Ratio Imaging of Cells with RatioWorks™ Probes

Intracelluar Fluorescence Ratiometric Imaging

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Many fundamental functions of a cell strongly depend on delicate, but nevertheless dynamic balances of ions (e.g. calcium, magnesium), voltage potentials and pH between the cell's cytosol and the surrounding extracellular space. Changes in these balances significantly alter a cell's behavior and function. Therefore, measurements of intracellular ion, voltage and pH dynamics in real time are of tremendous interest for researchers in neuroscience, cell biology and cell physiology in general. In many cases, however, exact estimations of actual ion concentrations or relative changes in different locations in a cell or a cellular network are difficult with conventional fluorescence methods. The reason is that these methods do not take account of the fact that differences in cell morphology within different parts of a single cell or between cell types in cellular networks might influence the quality and quantity of emitted light. This can lead to substantial misinterpretations when dynamic changes of ion concentrations, voltage or pH are investigated. Ratiometric imaging techniques bypasses these issues by observing emission or excitation wavelength shifts of fluorophores or by comparing the emission or excitation intensity of a fluorophore combination instead of measuring mere intensity changes.

Research activities are increasingly focusing on the identification and the spatial and temporal distribution of e.g. local "hot spots" for dynamic changes in ion concentration, voltage or pH in a cell or a cellular network. Such "hot spots" are often localized in specialized parts of a cell or in certain cells in a cellular network. Additionally, these areas often have different properties compared to the rest of the specimen in terms of cell metabolism or structure. Conventional fluorophores used to investigate dynamic physiological states change their emission intensity upon ion binding, pH change or voltage change (e.g. fluo-4 has increased emission upon calcium binding). However, these markers do not take into account that differences in structure, diameter or marker uptake/expression can cause changes in the quantity of emitted light that are not in correlation with the actual ion concentration, voltage or pH. To quantitatively and comparably detect the changes in cellular structures or different cells, a method insensitive to structure diameter and fluorophore concentration is needed. Ratiometric imaging offers the opportunity to reproducibly measure absolute intracellular ion, voltage and pH levels/changes with respect to cell diameter, fluorophore concentration and optical properties of the imaging setup. However, ratiometric imaging depends on a fast change of excitation wavelength or the detected wavelength, a strong light source, excellent transmission of optical components and fast signal detection. The recent development of ultrafast filter wheels, UV-light optimized objectives, highly sensitive fluorophores and new CCD cameras allows affordable quantitative high speed live cell imaging in high spatial resolution.

As mentioned above, in ratiometric imaging an emission shift instead of mere intensity change is imaged. To measure emission shifts, intensity changes of a fluorophore or fluorophore combination have to be measured either by using two different excitation wavelengths or by detecting at two different emission wavelengths. In the case of the commonly used calcium imaging dye Fura-2, the dye has to be excited with light at wavelengths of 340 nm and 380 nm and the detection wavelength is 510 nm. In contrast to that, the calcium imaging dye Indo-1 is usually excited with light at 350 nm wavelength and the detection wavelengths are 405 nm and 485 nm.

Fura-8™ for Ratiometric Calcium Detection

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Although Fura-2 has been widely used as the preferred excitation ratioable calcium indicator, it has certain limitations, e.g., lower sensitivity compared to the single wavelength calcium dyes such as Fluo-8^® and Cal-520™. AAT Bioquest has recently developed Fura- 8™ to improve the calcium response of Fura-2. As demonstrated in Figures 3.1 and 3.2, Fura-8™ AM is more sensitive to calcium than Fura-2 AM. In addition, Fura-8™ has its emission shifted to longer wavelength (Em = 525 nm). Fura-8™ might be also used for the flow cytometric analysis of calcium in cells due to its excellent excitation at 405 nm of violet laser.

Key Features of Fura-8™ Calcium Indicator

• Fura-8™ responses to calcium the same way as Fura-2 does
• Red-shifted dual excitation wavelengths (354 nm/415 nm)
• Better excited at 405 nm for flow cytometric applications
• Compatible with common filter sets.
• Higher signal/background ratio than that of Fura-2

PDMPO, an Unique Dual Excitation and Dual Emission Ratiometric pH Indicator

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The existing pH probes are unsuitable to study acidic organelles such as lysosomes, endosomes, spermatozoa and acrosomes because their fluorescence is significantly reduced at lower pH. In addition, most of the existing pH probes (such as BCECF and SNARF) are not selectively localized in acidic organelles. The growing potential of ratio imaging is significantly limited by the lack of appropriate fluorescent probes for acidic organelles although ratio imaging has received intensive attention in the past few decades. PDMPO [2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl) methoxy)phenyl)oxazole] is characterized as an acidotropic dual-excitation and dual-emission pH probe. It emits intense yellow fluorescence at lower pH and gives intense blue fluorescence at higher pH. This unique pH-dependent fluorescence makes PDMPO an ideal pH probe for acidic organelles with pKa = 4.47. PDMPO selectively labels acidic organelles (such as lysosomes) of live cells and the two distinct emission peaks can be used to monitor the pH fluctuations of live cells in ratio measurements. Additionally, the very large Stokes shift and excellent photostability of PDMPO make it an excellent fluorescent acidotropic reagent for fluorescence imaging. The unique fluorescence properties of PDMPO might give researchers a new tool with which to study the acidic organelles of live cells. PDMPO can be well excited by the violet laser at 405 nm for flow cytometric applications.

Although BCECF and BCFL dextrans are useful for detecting translocation into compartments that have an acidic pH; their relative insensitivity to fluorescence change below pH ~6 limits quantitative pH estimation. The lower pKa values of the PDMPO dextran conjugate make it a more suitable indicator for estimating the pH of relatively acidic lysosomal environments. Moreover, the shift in its excitation and emission spectra in acidic media permits ratiometric pH measurements.

Our PDMPO dextran conjugates can be used to quickly and accurately estimate the pH of lysosomes. As the labeled dextran is taken up by the cells and moves through the endocytic pathway, the PDMPO fluorescence changes from blue in the near-neutral endosomes to yellow in the acidic lysosomes. The greatest change in fluorescence emission occurs near the pKa of the dye at pH ~4.2. The pH in lysosomes can be measured with PDMPO dextrans using fluorescence microscopy or flow cytometry.

BCFL AM, a Superior Replacement to BCECF AM

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BCFL AM is developed to overcome the isomer difficulty associated with BCECF AM. As BCECF AM, BCFL AM exhibits pH-dependent dual excitations, essentially identical to those of BCECF AM. It has a pKa of ~7.0, identical to BCECF AM too. As with BCECF AM, the dual excitation spectrum of BCFL AM with an isosbestic point at 454 nm should make BCFL AM a good excitation-ratiometric pH indicator. BCFL ratiometric imaging makes intracellular pH determination essentially independent of several variable factors, including dye concentration, path length, cellular leakage and photobleaching rate. BCFL AM is a single isomer, making the pH measurement much more reproducible than BCECF AM, which is consisted of quite a few different isomers.