Cellvizio® LAB Fibered Fluorescence Microscope and Microdialysis Research

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Microdialysis is an integral part of preclinical research to determine extracellular fluid and blood concentrations of metabolites, hormones, drugs, etc, and is often used in quantifying the biochemistry of brain and peripheral tissues. However, it is a molecular-only technique and other imaging modalities are needed to provide the researcher with functional and anatomical information of the animal in vivo.

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Cellvizio® LAB Fibered Fluorescence Microscope and Microdialysis Research

  1. 1. Application Brief: Cellvizio® LAB Fibered Fluorescence Microscope and Microdialysis ResearchExecutive SummaryMicrodialysis is an integral part of preclinical research to determine extracellular fluid and bloodconcentrations of metabolites, hormones, drugs, etc, and is often used in quantifying thebiochemistry of brain and peripheral tissues. However, it is a molecular-only technique and otherimaging modalities are needed to provide the researcher with functional and anatomicalinformation of the animal in vivo. Because of the similarity of fibered fluorescence microscopy(FFM) to microdialysis in terms of probe size and insertion, it is the perfect complement as animaging modality. The Cellvizio LAB system has been demonstrated to be a highly accurate way ofvisualizing peripheral nerves, deep brain, individual nerve regeneration, calcium dynamics etc inreal-time, with minimal invasiveness, and longitudinally in the same animal. Furthermore, astereotaxic imaging platform setup can guide the insertion of microdialysis and FFM probes to theexact location of interest. The combination of these systems provide a powerful method of studyingbrain and nerve dynamics in real-time, in vivo and longitudinally. Calcium monitoring in the brain at the level of a Nerve root from the spinal cord in a Thy1-YFP mouse single neuron after surgery to expose the nervous tissueApplication Brief: Cellvizio LAB and Microdialysis Research ver1.0 1
  2. 2. Background on MicrodialysisMicrodialysis is an in vivo, minimally invasive procedure used in preclinical research to measure theconcentration of unbound markers in fluids of tissues and organs in animals. A thin dialysis tube isimplanted in to the tissue of interest, with the semi-permeable membrane of the tube in contactwith bodily fluid. The tube is perfused with a physiological liquid and molecular markers in thebodily fluid are exchanged across the membrane. This allows the perfusate to be chemicallyanalyzed and reflects the composition of the fluid in contact.1 Microdialysis allows sampling ofextracellular fluid and it simplifies chemical analysis by excluding large molecules from theperfusate.1 For this reason, it is extremely important in observing drug pharmacokinetics andpharmacodynamics. Some of the most commonly detect markers include:1,2  Glucose  Lactate  Pyruvate  Glycerol  Glutamate  Dopamine  Peroxynitrite  Hormones  Small molecule drugs, etc.Microdialysis can be performed in peripheral tissues such as muscle, lung, kidney, liver, etc.1 but ismost often used in neurobiology research.2,3 However, there are some problems associated withmicrodialysis, which can be ameliorated by use in combination with imaging modalities. In vivo neurons in the cortex of a Thy1 YFP CA1 layer of a Thy1-YFP mouse hippocampus mouseApplication Brief: Cellvizio LAB and Microdialysis Research ver1.0 2
  3. 3. Applicability of the Cellvizio LAB and Vevo® Imaging SystemOne significant drawback of microdialysis is that although it provides a plethora of molecularinformation, it does not detect any functional information at the tissue of interest. Furthermore, theinformation captured by microdialysis is always an estimate of the actual concentration of the fluidin question because equilibrium across the membrane is not complete, depending on themembrane pore size, area, rate of flow, diffusion speed, etc.2 Thus other imaging modalities shouldbe used to validate the data. This can be complemented by the Cellvizio LAB system, a high speedfluorescence microscope with miniaturized microprobes capable of high-resolution, real-timeendoscopic imaging of fluorescent tissues, cells and markers at a micron level. The system iscomposed of 300 µm probes attached to optical fibers, which are inserted into the tissue ofinterest, thus operational-wise is very similarly to a microdialysis device. Minimally invasivebecause of their diameter size, the microprobes have applications for surface, internal and deepimaging of any organ, including brain. Furthermore, image acquisition happens in real-time,making it a perfect complement to be used in conjunction with microdialysis.For example, Vincent et al. used the Cellvizio LAB system to image the peripheral nerves and deepbrain of a Thy-1 eYFP (enhanced yellow fluorescent protein) mouse.4 Specifically, axonal changesand nerve regeneration was studied longitudinally following crush injury of the saphenous nerve.4The non-invasive Cellvizio LAB system allowed the degeneration-regeneration sequence to berecorded daily for a total period of 15 days. The authors then used a 300 µm probe to image thestriatum and ventral tegmental area (VTA) of a mouse brain.4 Finally, the authors used bicucullineand electrical stimulation to stimulate the mouse brain, to visualize and quantify calcium changesover time.4 The authors concluded that fibered fluorescence microscopy (FFM) system proved to beminimally-invasive, direct, rapid and accurate in measurement of neuronal activity in vivo. Sincethe Cellvizio LAB provided real-time information, it was correlated in parallel to EEG and could alsobe complemented with microdialysis to provide the researchers with a more comprehensive dataset.The accuracy of results from microdialysis research is integrally linked to the location of the probeplacement. Microdialysis reflects the tissue biochemistry and metabolic dynamics only at the exactlocation where the probe is placed.2,5 It has been suggested that tip position after placement of theprobe be checked with micro-CT,2 but this exposes the animal to radiation which can compromiseresults (especially in tumor models) and is more of a confirmation of location rather than aguidance system to place the probe. On the other hand, the stereotaxic guidance system used inconjugation with the Cellvizio LAB imaging system makes use of accurate 3D coordinate systems toApplication Brief: Cellvizio LAB and Microdialysis Research ver1.0 3
  4. 4. automatically place the microscopy probe at specific brain locations. This was demonstrated byDavenne et al, who used the stereotaxic apparatus to achieve accurate positioning of theCellvizio LAB probe within the mouse brain.6 This allowed the authors to visualize neurons indifferent locations of the rostral migratory stream (RMS), and also inside the olfactory bulb (OB).Furthermore, the authors tracked migration of the GFP-labeled cell bodies over a period, withmigration speeds ranging from 40-80 μm/h.6 The authors concluded that the Cellvizio LAB systemwas uniquely suited for imaging dynamic processes in the brain, because of its real-time imageacquisition, and that it could be used in the brain for 6 hours or more with minimal bleaching andphototoxicity.6Overall, FFM fibred fluorescence microscopy provides neurobiologists with a powerful tool of real-time, in vivo, in situ, minimally invasive functional imaging of the brain.Neuromuscular junction of a Thy1-YFP mouse with skeletal Motor nerve terminals at the neuromuscular muscle fibers marked by topical application of Sytol3 junction of Thy1-YFP mouseApplication Brief: Cellvizio LAB and Microdialysis Research ver1.0 4
  5. 5. References 1. Peña A, Liu P, Hartmut D. Microdialysis in peripheral tissues. Adv Drug Deliv Rev 2000;45:189-216. 2. Smith M, Tisdall MM. Cerebral microdialysis: research technique or clinical tool. Br J Anaesth 2006;97(1):18-25. 3. Zielke HR, Zielke CL, Baab PJ. Direct measurement of oxidative metabolism in the living brain by microdialysis: a review. J Neurochem 2009;109(Suppl. 1):24-29. 4. Vincent P, Maskos U, Charvet I, Bourgeais L, Stoppini L, Leresche N, et al. Live imaging of neural structure and function by fibred fluorescence microscopy. EMBO Rep 2006;7(11):1154-61. 5. Engström M, Polito A, Reinstrup P, Romner B, Ryding E, Ungerstedt U, et al. Intracerebral microdialysis in severe brain trauma: the importance of catheter location. J Neurosurg 2005;102:460-9. 6. Davenne M, Custody C, Charneau P, Lledo PM. In vivo imaging of migrating neurons in the mammalian forebrain. Chem Senses 2005 Jan;30 Suppl 1:i115-6.Application Brief: Cellvizio LAB and Microdialysis Research ver1.0 5

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