One of the most important applications of Pokegama biosensors is measuring free zinc levels in cells, usually by fluorescence microscopy. Users may simply wish to map the concentrations of free zinc inside the cell(s) of interest, or look at the time rate of change of free zinc inside the cell or a particular organelle, perhaps due to transporter activity.  This Application Note describes how to do that.  

Introducing the Sensor into the Cell: 

For this Application Note we will focus on Pokegama’s excitation ratiometric zinc biosensors, which consist of two components, a fluorescent-labeled apocarbonic anhydrase variant, and a fluorescent sulfonamide (see  Pokegama Web Resources: "Why use biosensors to measure free metal ions?" and "Introduction to Zinc Sensing").  The fluorescent-labeled sensor protein can be introduced into the cell either by expressing it (having inserted the DNA for the fluorescent-protein –carbonic anhydrase fusion protein as a plasmid or other vector into the cell), or by using a TAT peptide to induce the cell to take up the sensor protein (see Products: "Fluorescent Biosensor Proteins").  The TAT peptide approach works with most cells, and is straightforward: the apo-sensor protein is introduced (for instance) as a 1 μM solution in zinc-free buffer to a cell monolayer in a dish; after 30 minutes incubation at 37 C., the protein solution is decanted (the uptake of the fluorescent protein sensor can be verified by fluorescence microscopy at excitation 530 nm, emission 600 nm.  For expression, the sensor DNA ligated into a suitable plasmid or vector such as a lentivirus is mixed with cells for two hours, then expression is induced by addition of IPTG or other suitable reagent; again, the success of the expression is judged by the appearance of the fluorescent protein label in the plate reader or microscope.  We note that with different cell types, conditions may need to be adjusted.  

Introduction of fluorescent sulfonamide into the cell

Calibration of Sensor in the Microscope or Plate Reader

It is essential to calibrate measurements of free zinc in cells (or wherever) using buffers of known free zinc concentration such as Pokegama MetalloBuffers.  The reason is that optical properties of filters, monochromators, detectors, and other components of the instruments differ, such that the actual measured fluorescence intensity ratio values  corresponding to particular free zinc concentrations also differ among microscopes, plate readers, and spectrofluorometers.   

Calibration inside the Cell 

Calibration within the cell (in situ calibration) is harder than external calibration, for a couple of reasons: the calibration buffers may be sufficiently unphysiological as to perturb the cell, the zinc concentrations may be toxic, and/or the ionophore may have deleterious effects  However, it may be desirable or essential to calibrate in situ if it is known or suspected that there is significant background fluorescence in the cell at the relevant wavelengths, or the pH is much different inside the cell or organelle of interest, or there is some effect of the cellular milieu on the sensor or its fluorescence.   The procedure is the same as measuring the free zinc inside the cell above, except that the cells are immersed in a metal ion buffer of known free zinc concentration (like our MetalloBuffer series), and the cells treated with the ionophore pyrithione (2-thiopyridine) at 5 uM for 30 minutes: the ionophore transfers zinc ion across the cell membrane such that the cytoplasmic free zinc concentration equilibrates with and matches the external free zinc concentration.  Pokegama has under development calibration buffers formulated in widely used basic salts media, and selected common growth media.  

For non-ratiometric sensors it is desirable to use the high affinity cell-penetrant zinc ligand TPEN to drastically lower the free zinc in the interior of the cell to measure the fluorescence of the unbound sensor.  

References

B. J. McCranor, H. Szmacinski, H-H. Zeng, A.K. Stoddard, T.K. Hurst,  C. A. Fierke,  J.R. Lakowicz,  and R. B. Thompson, "Fluorescence Lifetime Imaging of Physiological Free Cu(II) Levels in Live Cells with a Cu(II)-Selective Carbonic Anhydrase-Based Biosensor," Metallomics 6, 1034-1042 (2014); DOI: 10.1039/c3mt00305a; NIHMS 585083 PMID: 24671220

D. Wang, T. K.  Hurst, R. B. Thompson, and C. A. Fierke “Genetically Encoded Ratiometric Biosensors to Measure Intracellular Exchangeable Zinc in Escherichia coli” J. Biomed. Opt. 16(8) 087011/1-11 (2011) [DOI: 10.1117/1.3613926. PMID: 21895338 PMCID: PMC3166341

R. A. Bozym, A. K. Stoddard, C. A. Fierke, and R. B. Thompson, “Measuring picomolar exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor,” ACS Chemical Biology 1(2) 103 – 111 (2006)  PMID: 17163650.