescence and wherein the method further comprises the step of:
 decreasing exposure of the cells to light at wavelengths that will cause photobleaching. 
 
     
     
         19 . A method according to  claim 18  wherein the step of decreasing exposure of the sample to light at wavelengths that will cause photobleaching comprises turning off an illumination source during fluorescence imaging. 
     
     
         20 . A method according to  claim 18  wherein the step of decreasing exposure of the sample to light at wavelengths that will cause photobleaching comprises using an illumination source that emits light at a wavelength that is not within the excitation or autofluorescence wavelength detection gates being employed. 
     
     
         21 . A method according to  claim 18  wherein the step of decreasing exposure of the sample to light at wavelengths that will cause photobleaching comprises shielding the sample from light during at least a portion of the procedure. 
     
     
         22 . A method according to  claim 18  wherein the cells are maintained in a cell culture chamber that maintains the living cells under controlled conditions during performance of the method. 
     
     
         23 . A method according to  claim 22  wherein the controlled conditions include at least one condition selected from the group of: temperature, humidity, pH, osmolarity, nutrient levels, ion levels and ambient gas composition. 
     
     
         24 . A method according to  claim 8  wherein the method is repeated at one or more time points to determine changes in the mitochondrial distribution and/or structure of the cells over said one or more time points. 
     
     
         25 . A method according to  claim 8  wherein the cells are subjected to a treatment and wherein the method is performed i) prior to the treatment and ii) after the treatment, to thereby determine a treatment-effect or a lack of treatment-effect on mitochondrial distribution and/or structure. 
     
     
         26 . A method according to  claim 25  wherein the treatment comprises exposing the cells to a drug, therapeutic agent or other test substance.