Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements.
A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.
Pan
Xiaotao
X
NUS Graduate Program in Bioengineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore.
Foo
Willy
W
Lim
Wanrong
W
Fok
Marcus H Y
MH
Liu
Ping
P
Yu
Hanry
H
Maruyama
Ichiro
I
Wohland
Thorsten
T
D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007424:Intracellular Fluid / Q000378:metabolism*; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted / Q000295:instrumentation*